Mikko Laitaoja: Structure-Function Studies of Zinc Proteins
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110/2011 TORVINEN Mika: Mass spectrometric studies of host-guest complexes of glucosylcalixarenes
111/2012 KONTKANEN Maija-Liisa: Catalyst carrier studies for 1-hexene hydroformulation: cross-linked
poly(4-vinylpyridine), nano zinc oxide and one-dimensional ruthenium polymer
112/2012KORHONENTuulia:Thewettabilitypropertiesofnano-andmicromodifiedpaintsurfaces
113/2012JOKI-KORPELAFatima:Functionalpolyurethane-basedfilmsandcoatings
114/2012 LAURILA Elina: Non-covalent interactions in Rh, Ru, Os, and Ag complexes
115/2012 MAKSIMAINEN Mirko: Structural studies of Trichoderma reesei, Aspergillus oryzae and
Bacillus circulans sp. alkalophilus beta-galactosidases – Novel insights into a structure-function
relationship
116/2012 PÖLLÄNEN Maija: Morphological, thermal, mechanical, and tribological studies of polyethylene
compositesreinforcedwithmicro–andnanofillers
117/2013LAINEAnniina:Elementaryreactionsinmetallocene/methylaluminoxanecatalyzedpolyolefin
synthesis
118/2013TIMONENJuri:Synthesis,characterizationandanti-inflammatoryeffectsofsubstitutedcoumarin
derivatives
119/2013 TAKKUNEN Laura: Three-dimensional roughness analysis for multiscale textured surfaces:
Quantitative characterization and simulation of micro- and nanoscale structures
120/2014 STENBERG Henna: Studies of self-organizing layered coatings
121/2014 KEKÄLÄINEN Timo: Characterization of petroleum and bio-oil samples by ultrahigh-resolution
Fourier transform ion cyclotron resonance mass spectrometry
122/2014 BAZHENOV Andrey: Towards deeper atomic-level understanding of the structure of magnesium
dichloride and its performance as a support in the Ziegler-Natta catalytic system
123/2014 PIRINEN Sami: Studies on MgCl2/ether supports in Ziegler–Natta catalysts for ethylene
polymerization
124/2014 KORPELA Tarmo: Friction and wear of micro-structured polymer surfaces
125/2014 HUOVINEN Eero: Fabrication of hierarchically structured polymer surfaces
126/2014 EROLA Markus: Synthesis of colloidal gold and polymer particles and use of the particles in
preparation of hierarchical structures with self-assembly
127/2015 KOSKINEN Laura: Structural and computational studies on the coordinative nature of halogen
bonding
128/2015 TUIKKA Matti: Crystal engineering studies of barium bisphosphonates, iodine bridged ruthenium
complexes, and copper chlorides
129/2015JIANGYu:Modificationandapplicationsofmicro-structuredpolymersurfaces
130/2015 TABERMAN Helena: Structure and function of carbohydrate-modifying enzymes
131/2015KUKLINMikhailS.:Towardsoptimizationofmetaloceneolefinpolymerizationcatalystsvia structuralmodifications:acomputationalapproach
132/2015SALSTELAJanne:Influenceofsurfacestructuringonphysicalandmechanicalpropertiesof polymer-cellulosefibercompositesandmetal-polymercompositejoints
133/2015 CHAUDRI Adil Maqsood: Tribological behavior of the polymers used in drug delivery devices
134/2015 HILLI Yulia: The structure-activity relationship of Pd-Ni three-way catalysts for H2S suppression
135/2016 SUN Linlin: The effects of structural and environmental factors on the swelling behavior of
Montmorillonite-Beidellite smectics: a molecular dynamics approach
136/2016 OFORI Albert: Inter- and intramolecular interactions in the stabilization and coordination of
palladium and silver complexes: DFT and QTAIM studies
137/2016 LAVIKAINEN Lasse: The structure and surfaces of 2:1 phyllosilicate clay minerals
138/2016 MYLLER Antti T.: The effect of a coupling agent on the formation of area-selective monolayers of
iron a-octabutoxy phthalocyanine on a nano-patterned titanium dioxide carrier
139/2016KIRVESLAHTIAnna:Polymerwettabilityproperties:theirmodificationandinfluencesupon water movement
Dissertations
Department of Chemistry
University of Eastern Finland
No. 140 (2016)
Mikko Laitaoja
Structure-Function Studies
of Zinc Proteins
Structure–Function Studies of Zinc Proteins
Mikko Laitaoja
Department of Chemistry
University of Eastern Finland
Finland
Joensuu 2016
Mikko Laitaoja
Department of Chemistry, University of Eastern Finland
P.O. Box 111, FI-80101 Joensuu
Supervisor
Prof. Janne Jänis, University of Eastern Finland, Joensuu, Finland
Referees
Prof. Risto Kostiainen, University of Helsinki, Helsinki, Finland
Assoc. Prof. Vesa Hytönen, University of Tampere, Tampere, Finland
Opponent
Prof. Mariusz Jaskólski, Adam Mickiewicz University, Poznań, Poland
To be presented with the permission of the Faculty of Science and Forestry of the
University of Eastern Finland for public criticism in Auditorium F100, Yliopistokatu 7,
Joensuu, on December 9th 2016, at 12 noon.
Copyright © 2016 Mikko Laitaoja
ISBN: 978-952-61-2352-3
ISSN: 2242-1033
Grano Oy
Joensuu 2016
3
ABSTRACT
Zinc is one of the most abundant metals in biology. A wide variety of proteins include a
zinc cofactor, having roles in protein folding, protein-protein interactions and enzyme
catalysis. Zinc proteins range from small zinc fingers to large multi-protein complexes,
and are present in all enzyme classes with zinc acting as an active site or a structural
metal. Their role in gene expression and regulation makes them especially attractive
targets for biomedical research. High bioavailability, redox-inertness and possibility for
different coordination numbers and geometries are the key aspects that allow zinc to hold
a prominent place in biological systems. In this work, different zinc proteins were studied
by using protein structures available in the Protein Data Bank (PDB) or by experimental
methods, especially high-resolution Fourier transform ion cyclotron resonance (FT-ICR)
mass spectrometry.
Currently, the PDB contains thousands of zinc protein structures. A detailed analysis of
these structures revealed that many previous database surveys suffer from serious flaws
and misinterpretations, such as exclusion of symmetry-related molecules in crystals or
incorrect assignment of ligands, resulting in an incorrect coordination number and/or
geometry. Thus, a more comprehensive analysis was conducted in this work, taking the
aforementioned factors fully into account. Interestingly, a clear dependence of the metalto-ligand bond lengths on the crystallographic resolution was observed, pointing to the
need for further analysis and validation protocols of the metalloprotein crystal structures
when assessing metal ion coordination.
SAP30L is a member of the Sin3A corepressor protein complex, which is involved in
transcriptional regulation. SAP30L contains a novel zinc finger motif, which mediates
the key protein, lipid and DNA interactions of the complex. By using high-resolution
FT-ICR mass spectrometry, we characterized redox-dependent disulfide formation in the
SAP30L ZnF motif and its structural and functional implications. Upon oxidative stress,
SAP30L undergoes formation of two disulfide bonds with a concomitant release of the
coordinated zinc ion. The oxidized SAP30L was shown to remain folded and to bind
signaling phospholipids with markedly higher affinity as compared to holo-SAP30L.
The results suggest that ZnF in SAP30L works as a redox switch, which may be essential
in controlling the repression activity of the Sin3A complex.
Small zinc finger motifs are promising molecular scaffolds for protein design, serving
as versatile building blocks for artificial nanocatalysts or specific biosensors. In the last
part of this work, structural robustness of a designed ZnF motif, named MM1, was
characterized with the use of FT-ICR mass spectrometry. The results showed that MM1
binds zinc specifically with sub-micromolar affinity. Additionally, only gold ions were
able to form a complex with the peptide. Surprisingly, MM1 was able to retain most of
its metal ion binding affinity in the presence of selective alanine mutations of the primary
zinc coordinating amino acid residues, indicating an exceptional structural stability.
4
LIST OF ORIGINAL PUBLICATIONS
This dissertation is a summary of the following original publications I–III.
I
Laitaoja, M.; Valjakka, J.; Jänis, J. Zinc Coordination Spheres in Protein
Structures. Inorg. Chem. 2013, 52, 10983–10991.
II
Laitaoja, M.; Tossavainen, H.; Pihlajamaa, T.; Valjakka, J.; Viiri, K.; Lohi, O.;
Permi, P.; Jänis, J. Redox-Dependent Disulfide Bond Formation in SAP30L
Corepressor Protein: Implications for Structure and Function. Protein Sci.
2016, 25, 572-586.
III
Laitaoja, M.; Isoniemi, S.; Valjakka, J.; Mándity, I.M.; Jänis, J. Deciphering
Metal Ion Preference and Primary Coordination Sphere Robustness of a
Designed Zinc Finger with High-Resolution Mass Spectrometry, Protein Sci.,
in press.
5
CONTENTS
ABSTRACT .................................................................................................................... 3
LIST OF ORIGINAL PUBLICATIONS ........................................................................ 4
CONTENTS .................................................................................................................... 5
ABBREVIATIONS ......................................................................................................... 6
1.
INTRODUCTION................................................................................................. 7
1.1. ZINC PROTEINS ............................................................................................ 7
1.2. ZINC FINGER MOTIFS ................................................................................. 7
1.3. MASS SPECTROMETRY .............................................................................. 9
1.3.1. GENERAL ............................................................................................... 9
1.3.2. ELECTROSPRAY IONIZATION ........................................................... 9
1.3.3. FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS
SPECTROMETRY ................................................................................ 10
1.3.4. PROTEIN MASS SPECTROMETRY ................................................... 12
2.
AIMS OF THE STUDY...................................................................................... 13
3.
EXPERIMENTAL .............................................................................................. 14
3.1. DATABASE SURVEY ................................................................................. 14
3.2. PROTEIN AND PEPTIDE MATERIALS .................................................... 15
3.3. MASS SPECTROMETRY ............................................................................ 16
4.
RESULTS AND DISCUSSION ......................................................................... 17
4.1. DATABASE SURVEY ON ZINC PROTEINS I .......................................... 17
4.2. NMR STRUCTURES .................................................................................... 20
4.2.1. CLASSIFICATION AND COORDINATION SPHERES .................... 20
4.2.2. COORDINATING LIGANDS AND BOND LENGTHS ...................... 21
4.3. X-RAY STRUCTURES ................................................................................ 22
4.3.1. CLASSIFICATION AND COORDINATION SPHERES .................... 22
4.3.2. COORDINATING LIGANDS AND BOND LENGTHS ...................... 24
4.3.3. INCOMPLETE SPHERES..................................................................... 26
4.4. CHARACTERIZATION OF THE SAP30L COREPRESSOR PROTEIN II .. 30
4.4.1. GENERAL ............................................................................................. 30
4.4.2. ZINC-INDUCED FOLDING OF SAP30L ............................................ 32
4.4.3. REDOX-DEPENDENT DISULFIDE FORMATION ........................... 33
4.4.4. PHOSPHOLIPID AND DNA BINDING .............................................. 35
4.4.5. SOLUTION NMR STRUCTURE OF SAP30L ..................................... 36
4.4.6. DISCUSSION ........................................................................................ 37
4.5. DESIGNED ZINC FINGER PEPTIDES III ................................................... 39
4.5.1. GENERAL ............................................................................................. 39
4.5.2. METAL ION BINDING OF ZINC FINGER PEPTIDES ..................... 40
4.5.3. ZINC COORDINATION SPHERE ROBUSTNESS OF MM1............. 42
5.
CONCLUSIONS ................................................................................................. 44
ACKNOWLEDGEMENTS .......................................................................................... 46
REFERENCES .............................................................................................................. 47
6
ABBREVIATIONS
CID
DFF2
EC
ECD
ESI
FT-ICR
HOAc
IUBMB
Kcx
Kd
m/z
MeCN
MM1
MS
NC
NH4OAc
NLS
NMR
PDB
ppm
SAP30
SAP30L
SPPS
ZnF
collision induced dissociation
Designed Functional Finger 2
Enzyme Commission
electron capture dissociation
electrospray ionization
Fourier transform ion cyclotron resonance
acetic acid
International Union of Biochemistry and Molecular Biology
carboxylated lysine
dissociation constant
mass/charge ratio
acetonitrile
Minimal Mutant 1
mass spectrometry
nucleocapsid
ammonium acetate
nuclear localization signal
nuclear magnetic resonance
Protein Data Bank
parts-per-million
Sin3A associated protein 30
Sin3A associated protein 30-like
solid-phase peptide synthesis
zinc finger
7
1.
INTRODUCTION
1.1.
ZINC PROTEINS
Approximately one third of all proteins require a metal cofactor for proper function. In
general, metal cofactors have important roles in protein folding and structural
stabilization, formation of protein–protein assemblies, and when acting as active site
metals in many enzymes.1–6 Zinc holds the prominent place among these metals. The
genomic studies have estimated that zinc is present in about 10% of all proteins
expressed, representing a vast amount of proteins among a broad range of protein
families.7 A steadily growing number of zinc containing protein structures in the Protein
Data Bank (PDB) seems to reflect this. At present, the PDB contains about 10 500
protein structures with coordinated zinc ions.
Zinc is the second most abundant trace metal in the human body after iron.7–10 Structural
zinc sites are made up by small protein motifs or larger protein domains, requiring one
or more zinc ions for correct folding and function.11–13 Zinc ions are typically coordinated
by four cysteine and/or histidine residues, although penta- and hexa-coordinated zinc
ions also exist with a variety of amino acid ligands. In catalytic and co-catalytic zinc
sites, one of the coordination sites is usually occupied by a water molecule, which is
easily displaced by an incoming substrate molecule to initiate the catalysis. Zinc
containing enzymes are present in all six enzyme classes, catalyzing various different
reactions. Notable examples of zinc metalloenzymes are carbonic anhydrases and
alcohol dehydrogenases.14,15 At protein interfaces, zinc ions assist formation of protein
subunit assemblies.2,9,16,17 In these sites, zinc ions may be essential for complex
formation or merely stabilize certain conformations. While some of these interactions
might be present only in a crystalline state, such transient intermolecular interactions
may also be important for the regulation of protein expression.9,18,19 A good example of
a zinc ion mediated protein assembly is insulin, where three protein dimers and two zinc
ions form a hexameric assembly, a main physiological form of insulin.4,20
The methods to study zinc proteins are quite limited due to the spectroscopically silent
nature of zinc. The completely filled d-orbital of Zn(II) ion (d10 electron configuration)
renders zinc diamagnetic and, thus, invisible in electron spectroscopy. In addition, zinc
complexes are colorless and have no absorbance in the ultraviolet or microwave spectral
regions.21 Therefore, indirect methods, e.g. metal substitution, have been used for
biochemical studies of zinc proteins.
1.2.
ZINC FINGER MOTIFS
Protein transcription factors that regulate gene expression and differentiation are
relatively difficult to study owing to their low abundance and dynamic behavior with
nucleic acids. The transcription factor IIIA (TFIIIA) from Xenopus laevis (African
8
clawed frog) was the first to be characterized to contain a series of zinc-dependent
structural motifs, able to “grip” RNA, hence the name “zinc finger” (ZnF). The first
three-dimensional structure of a ZnF motif was determined from the 31st zinc finger of
the Xfin protein (PDB 1ZNF) by using nuclear magnetic resonance (NMR)
spectroscopy. The term “zinc finger” is broadly used to describe any small and compact
protein motif capable of binding zinc ions in a tetrahedral arrangement by a combination
of four cysteine and/or histidine residues. In recent years, the rational design of short
non-native amino acid sequences, capable of performing specific functions, such as
metal binding, oligomerization or genome editing with nucleases has gained
considerable interest.22–25 ZnF motifs provide an excellent starting point for such
endeavors due to their small size, straightforward production and easily tailorable
structures.
Figure 1 shows the three-dimensional structures for different types of zinc fingers. The
first structure is a canonical Cys2His2 ZnF motif from the Xfin protein (PDB 1ZNF),11
in which the zinc ion is bound in a tetrahedral geometry by four amino acid ligands, two
cysteine and two histidine residues.12 In the middle is a structure of the CREB binding
protein (CBP) (PDB 1U2N), where three zinc ions coordinate to the same polypeptide
chain, forming a scaffold for protein binding.26 When the terminal ligands of the two
“zinc bundles” are removed, the region shown in blue folds independently to a stable
non-natural structure.27,28 On the right is the N-terminal zinc finger from the HIV-1
nucleocapsid protein 7 (NCp7) (PDB 1HVO) bound to the pentanucleotide.29
Figure 1. Cartoon representation of structures of different types of zinc finger (ZnF) motifs. a)
Canonical Cys2His2 zinc finger from X. laevis Xfin protein (PDB 1ZNF), b) CREB binding
protein (CBP), containing three consecutive zinc finger motifs (PDB 1U2N) and c) N-terminal
zinc finger of the HIV-1 nucleocapsid protein 7 (NCp7) bound to the pentanucleotide d(ACGCC)
(PDB 1HVO).
9
1.3.
MASS SPECTROMETRY
1.3.1.
GENERAL
Mass spectrometry (MS) is an analytical technique that aims to determine the neutral
molecular mass of an analyte (an atom or a molecule) by measuring the ratio of the mass
and the charge (m/z) of the corresponding ion. The current definition of m/z ratio defines
it as a dimensionless quantity, calculated by the mass of the ion (given in unified atomic
mass units, u or Da) divided by its charge number (z).30 The analyzed ions can be
positive, negative or radical ions. Since mass spectrometry is independent on the
spectroscopic nature of the analyte, it provides excellent means to characterize various
different types of molecules, reactions and interactions.31–33 Besides analysis of intact
molecules, mass spectrometry can be used to further identify or characterize ions by
fragmenting them into smaller components.34,35 Various types of instruments have been
developed, each with its own strengths and weaknesses, including ionization methods
available, mass range (the lowest and the highest detected m/z), mass resolution and mass
accuracy, sensitivity, dynamic range, speed, and the capability of performing tandem
mass spectrometry (MS/MS) experiments.
1.3.2.
ELECTROSPRAY IONIZATION
A mass spectrometer is only able to analyze charged atoms or molecules and therefore
the analyte molecules have to be ionized and transferred into the gas-phase. The most
common ionization method used in the biomolecule analysis is electrospray
ionization (ESI).36–38 ESI is a “soft” ionization method causing very little fragmentation
of the analyte molecules or even their non-covalent complexes, thus making it suitable
for analysis of interactions between biomolecules, e.g. proteins, nucleic acids,
carbohydrates, metal ions or other small ligands.39
In ESI, a polar volatile solvent containing analyte molecules, typically at low micromolar
concentrations, is sprayed through a narrow-bore metal capillary placed in a high electric
field in ambient conditions.40 The electrical field causes a formation of a Taylor cone
and a jet at the apex of the cone. Due to electrostatic repulsion of the ions, the jet breaks
into small non-spherical droplets carrying excess ionic charge (Figure 2). The charge
imbalance of the ions in the droplets is one of the key aspects of ESI, which eventually
leads to the formation of gas-phase ions. The electrospray can be regarded as an
electrochemical cell where the current is transported by the droplets. The evaporation of
the solvent molecules from the droplets causes repeated shrinking and coulombic
fissions of the droplets, ultimately forming gas-phase ions. Current instruments use a
pneumatic nebulizer, where the inner metal capillary carries the solution and the outer
capillary carries pressure-controlled nitrogen gas to produce uniformly sized droplets
and to improve the evaporation of the droplets and the ionization process. A heated
countercurrent drying gas is used to further aid the evaporation process and to block the
neutrals from entering into the ion source.
10
Another key aspect of the ionization process of large biomolecules in ESI is a multiple
charging phenomenon, which means that the same analyte may be present in the
spectrum with several signals, corresponding to different amounts of charge carriers
(e.g., protons) attached.41,42 Most biomolecules are either polybasic or acidic in nature
and have multiple sites of charging, which usually complicates the spectral
interpretation. The main advantage of the multiple charging phenomenon is that it lowers
the requirement for the highest detected m/z for a given mass. In addition, mass accuracy
is statistically increased when the mass is calculated from several peaks.
Figure 2. Schematic representation of the electrospray process. In the presence of a high electric
field the solution creates the typical cone-jet plume from the Taylor cone upon exiting the
capillary.
1.3.3.
FOURIER TRANSFORM ION CYCLOTRON RESONANCE
MASS SPECTROMETRY
In Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, detection of
the externally generated ions occurs in an ICR cell, placed in the center of a large
superconducting magnet.43–45 The ICR cell consists of pairs of trapping, excitation and
detection plates (Figure 3a). In the presence of a high magnetic field, the ions start to
circulate around the magnet axis, at a frequency which is dependent only on the m/z of
the ions and the strength of the magnetic field. This phenomenon is called the cyclotron
motion. The magnet is able to confine ions only radially; the axial confinement is
accomplished by using a static electric field (a few volts) set to the trapping plates. The
radius of the natural cyclotron motion is too small and incoherent for detection, so in
order to produce a coherent measurable signal the ions are excited to a larger radius by
using a short radio-frequency (RF) pulse, applied to the excitation plates (Figure 3b).
After the excitation, the ions circulate in a larger cyclotron radius and induce a small
11
alternating current (image current) between the detection plates, known as the timedomain transient (Figure 3c). In the presence of an ultra-high vacuum (∼10-10 mbar)
inside the cell, the ions travel tens of kilometers in one second, a typical transient time.
Modern FT-ICR instruments have the capability of measuring millions of data points
during the data acquisition period, providing unparalleled resolution and mass accuracy.
In principle, mass resolution can be increased by acquiring a longer transient or by using
a higher field magnet. The higher the field, the higher the cyclotron frequency and faster
the data acquisition is for a given resolution.46 For the same post-excitation radius, the
theoretical maximum resolution does not increase with the field strength; however, the
higher field allows longer transients to be measured without a signal decay, resulting in
higher resolution.34 The time-domain transient is then Fourier transformed into a
frequency-domain signal, followed by magnitude calculation and frequency-to-mass
conversion. Figure 3d shows the ESI FT-ICR mass spectrum of a protonated arginine
(monoisotopic mass 175.11895 Da) at 12-T field. Due to the obtained ultra-high
resolution (∼300 000), the arginine 15N and 13C isotopologue ions (176.11599 and
176.12231 Da, respectively) can be fully resolved. Note that the mass difference between
these two ions is only ∼6.3 mDa, which corresponds to the mass of a dozen electrons.
Figure 3. a) Geometry and construction of a cylindrical ICR cell. b) Principles of ion excitation
and detection in the ICR cell. c) Time-domain transient. d) 12-T ESI FT-ICR mass spectrum of
protonated arginine (the inset shows the magnified view at the first isotopic peak pattern).
12
1.3.4.
PROTEIN MASS SPECTROMETRY
High sensitivity and small sample requirement associated with mass spectrometry have
made it an indispensable tool in proteomics, and the advent of soft ionization methods,
has enabled measurements of intact proteins and even their non-covalent complexes.47-53
Figure 4a shows an ESI-MS spectrum of a recombinant non-glycosylated form of an
avidin protein, expressed in E. coli, measured in a 500 mM ammonium acetate buffer
(pH 6.8) at 1.5 µM concentration.54–56 The signals correspond to the charge states of 16+,
17+, and 18+ of the intact avidin tetramer, whose crystal structure (PDB 2AVI) is shown
above. The approximate mass of the protein can be obtained by spectral deconvolution
(see inset in Figure 4a). Figure 4b shows the same protein measured in a mixture of
acetonitrile (MeCN) and water (50:50, v/v) with 1% of acetic acid (HOAc), showing
only an unfolded protein monomer. Since the spacing of the baseline-resolved isotopic
peaks directly provides the charge state as 1/z, the mass of the protein can be accurately
calculated from any signal. Figure 4c shows a signal for a single fragment ion,
corresponding to a C-terminal peptide of the avidin protein. Fragment ions can be
obtained through MS/MS experiments, in which intact protein ions are fragmented, e.g.
by collision induced dissociation (CID) or electron capture dissociation (ECD)
techniques (a method known as top-down mass spectrometry).57–59 Such experiments can
be used for determination of the amino acid sequences or post-translational
modifications.60–63
Figure 4. Protein mass spectra at different structural levels (see text for details). a) Native
tetrameric avidin. The inset shows a deconvoluted mass spectrum. b) Avidin monomer and
c) C-terminal fragment ion, corresponding to the residues of D109–E128. The structural models
above the spectra are based on the crystal structure of egg-white avidin (PDB 2AVI).54
13
2.
AIMS OF THE STUDY
Zinc proteins play essential roles in many physiological processes as enzymes, storage
proteins, transcription factors, and replication proteins. Therefore, knowledge of their
structure–function relationships is of importance for understanding these processes at
the molecular level. Moreover, designed metal binding protein scaffolds, based on zinc
proteins, hold great promise as versatile building blocks for a variety of purposes (e.g.,
artificial catalysts or specific biosensors). In this work, the structure and function of
several different zinc proteins were studied. The specific aims were as follows:
1. Analysis of all zinc protein structures available in the PDB with respect to their
structural classification, coordination geometry, ligand types, and metal-to-ligand
bond lengths. These structures were manually analyzed to overcome some softwarerelated limitations. The study aimed at addressing some of the shortcomings of the
previous database surveys, and providing a more complete, up-to-date view on the
zinc coordination environments in proteins.
2. Structural characterization of the SAP30L corepressor protein, especially its redoxdependent disulfide bond formation, by using high-resolution ESI FT-ICR mass
spectrometry. SAP30L has been previously shown to contain an N-terminal Cys3His
zinc finger motif, a key structural element for its folding and function. In addition,
the aim was to determine the three-dimensional structure for holo-SAP30L and to
study its DNA and lipid binding by using NMR spectroscopy.
3. Analysis of metal ion preference and structural robustness of the designed zinc finger
motif (named MM1) by using ESI FT-ICR mass spectrometry. The main aim of the
study was to characterize the primary coordination sphere robustness of MM1
towards efficient metal ion binding upon selective alanine substitutions of the
primary zinc coordinating amino acid residues.
14
3.
EXPERIMENTAL
3.1.
DATABASE SURVEY
The Protein Data Bank (as of January 18, 2012) was queried for molecules containing
zinc ions.10 In this study, only the protein structures without nucleic acids or their
complexes were analyzed. Further analyses and handling of the data were conducted
separately for the NMR and crystal structures. To avoid redundancy, removal of
structures with 95% sequence identity was accomplished via the BLASTClust algorithm.
No other restraints, such as resolution, molecular mass or atom-specific cut-offs, were
used in collecting the dataset. Since a large part of zinc proteins are enzymes, a separate
search was performed on each enzyme class and the enzyme datasets were compared
with the total number of zinc proteins. The final working dataset contained a total of
2616 structures (590 NMR and 2026 crystal structures). The structures were classified
according to the Enzyme Commission (EC) numbers, assigned to each structure based
on the reaction it catalyzes. The structures without enzymatic activity were classified as
structural sites.
The structures were initially analyzed using Ligand Explorer 3.9 (available in the PDB),
for zinc coordinating ligands, metal-to-ligand bond lengths and coordination geometries.
In cases where the coordination sphere showed a largely distorted geometry or was
clearly missing some of the coordinating ligands, the corresponding original publications
(if available) were inspected for clarification. Further analyses of the structures were
performed by using PyMOL 1.3 software.64 Protein figures in the original publications
and in this thesis were created using the same software.
Coordinating ligands were listed in order of appearance and marked by their three letter
codes, e.g. Cys for cysteine and Kcx for carboxylated lysine. Water was marked as Wat
and the other exogenous ligands, inhibitors and solvent molecules by their coordinating
atoms, e.g. O for oxygen, N for nitrogen, and Cl for chloride. Histidine can bind via
either of its nitrogen atoms; however, the structural and functional significance of these
two binding modes remains elusive, thus the binding modes were not treated separately.
Carboxylates, such as aspartate, also possess multiple binding modes. In this study,
aspartate and glutamate residues were treated as monodentate ligands, i.e., possessing a
two-electron donor to a single zinc ion. The bidentate mode observed for carboxylates
would form a four-membered ring, with two highly distorted orbitals and is represented
by resonance structures of single coordination bonds. In the coordination spheres with
four ligands, where a bidentate mode was observed, the bond angles of the other ligands
were consistent with the tetrahedral geometry, thus supporting the analysis. When
bridging two different metal ions, a carboxylate group can coordinate with both of its
oxygen atoms. Since the coordination spheres usually have some distortions from ideal
geometries, the structures were broadly categorized into tetrahedral, trigonal
bipyramidal/square pyramidal, octahedral and incomplete geometries.
15
3.2.
PROTEIN AND PEPTIDE MATERIALS
The N-terminal zinc finger motif of SAP30L (residues 25-92) was produced as a GST
fusion protein by using a modified expression construct in the pGEX-4T1 vector in
Escherichia coli Rosetta (DE3) cells.65 Thrombin and GST protein were removed by
using benzamidine and glutathione affinity columns (GE Healthcare) and the SAP30L
protein was further purified by using cation exchange (Resource S column; GE
Healthcare) and size exclusion chromatography (Superdex 75 column; GE Healthcare).
The construct used contained two additional N-terminal amino acids (GS) from the
expression vector construct (a thrombin cleavage site).
For mass spectrometric experiments, SAP30L was buffer-exchanged to 20 mM
ammonium acetate (NH4OAc) buffer (pH 6.8) by using PD-10 desalting columns
(GE Healthcare). Concentrations of the eluates were determined by UV absorbance at
280 nm, based on the extinction coefficient calculated from the amino acid sequence.
The coefficient values used were 2560 M–1 cm–1 for the apo (without zinc) and holo (with
zinc) forms and 2800 M–1 cm–1 for the oxidized form (ox).66 The protein eluates were
concentrated with 3K MWCO (molecular weight cut-off) centrifugal concentrators
(Amicon Ultra or Vivaspin2; Millipore) and were stored at 4 °C until used. Water,
acetonitrile and acetic acid were HPLC or the highest quality available. Chelation of zinc
ions was performed with 1,10-phenanthroline, the in vitro oxidation was carried-out with
hydrogen peroxide (H2O2) and disulfide reduction was done with dithiothreitol (DTT).67
For disulfide bond analysis of SAP30L, trypsin digestion was performed at a 1:40 (w/w)
protease-to-substrate ratio in 20 mM NH4OAc (pH 6.8) at room temperature, from 15
minutes to overnight. Prior to the measurements, acetonitrile and acetic acid were added
to the solutions to quench the reaction and enhance ionization. Online (pepsin) digestion
was performed using a previously published protocol.68 The resulting peptides from all
digestions were analyzed by using GPMAW 7.10 software (Lighthouse Data, Denmark)
or online ProteinProspector tools (available at http://prospector.ucsf.edu). The C8phosphoinositides (PIPs) were dissolved in a 20 mM NH4OAc buffer and used without
further purification. The lipids were stored in a freezer until used. The binding
experiments were performed by mixing appropriate amounts of SAP30L and lipid
samples and the mixtures were heated at 50 °C for 15 minutes.
The zinc finger peptides (MM1, DFF2, NC and the MM1 mutants) were synthesized
either manually by solid-phase peptide synthesis (SPPS) at the University of Szeged
(Szeged, Hungary) or purchased from Genecust (Dudelange, Luxembourg) as a
lyophilized powder (≥ 95% purity) and used without further purification. The
commercial peptides were identical to those made by SPPS, except that their C-terminus
was a free acid compared to the amide from the SPPS, due to the Tentagel R RAM resin
used in the synthesis. The oxidized peptides were reduced by heating the stock solutions
at 70 °C for 10 minutes in the presence of 1 mM DTT and stored at 4 °C until analyzed.
The metal ions (Ag+, Cd2+, Co2+, Cu2+, Hg2+, Mg2+, Mn2+, Ni2+ and Zn2+) were added as
acetate salts, except for Au+, where a chloride salt was used (all from Sigma-Aldrich).
16
3.3.
MASS SPECTROMETRY
All mass spectrometry experiments were performed with a Bruker Apex-Qe FT-ICR
mass spectrometer, equipped with a 4.7-T superconducting magnet, an Infinity ICR cell,
an Apollo II electrospray ionization (ESI) source and a mass selective quadrupole. The
positive ionization mode was used in all measurements. The ultra-high vacuum
(~10-10 mbar) needed was generated with two rotary pumps and four turbomolecular
pumps. The mass range of m/z 387–4000 and a 512-kWord time-domain transient size
were used in all measurements, providing a maximum theoretical mass resolution of
∼170 000 at m/z 500. The mass spectra were externally calibrated with respect to the ions
of the ESI Tuning Mix calibration mixture (Agilent Technologies). The data were
measured and further processed with XMASS 7.0.8 software.
For intact protein mass analysis in denaturing solution conditions, SAP30L was diluted
with a MeCN/H2O/HOAc (49.5:49.5:1.0, v/v) solvent mixture. For measurements in
native conditions, 20 mM ammonium acetate (pH 6.8) was used as a solvent instead.
The samples were directly infused using a syringe pump at a flow rate of 1.5 µL/min
with dry nitrogen gas serving as the nebulizer gas, at a pressure of 1.0 bar. Heated
nitrogen gas was used as the drying gas with a flow rate of 5.0 L/min and temperature
of 240 °C. Electron capture dissociation (ECD) measurements were performed by
isolating the peptide ion of interest in the quadrupole with an isolation window of 5 m/z
units and subjected to low-energy electrons inside the ICR cell. For the lipid binding
experiments, approximately ~70 spectra were measured from a single sample using a
serial measurement program. The binding affinities were calculated from the chargenormalized intensities over all the observed charge states.69
Similar parameters and solvent conditions were used for the measurements of the zinc
finger peptides. A mass range of m/z 184–2000 was used in the measurements, and the
instrument parameters were adjusted for optimal detection of the peptide signals and to
prevent unintentional collisional activation of the peptide–metal ion complexes. The
solvent used for the metal binding experiments was MeCN/H2O (50:50, v/v; pH 7.0),
since pure water or an acetate buffer resulted in rapid peptide oxidation, and the
excessive use of DTT (for reduction) was not possible due to its zinc ion affinity. The
determination of the zinc binding affinity of MM1 was performed using a microchipbased nanoESI ion source (Advion Triversa Nanomate), where the flow rate is
approximately 0.2 µL/min. In these measurements, the MM1 peptide concentration was
1.0 µM while the zinc concentration varied between 0.25 and 5.0 µM. The positions of
the disulfide bonds in the oxidized peptides were determined by using collision-induced
dissociation (CID) experiments.
17
4.
RESULTS AND DISCUSSION
4.1.
DATABASE SURVEY ON ZINC PROTEINS I
At the time of the database survey, the PDB (http://www.rcsb.org/pdb) contained about
76000 structures, with zinc present in around 7100 of them, reflecting the amount of zinc
proteins estimated from genomic studies (ca. 10%). Zinc has the highest amount of
structures and binding sites, and is only surpassed by magnesium in the number of
individual ions.70 Most protein structures present in the PDB are determined from
human, mouse, E. coli and yeast proteins. Highly similar structures were removed from
the initial search results to avoid data redundancy (see, Experimental section for details).
Thus, the working dataset contained about 35% of all deposited zinc protein structures.
Since the search was conducted separately for NMR and crystal structures, the same
protein structure may be present in both datasets, such as the zinc finger of histone lysine
demethylase JARID1A (PDB entries 2KGI and 3GL6).71 Figure 5 shows the most
common metal ions in protein structures and the growth of the unique zinc protein
structures available in the PDB over the past twenty years.
Figure 5. a) Number of protein structures containing different metal ions in the Protein Data Bank
and b) growth of the unique zinc protein structures, determined by NMR spectroscopy (red) and
X-ray crystallography (blue).
The analyzed structures amounted to around 85% of zinc proteins determined by NMR.
It must be noted that around 60% of the structures do not have a journal article associated
to them, as most of these structures are from structural genomics or proteomic initiatives.
Figure 6 shows the molecular mass distributions of zinc proteins determined by NMR
and X-ray. The NMR structures have an average molecular mass of 8.6 kDa, thus the
majority of these structures are from small zinc fingers. A classical zinc finger contains
one zinc ion tetra-coordinated by four amino acid residues, whereas PHD-, LIM- and
RING-type zinc fingers contain two zinc ions. The presence of zinc has to be known
beforehand, as zinc is “invisible” in NMR spectroscopy and has to be determined by
using distance and bond angle restraints, and the models are calculated based on these
values. The structures are highly similar as the short sequence motifs require less
18
changes in the sequence to fulfill the identity criteria (<95%, i.e. 1/20 amino acids). This
can also be seen from the molecular mass distribution (bimodal; centered around 5 and
10 kDa) (Figure 6a). The structures beyond 10 kDa are metallothioneins, enzymes and
repeats of the single zinc finger motifs. The structure with the highest molecular mass
determined with NMR is hexameric insulin with a mass of 35.6 kDa (PDB 1AI0).72 The
NMR structures contained a total of 922 zinc ions.
Figure 6. Molecular mass distributions of zinc proteins determined by a) NMR and b) X-ray
crystallography. Red bars in b) correspond to proteins where zinc ions are merely crystallization
artifacts.
Among the crystal structures, numerous zinc proteins have been determined several
times in different space groups, as different expression constructs or mutant structures,
different protein–ligand complexes, or simply at higher resolution, and these duplicate
structures were removed from the working dataset. For example, the database query for
carbonic anhydrase II results in over 400 nearly identical structures.73 The largest zinc
protein crystal structure is from RNA polymerase II (PDB 3H0G) with the structural
molecular mass of 996.1 kDa. Figure 6 emphasizes the main difference between NMR
and X-ray crystallography; ca. 90% of the zinc protein structures determined with NMR
are spanning the mass range of 2–20 kDa, whereas the mass range for X-ray extends up
to 1000 kDa. Thus, NMR is still, in practice, limited to the study of only small proteins
(with a few exceptions), despite major progress in the field in recent years.
The resolution range for zinc proteins determined by X-ray crystallography is from
0.79 Å (atomic resolution) up to 4.30 Å with a weighted average of 2.06 Å (Figure 7a).
The average resolution has remained the same for the past 20 years, despite improvement
in the crystallization techniques and the use of synchrotron radiation sources (data not
shown). About 96% of the structures have a resolution better than 3.00 Å and the rest
are very large protein complexes, where the size sets an obvious limit to the achievable
resolution. In addition, the average structural molecular mass increases quite linearly
with the achieved crystallographic resolution (Figure 7b). In total, the 2026 analyzed
X-ray structures contained 6950 zinc ions.
19
Figure 7. a) Distribution of crystallographic resolution for zinc proteins (0.1 Å bins) and b)
average structural molecular mass as a function of resolution. Red bars/dots correspond to the
structures where the zinc ion is considered a crystallization artifact.
A large number of structures contained zinc ions bound to the protein surface, where the
number of ligands ranged from zero to six with zero being the most common occasion.
Usually the binding was accompanied by more than one water molecule as a result of a
nearby solvent channel. By inspecting these structures and the corresponding
publications, it was realized that these zinc ions are not required for the protein function
and are merely crystallization artifacts resulting from high concentrations of zinccontaining buffers used upon crystallization experiments (Figures 6b and 7a). Many
publications indicated that higher quality crystals having better X-ray diffraction could
be obtained in the presence of zinc. Zinc ions presumably aid the crystal formation by
stabilizing intermolecular contacts.
20
4.2.
NMR STRUCTURES
4.2.1.
CLASSIFICATION AND COORDINATION SPHERES
The coordination spheres in the NMR structures mainly comprise of tetrahedral zinc
sites (98%) with cysteine and histidine residues. As most of them are from small zinc
fingers they possess only a structural role. The total number of enzymes among the NMR
structures is 84 (of 590 structures). The majority of enzymes are hydrolases (EC 3.x.x.x)
and transferases (EC 2.x.x.x) with minor contributions from ligases (EC 6.x.x.x) and
oxidoreductases (EC 1.x.x.x). Only one lyase (EC 4.x.x.x) and no isomerases (EC
5.x.x.x) were found among the NMR structures. However, as the molecular mass of these
enzymes is less than 22 kDa, it can be assumed that the zinc ions are structural rather
than active site metals, the number of active site metals is less than 2%. These domains
most likely have a role in substrate recognition or in regulating the enzyme activity, as
can be seen from the zinc functions in transferases and ligases. Figure 8 shows
classification and the coordination geometry of zinc ions in the analyzed NMR
structures.
Figure 8. a) Classification and b) coordination geometry for zinc ions in protein structures
determined by NMR. In a), the two numbers for enzyme classes indicate the distribution of zinc
ions in active sites and structural sites, respectively.
In the NMR structures, all structural zinc sites are tetrahedral (98% share), reflecting the
high amount of zinc fingers and the low amount of zinc enzymes deposited. Trigonal
pyramidal spheres were found in the enzyme-inhibitor complexes. Moreover, a few
tricoordinated patterns were found in enzyme active sites, where the geometry clearly
pointed to a tetrahedral coordination. Indeed, original publications indicated that the
“empty” coordination sites in these structures are occupied by water molecules,
increasing the actual coordination number to four. The most common coordination
spheres in NMR structures are Cys3-His (36.4%), Cys4 (29.6%) and Cys2-His2 (26.6%),
with some positional variations. As most of the NMR structures are small zinc fingers,
cysteine and histidine residues account for 97.5% of coordinating ligands, in an
approximate ratio of 3:1. Other coordinating amino acids are aspartate and glutamate
residues, both having a ∼1% share. The rest of the ligands in these structures are oxygenor sulfur-containing small molecules (inhibitors) or water molecules.
21
4.2.2.
COORDINATING LIGANDS AND BOND LENGTHS
The bond lengths in the NMR structures were typically within the accepted values for
zinc-ligand distances (Table 1). However, some unrealistic bond lengths were also
found. The shortest bond length found was only 1.23 Å for a Zn–Cys bond (PDB 2FUU).
In contrast, the longest bond found was 4.12 Å, observed for a Zn–His bond (PDB 2JMI).
In these NMR ensembles, the quality of the calculated models is very low, resulting in
clearly incorrect coordination geometries and bond lengths.74,75 In general, Zn-Cys bond
lengths in the NMR structures display a rather symmetrical and narrow distribution,
centered at 2.35 Å (Figure 9). For Zn-His bond lengths, a bimodal distribution can be
observed instead, peaking at 2.05 Å and 2.35 Å, the latter being roughly the same as the
zinc–cysteine bond length. This results when the cysteine and histidine residues are not
treated separately in the structure calculation. A bimodal distribution is observed for
glutamate, and the average bond length (1.83 Å) is markedly shorter than for aspartate
(2.10 Å), although the coordination should be identical. The low counts for Zn–Glu and
Zn-Asp bonds, however, prevent good statistical comparison of these ligands.
Table 1. Coordinating ligands in zinc structures determined with NMR spectroscopy.
Coordinating ligand
Occurrence
Bond length (Å)a
Relative share (%)
Cysteine (Cys)
2659
2.32 ± 0.16
72.16
Histidine (His)
936
2.09 ± 0.14
25.40
Glutamate (Glu)
36
1.83 ± 0.16
0.98
Aspartate (Asp)
33
2.10 ± 0.24
0.90
Other oxygen (O)
17
2.20 ± 0.13
0.46
Water (Wat)
3
2.18 ± 0.03
0.08
Other sulfur (S)
1
2.63
0.03
Total
3685
a
Reported as average ± standard deviation
Figure 9. Distributions of zinc-ligand bond lengths (0.05 Å bins) in NMR structures for
a) cysteine, b) histidine, c) glutamate and d) aspartate.
22
4.3.
X-RAY STRUCTURES
4.3.1.
CLASSIFICATION AND COORDINATION SPHERES
Protein structures determined by X-ray crystallography dominate in the PDB database
and this also applies to zinc proteins. When the search was limited to enzyme classes,
some additional structures were also found, as compared to the full database search. The
additional structures are high-resolution structures that have not been classified as
enzymes or are highly similar structures without enzymatic function, which are then
excluded from the search. Furthermore, some enzymes have not been given an EC
number, although the publications clearly state enzymatic function for these proteins.
These structures were added to their corresponding enzyme classes or to the “other
enzymes” class if the reaction catalyzed was uncertain. Similar to the NMR structures,
for oxidoreductases, hydrolases, lyases and isomerases, a majority of the zinc ions are
active site metals (Figure 10). On average, enzyme structures contained three zinc ions,
which indicates a tendency to form higher oligomers as the functional unit, such as
tetrameric D-hydantoinase or trimeric γ-carbonic anhydrase.3,76 In the X-ray structures,
the coordination sphere was more diverse, but mostly tetrahedral geometries were found
(Figure 10). Most structural proteins and mononuclear enzymes are tetrahedral, while
many binuclear enzymes and inhibitor complexes display trigonal bipyramidal
coordination geometry.
Figure 10. a) Classification and b) coordination geometry for zinc ions in protein structures
determined by X-ray crystallography. In a), the two numbers for enzyme classes indicate the
distribution of zinc ions in active sites and structural sites, respectively.
The most common coordination spheres in the X-ray structures are the same as in the
case of NMR structures, with Cys4 having a prominent share (ca. 20% out of all spheres,
or 842 out of a total of 4415 zinc ions). Table 2 summarizes the most common
coordination spheres present in different classes of zinc proteins determined by X-ray.
As nearly 50% of the structures are from enzymes, the coordination is much more diverse
as compared to the NMR structures (more than 500 different spheres found).
23
Table 2. The most common coordination spheres in different zinc protein classes determined by
X-ray crystallography.
Functional class
Common coordination spheres (share-%)a
Structural
Cys4 (31.4%), Cys2-His-Cys (10.9%), Cys2-His2 (4.7%),
His-Cys3 (4.5%), His3-Asp (4.0%), Glu2-His-Glu (2.0%)
Oxidoreductase
Cys4 (27.7%), His3-Asp (13.3%), Cys-His-Cys (12.5%),
Cys-His-Asp, (6.3%), Cys-His-Glu (3.9%), Asp-His-His (3.7%)
Transferase
Cys4 (45.8%), Cys3-His (6.9%), His-Cys3 (4.9%),
Cys2-His-Cys (3.8%), Cys3 (3.6%), Cys-His-Cys2 (3.3%)
Hydrolase
Cys4 (7.1%), His3 (5.8%), His2-Glu (5.7%), His2-Kcx-Asp (4.3%),
Kcx-His2 (4.3%), His3-Asp (4.2%), His2-Glu; (4.0%),
Asp-His2 (3.3%), His-Glu-His (3.1%), Asp-Glu-His (3.0%),
His2-Asp2 (2.9%)
Lyase
His3 (28.6%), Cys-His-Cys (12.0%), Asp-His2 (8.1%),
Cys-Asp-His-Cys (7.3%), Glu2-His2 (5.1%), Cys3 (3.8%)
Isomerase
His-Asp-His-Asp (17.0%), Cys4 (9.6%), Glu-Asp-His-Asp (8.5%),
His3 (6.4%), His2-Glu-His (5.3%)
Ligase
Cys4 (42.9%), Cys-His-Cys2 (13.8%), Cys2-His-Cys (11.7%),
Cys3-His (6.1%), Cys-His2 (3.6%), Cys2-His2 (3.1%)
Unclassified enzyme
His2-Glu (13.7%), His3 (9.2%), His2-Glu-Asp (5.9%),
His2-Kcx-Asp (5.2%), Kcx-His2 (5.2%), Glu-Asp-His-Glu (5.2%),
His3-Asp (4.9%), His-Glu-His (4.2%), Asp-His2 (4.2%)
Artifact
His (8.3%), His-Glu (5.7%), Glu2 (5.6%), Asp (4.4%), Glu (4.1%),
His-Asp (4.1%), Asp2 (4.0%), Glu-His (3.6%), His2 (3.6%),
Glu3 (3.2%), Asp-His (3.1%)
a Note that these contain only the incorporated protein ligands and the actual structures may also contain
various external ligands, especially at low coordination numbers (incomplete spheres).
In enzymes, typically three amino acid residues are involved in the zinc ion binding and
the fourth coordination site is occupied by a water molecule, substrate or an inhibitor
molecule. In oxidoreductases, the most common sphere is Cys4, followed by His3-Asp,
due to the high number of Cu/Zn superoxide dismutases, and Cys-His-Cys, which is
typical in alcohol dehydrogenases and where the fourth coordination site is occupied by
various oxygen ligands. Most inhibitors are based on sulfonamide, hydroxamic acid or
phosphonate functional groups. Hydrolases have the most diverse coordination, most
likely owing to the higher number of structures. Lyases, isomerases and the unclassified
enzymes have similar spheres as in hydrolases. Transferases and ligases have mostly
classical zinc finger coordination spheres (~85 %), which suggests that these enzymes
use zinc merely for stabilization of the structures rather than the active site metal.
24
4.3.2.
COORDINATING LIGANDS AND BOND LENGTHS
Cysteine and histidine are the most frequent coordinating residues in X-ray structures,
followed by aspartate, glutamate and a water molecule (Table 3). Other oxygencontaining (non-amino acid) ligands have the next biggest share, including a variety of
molecules (e.g., substrates and inhibitors). Carboxylated lysine (Kcx) is also a notable
ligand which has not been categorized in the previous studies. Kcx is an important ligand
in binuclear zinc enzymes and is frequently found in hydrolases. Interestingly,
carboxylated lysine residues were not observed in the coordination spheres of artifact
zinc ions.
Table 3. Coordinating ligands in zinc protein structures determined with X-ray crystallography.
Coordinating ligand
Cysteine (Cys)
Histidine (His)
Aspartate (Asp)
Water (Wat)
Glutamate (Glu)
Other oxygen (O)
Carboxylated lysine (Kcx)
Other nitrogen (N)
Chlorine (Cl)
Lysine (Lys)
Asparagine (Asn)
Other sulfur (S)
Serine (Ser)
Threonine (Thr)
Tyrosine (Tyr)
Glutamine (Gln)
Phosphoserine (Sep)
Selenomethionine (Mse)
Methionine (Met)
Formylglycine (Fgl)
Arginine (Arg)
Bromine (Br)
Tryptophan (Trp)
Total
Functional
Artifact
Total
6102
5716
2026
1753
1293
974
161
137
65
54
51
49
24
24
22
20
7
5
4
4
18491
122
1810
1415
2586
1952
546
193
115
46
50
1
42
36
14
37
2
7
6
2
2
8984
6224
7526
3441
4348
3245
1521
161
330
180
100
101
50
66
60
36
57
9
5
11
4
6
2
2
27475
The bond length analysis of all coordinating residues shows that cysteine has a quite
symmetrical and narrow length distribution, while the others have clearly broader
distributions, which are tailing towards the longer bond lengths (Figure 11). For some
residues, especially nitrogen and carboxylated lysine, the occurrence counts are low, thus
the distributions are not well defined.
25
Figure 11. Distributions of zinc-ligand bond lengths (0.05 Å bins) in the X-ray structures for a)
cysteine, b) histidine, c) aspartate, d) glutamate, e) nitrogen, f) carboxylated lysine, g) water and
h) oxygen ligands.
Figure 12 shows average zinc–ligand bond lengths for cysteine, histidine and water as a
function of crystallographic resolution. The average bond lengths clearly increase when
the resolution lowers. This is rather surprising, since the bond length should be
independent of the achieved resolution. Furthermore, the data shows that the bond length
is only dependent on the crystallographic resolution, and not affected by the date of the
data acquisition, molecular mass of the protein or the refinement method applied.
Figure 12. Zinc-ligand bond lengths and average bond length per crystallographic resolution
(0.1 Å bins) for cysteine (a,b), histidine (c,d) and water (e,f) ligands.
26
Interestingly, the Zn–His bond length is 2.03 ± 0.04 Å and Zn–Cys bond length is
2.31 ± 0.03 Å on average for atomic resolution structures, which are very close to the
average values determined from small molecule complexes. The variation of the average
bond lengths with resolution is not well understood but a similar phenomenon has been
observed with other metals, too.77,78
4.3.3.
INCOMPLETE SPHERES
Zinc-specific coordination spheres have been previously analyzed in several database
surveys, which have mostly dealt with high to medium-resolution crystal structures and
have used different computer algorithms to obtain statistical data. In these surveys,
coordination numbers from two to eight have been reported.79–82 Thus, a large number
of di- and tricoordinated zinc ions have been reported in the crystal structures of zinc
proteins. Finding di- or tricoordinated metal ions in proteins is very surprising given the
rarity of any transition metal complex to possessing required linear or trigonal planar
coordination geometry.77,78,83–85 Re-examination of these structures revealed several
factors which have been overlooked in the past, such as the exclusion of the symmetryrelated molecules in crystals or missing electron densities for ligands, leaving the
coordination spheres incomplete.86–89
In compounds, zinc appears exclusively in the oxidation state +II. In some very rare
compounds, having Zn–Zn bonds, the oxidation state +I is observed.18 Due to a fully
filled d-subshell, covalent coordination bonds are formed by sp3 hybrid orbitals, with the
Zn(II) ion acting as a strong electron acceptor. In the case of tetrahedral coordination, all
four sp3 orbitals are fully occupied in accordance with the 18-electron rule. Removal of
electrons from these orbitals greatly destabilizes the complex, leaving an electronically
unsaturated sphere. 16-electron complexes are possible with certain low-spin d8 metals,
with ligands having both σ-donor and π-acceptor properties. These complexes are square
planar with an empty dx²–y² orbital, which is not possible with zinc due to a d10 electron
configuration. Higher coordination numbers for Zn(II) are also possible with the
incorporation of empty 4d orbitals (i.e., penta- and hexacoordinate zinc ions). It must be
noted that di- and tricoordinate zinc ions do exist in some organozinc compounds, but
they are electron-deficient and highly reactive in ambient conditions. Thus, it is obvious
that di- or tricoordinated zinc ions cannot exist in a biological environment.
To correct some of the misinterpretations of the coordination spheres in zinc proteins,
made in the previous database surveys, all the structures were manually validated in this
study by taking symmetry molecules into account or by inspecting the actual electron
density maps to find unassigned ligands, incorrect side-chain conformations or unusually
long metal-to-ligand bonds. In addition, in many occasions, the sole inspection of the
original publications revealed details, which pointed to the unassigned ligands present
in the structures, while being fully absent in the actual coordinate files. It must be
emphasized that no structures with di- or tricoordinated zinc ions were found in this
study upon taking the above-mentioned factors into account.
27
An incomplete sphere is a rare occasion in the NMR structures, ca. 1% out of all spheres,
and is mostly due to missing exogenous ligands or unresolved binding conformations.
In the crystal structures, incomplete spheres are far more frequent, counting up to 40%
of all zinc sites, even though a majority of them appear in the artifact sites. The following
are the main reasons for incorrect assignments of the coordination spheres found in zinc
protein structures:
a) Symmetry-related molecules in crystals. The coordinating ligands come from
different polypeptide chains related to each other by crystallographic symmetry.
These are easily ignored for the simple reason that the asymmetric unit does not
necessarily represent these interactions and the symmetry molecules need to be
manually generated. Insulin provides an excellent example of this. In the hexameric
insulin assembly, the two zinc ions are coordinated by three histidine residues
(Figure 13a) from three insulin monomers that reside on the same three-fold
symmetry axis.4
b) Missing or unassigned electron density for ligand. The placement of atoms is based
on the electron density map, where either missing or unambiguous density prevents
the exact placement of the atoms. This is usually true for water molecules, ligands
(substrates or inhibitors) in the enzyme active sites or even the entire side-chains
(Figure 13b). For example, water molecules cannot be placed with certainty to the
electron density in the low-resolution enzyme structures, even though the bond angles
of the other three ligands would clearly indicate a tetrahedral coordination.90,91
c) Bond-lengths over the used cut-off values. Even though the bond lengths between the
metal and the ligand should be close to the values determined, on average, for small
compounds, protein structures exhibit variations from these values due to limited
conformations of the main and side-chains. Due to differences in resolution, some
bond lengths may be longer than the others (Figure 13c). If specific cut-off values are
used, such deviations from the average values may result in false interpretation of the
coordination numbers. The low resolution limits the accuracy of the electron density
refinement, which becomes clearly evident in very large structures, such as RNA
polymerases.92
d) Multiple or incorrect side-chain conformations. In structures, where the ligands have
multiple conformations or incorrectly placed atoms, the coordination sphere may be
interpreted as duplicate or incomplete. (Figure 13d). For example, in some structures,
the imidazole ring of histidine may be flipped, so the carbon is coordinating to the
metal, resulting in a seemingly vacant coordination site. Similar flips have been seen
in the amide coordination. On the other hand, some structures have very distorted and
clustered ligands that overlap with each other.89
e) Overlapping or an unknown metal. Metal ions are usually added to the structure,
when an unexpected, large electron density is found (Figure 13e). Good refinement
statistics for a certain metal does not necessarily mean that the metal ion is correct.
28
The atomic (van der Waals) radii for zinc and other transition metals are very similar
and the identification of the correct metal ion can be problematic without additional
experiments, as the refinement may give better values for incorrect metals.
The first three reasons count for the majority of errors observed in this study. However,
the latter two also pose a need for re-refinement of the structures involved (Table 4).
Since the protein crystal is a repeating unit, interactions between the molecules are not
necessarily described by the asymmetric unit, especially when the surface contacts are
involved. In many crystal structures, metal ions are present on the surface of the protein,
resulting e.g. from high concentrations of metal ion containing buffers used in
crystallization. Thus, the coordination is frequently misinterpreted if symmetry-related
molecules are not taken into account, as in the case of hexameric insulin.
Table 4. Factors leading to incomplete zinc coordination spheres in crystal structures
Reason
Symmetry-related molecules
Missing solvent molecules
Missing water from active site
Symmetry with missing solvent
Missing sidechain or ligand
Metal placed to fit electron
density
Sidechain conformation
Metal or ligand occupancy
Unknown or missing metal
Sidechain flip (His/Asn/Gln)
Total
Fraction of
artifact sites
(%)
39.2
33.1
6.8
14.9
0.4
Functional
Artifact
90
21
467
116
760
642
132
288
8
Fraction of
functional sites
(%)
10.7
2.5
55.5
13.8
-
70
-
3.6
60
58
12
17
841
10
11
17
2
1940
7.1
6.9
1.4
2.0
19.1
0.5
0.6
0.9
0.1
76.5
29
Figure 13. Factors leading to misinterpretation of zinc coordination spheres in some protein
crystal structures deposited in the PDB. Left: overall structure; right: zinc coordination site in
detail. (a) Symmetry related molecule in the crystal (PDB 1OHT). (b) Missing ligand/electron
density for ligand (1Z5R). (c) Bond lengths over cut-off values (2FE8). (d) Multiple occupancy
or erroneous atom deposition (2APO). (e) Unknown metal ion in the active site (3QC3).
30
4.4.
CHARACTERIZATION OF THE SAP30L COREPRESSOR
PROTEIN II
4.4.1.
GENERAL
The Sin3A-associated protein 30-like (SAP30L) is the newest member of the Sin3A corepressor complex, a multi-component regulatory element of gene expression in
mammalian cells. (Figure 14a)93,94 The Sin3A complex contains at least ten different
proteins, including SAP30, which is highly homologous to SAP30L (∼70% sequence
identity). Thus, SAP30 and SAP30L are collectively known as SAP30 proteins. Sin3A
itself is an acidic protein involved in transcriptional repression, but is unable to bind
DNA, and therefore requires the recruitment and interaction of the other DNA-binding
proteins. It has been suggested that SAP30 and SAP30L serve as bridging and stabilizing
molecules between Sin3A and other co-repressors and transcription factors.95–101
Figure 14. a) Overview of the Sin3A multiprotein complex (for details, see original
publication II). b) Known functional motifs of SAP30L: ZnF, zinc finger motif; NLS, nuclear
localization signal; NoLS, nucleolar localization signal. c) Primary structure of the ZnF motif of
SAP30L.
31
SAP30L has been previously shown to contain several structural motifs, which have
different functions in the corepressor complex (Figure 14b,c).102 The N-terminal zinc
finger motif is necessary for the folding and function of the protein, mainly responsible
for the DNA and phospholipid binding. The ZnF motif is almost identical in SAP30 and
SAP30L.98,102,103 In addition to the ZnF motif, SAP30L also contains an N-terminal low
complexity region (residues 1–25), with a yet unknown function. These are followed by
a central nuclear localization signal (NLS), which also mediates DNA binding.104–108 The
C-terminal part contains an acidic region for histone 2A/2B binding and a Sin3A binding
region along with a nuclear matrix targeting signal.65,102,103,109
In the previous study, native mass spectrometry was used to demonstrate that SAP30L
contains a novel N-terminal Cys3His type ZnF motif with Cys29, Cys38, Cys74 and
His77 serving as the primary zinc coordinating amino acid residues.102 In addition, the
ZnF motif also contains a non-coordinating Cys30 residue, which is highly conserved
through evolution. Previous mutation analyses indicated that this residue is not directly
involved in the zinc ion binding. Later, the solution NMR structure was determined for
the homologous SAP30 (PDB 2KDP), which indicated that the ZnF motif adopts a
totally new fold with two anti-parallel β-sheets and a pair of α-helices, which form a
well-defined hydrophobic core where the zinc binding site is situated.110 Although
lacking any sequence similarity to other zinc fingers, the three-dimensional structure
bears resemblance to the treble clef zinc finger motif.12,110,111 The coordinating residues
and the additional non-coordinating cysteine (Cys30) are highly conserved among the
SAP30 family proteins, suggesting that they are critical for the protein folding and
function. Similar structural motifs can be found in the zinc-binding THAP domains,
which are also involved in nucleic acid binding and transcriptional regulation.111,112
In this study, we characterized a redox-dependent disulfide bond formation in SAP30L
as a regulatory mechanism for its structure and function by using high-resolution ESI
FT-ICR mass spectrometry. The main hypothesis was that the ZnF motif in SAP30L
works as a redox switch, which controls the DNA and phospholipid binding and, thus
the repression activity of the whole Sin3A complex. In addition, we determined the
three-dimensional structure for the SAP30L ZnF motif (hereafter simply referred to as
SAP30L) by NMR spectroscopy. The construct used for the structure determination
comprised residues 25–92, since the largely unstructured N-terminal region has not been
shown to possess any function or importance for the folding.
32
4.4.2.
ZINC-INDUCED FOLDING OF SAP30L
In denaturing solution conditions, the mass spectrum of SAP30L showed a wide charge
state distribution (CSD), from 6+ to 14+ (centered around 10+), typical for a fully
unfolded protein (Figure 15a). The deconvoluted spectrum shows the experimental
isotopic distribution, with the most abundant isotopic mass (8100.291 Da) matching
perfectly with the mass calculated from the amino acid sequence (8100.267 Da for
C350H580N112O101S4). No zinc binding was observed in these conditions. In contrast, the
mass spectrum measured in near-native conditions (Figure 15b) showed a much
narrower CSD (5+ to 7+) as compared to the denaturing conditions, accompanied with
the mass increment of ∼64 Da, which is consistent with the binding of a single zinc ion.
The most abundant isotopic mass (8164.160 Da) agrees well with the theoretical mass
of holo-SAP30L (8164.181 Da for C350H578N112O101S4Zn1), assuming removal of two
protons upon zinc ion binding.113,114 A complete saturation of the protein with zinc was
observed, indicating very high affinity. Zinc finger motifs are known to lose their tertiary
structure upon demetallation, due to the lack of a large hydrophobic core. The mass
spectrum of SAP30L in 20 mM NH4OAc (pH 6.9) in the presence of 1,10-phenantroline
(Figure 15c), a strong zinc chelator, was essentially the same as upon denaturing
conditions, indicating a lack of a stable fold in the absence of zinc.
Figure 15. ESI FT-ICR mass spectra of SAP30L measured in a) denaturing solution conditions
(MeCN/H2O/HOAc 49.5:49.5:1.0, v/v) and in near-native conditions (20 mM NH4OAc, pH 6.8)
in the b) presence or c) absence of 1,10-phenanthroline, and d) after in vitro oxidation for 10 min
with H2O2. The numbers indicate different charge states. The insets show deconvoluted mass
spectra with the arrows denoting the peaks representing the most abundant isotopologues.
33
4.4.3.
REDOX-DEPENDENT DISULFIDE FORMATION
Cysteine residues in proteins are redox-active and can respond to the oxidative stress by
formation of disulfide bonds or other oxidative modifications.115–120 Thus, they can act
as “redox switches” to sense changes in the redox status of the cell. These structural
changes may lead to conformational changes, affecting protein function, e.g. DNA
binding. As ZnF proteins are known to be redox-regulated, we sought to investigate
redox-dependent structural changes in SAP30L.
Figure 15d shows the mass spectrum of SAP30L, measured in 20 mM NH4OAc (pH 6.9)
after the in vitro oxidation with hydrogen peroxide (H2O2). Interestingly, the charge state
distribution was very similar to that observed with holo-SAP30L (Figure 16b). However,
the most abundant isotopic mass was determined to be 8096.219 Da, suggesting that two
disulfide bonds had been formed with a concomitant release of the coordinated zinc ion
(theor. 8096.236 Da for C350H576N112O101S4). This is an interesting finding, since
SAP30L has only three cysteine residues coordinating to the zinc ion (Cys29, Cys38 and
Cys74). Therefore, it is evident that the unliganded Cys30 was also involved in the
oxidation. No dimeric forms of the protein were observed, indicating that only
intramolecular disulfide bonds were formed upon oxidative stress. Very similar CSD
observed for the oxidized SAP30L as compared to holo-SAP30L suggests that it
remained folded in solution, having a stable tertiary structure. This is plausible, as the
cysteine residues in SAP30L are in close proximity in the zinc-bound form, and it is
assumed that only minor conformational changes are needed to form the two observed
disulfide bonds.
To determine the reversibility of the oxidation, disulfide bond reduction was performed
with the oxidized SAP30L, which resulted in full reduction and instant re-coordination
of the zinc ion. This shows that the folding of SAP30L is initiated by the zinc ion binding
and the oxidation is a fully reversible process. In the presence of a zinc-chelating agent
(1,10-phenantroline), the protein did not reduce at all, suggesting a combined effect of
zinc and the reducing agent. Zinc and cysteine is a known redox pair involved in the
regulation of the function of many cellular proteins. The zinc protein oxidation causes
two major effects, a consumption of the oxidative molecules and the release of the
coordinated zinc ion, which may act as a secondary messenger in cellular environments.9
In order to determine the pairing of the cysteines, the oxidized and reduced SAP30L
were digested with trypsin (in solution digestion) or pepsin (online digestion). HoloSAP30L digested with trypsin in the presence of DTT showed all cysteine containing
tryptic peptides in their reduced form (Figure 16a; for the list of all identified tryptic
peptides, see Table II in the original publication II). The high mass accuracy of the FTICR instrument allowed unambiguous identification of the resulting peptides and full
sequence coverage was obtained for the holoprotein. In contrast, the tryptic digest of the
oxidized SAP30L revealed the presence of a number of oxidized peptides having
disulfide bonds (Figure 16b; for the list of peptides, see Table 2 in II). Among the
identified peptides, a peptide with an intramolecular disulfide bond between Cys29 and
34
Cys30 (1613.64 Da) was observed, demonstrating that the disulfide bond is formed
between the adjacent cysteine residues. Such vicinal disulfide bonds have been
extensively studied in the past.119–122 Also, two other tryptic peptides were observed
(2408.15 and 2564.25 Da), both having a disulfide bond between Cys38 and Cys74.
Moreover, three larger disulfide-linked peptides were detected, containing both
disulfides (e.g. 4003.78 Da, see Table II in I). The online pepsin digestion resulted in
essentially the same results (see I for details). The combined results from both protease
digestions indicated that the two specific disulfide bonds, Cys29-Cys30 and
Cys38-Cys74, are formed in SAP30L upon oxidative stress.
Figure 16. ESI FT-ICR mass spectra measured from tryptic digests of a) holo-SAP30L and b)
oxidized SAP30L. The tryptic peptides carrying Cys residues have been assigned (for details, see
original publication II). The insets show isotopic patterns for Cys-containing tryptic peptides.
The disulfide-containing peptides were subjected to ECD experiments for additional
verification (see, Supplementary Figure S5 in II), which further confirms their identity
and sequence.
35
4.4.4.
PHOSPHOLIPID AND DNA BINDING
Cell signaling phospholipids are known to mediate the DNA binding of SAP30L by
targeting the same binding site. Therefore, nuclear phospholipids can regulate chromatin
association of SAP30L and decrease the repression activity of the whole Sin3A
complex.102 SAP30L binds nucleic acids and phospholipids primarily by its polybasic
region (Figure 14b,c). The phospholipid binding competes with the binding of the
nucleic acids and leads to the detachment of the protein from the chromatin.
Therefore, the lipid binding affinity of SAP30L in different redox states was studied by
using three phosphatidylinositol monophosphates (PIPs), PI5P, PI4P and PI3P (bearing
distal phosphate units at the 5-, 4-, or 3-position of the inositol ring, respectively), which
are among the main cell signaling phospholipids, anchored to the intracellular
membranes. Native mass spectra for the holo and the oxidized SAP30L were measured
in the presence of an equimolar amount of each lipid to avoid extensive non-specific
binding or oligomerization typically encountered with lipids (see, Figure S7 in II).
Holo-SAP30L was found to have the highest affinity towards PI5P, having a dissociation
constant (Kd) in the low micromolar range (Table 7). PI3P was found to bind to SAP30L
with somewhat lower affinity than PI5P, although the structures are very similar and
differ only by the orientation of the phosphate and hydroxyl groups. These lipids also
have the closest structures compared to the sugar–phosphate chain of DNA and based
on their structures, would bind with similar affinity.109 Surprisingly, PI4P was found to
bind to holo-SAP30L with very similar affinity to that of PI5P, despite the structural
differences of these lipids. The similar binding affinities of these lipids could be
explained by a conformational change in the lipid sugar-phosphate ring, from axial to
equatorial, upon the binding. The length of the acyl chain is not expected to have a role
in the binding, since they are hidden in the cell membranes and only the head groups are
accessible to proteins.
Interestingly, it was observed that all PIPs bind to the oxidized SAP30L, with the
determined Kd values indicating ca. 4-fold higher affinity compared to the holoprotein
(Table 5). This further supports the finding that the oxidized SAP30L adopts a folded
structure in solution, as suggested by the CSD analysis (Figure 15).
Table 5. Dissociation constants for phosphoinositide binding to SAP30L.
Dissociation constant Kd (µM)a
Protein form
PI5P
PI4P
PI3P
Holo
61 ± 4
66 ± 4
83 ± 6
Oxidized
16 ± 1
22 ± 1
32 ± 2
a
Values reported as average ± standard deviation
36
4.4.5.
SOLUTION NMR STRUCTURE OF SAP30L
NMR spectroscopy was used to characterize zinc-dependent folding and DNA/lipid
binding of SAP30L and to determine its three-dimensional structure. The 1H,15N HSQC
spectrum recorded for holo-SAP30L showed characteristics of a well-folded protein in
solution with good signal dispersion and roughly equal cross-peak intensities (see Figure
3 in II). The slightly downfield Cβ shifts of Cys29, Cys38 and Cys74 (32.8, 31.3, and
33.3 ppm, respectively), compared to that of Cys30 (28.9 ppm), are in accordance with
these three cysteines being coordinated to the zinc ion.
The final NMR ensemble of the fifteen lowest-energy structures for SAP30L is shown
in Figure 17a. The residues Ser28–Arg85 form the core structure, while the N and Cterminal residues, Gly23–Gln27 and Asn86–Thr92, respectively, are disordered. The
regular secondary structure elements are formed by the residues Leu31–Glu33 and
Leu62–Ile64, making a short antiparallel β-sheet, and the residues Val51–Ser56 and
Asp75-Ser85, forming the two α-helices (Figure 17b). The overall three-dimensional
structure of SAP30L is remarkably similar to that of SAP30 determined earlier
(Figure 17c) with the backbone atom RMSD of 0.58 Å only.
Figure 17. Solution NMR structure of SAP30L. (a) Ensemble of 15 lowest-energy structures.
(b) Ribbon model of SAP30L with secondary structure elements and zinc binding site
highlighted. (c) Overlay of the best matching structures of SAP30L (tan; PDB 2N1U) and SAP30
(light blue; PDB 2KDP) NMR ensembles.
The phospholipid binding of SAP30L was examined with NMR by a reverse titration
because of the aggregation phenomenon occurring in the direct titration with PI5P, at the
concentration exceeding that of SAP30L. The cross-peaks shifted approximately linearly
with an increasing concentration of the ligand, indicative of low, micro- to millimolar
binding affinity that is consistent with the mass spectrometry results. All attempts to
37
determine the DNA binding with native mass spectrometry, either with the holo or the
oxidized protein, were unsuccessful, probably due to the low ionization efficiency of the
protein–DNA complex or protein aggregation. However, by using NMR we were able
to map chemical shift perturbations induced by the binding of the 8-bp DNA to the holoprotein. The protein aggregation compelled the use of a reverse titration in this case. The
binding affinity was notably higher for DNA than for PI5P, given that the cross-peaks
did not significantly shift after an equimolar concentration ratio had been reached. A
pattern of perturbations similar to that with PI5P was observed, namely, the most
affected residues located in the C-terminal region and in the first α-helix. No attempts
were made to characterize the DNA binding to the oxidized SAP30L.
4.4.6.
DISCUSSION
On the basis of the present results, the zinc finger motif of SAP30L undergoes a redoxdependent disulfide bond formation upon oxidative stress. The two disulfide bonds,
Cys29-Cys30 and Cys38-Cys74, are formed, involving Cys30, the non-coordinating but
a highly conserved cysteine residue. The two adjacent cysteines, Cys29 and Cys30, form
a well-known, redox-active Cys-Cys protein motif, which has been frequently reported
to occur, especially in ribonucleases.119,123,124 The disulfide bond between two adjacent
cysteine residues (i.e., a vicinal disulfide bond) is referred to in the literature as a
“forbidden” disulfide, involving an eight-membered ring structure in which the peptide
bond typically adopts a highly distorted, non-planar trans-conformation.
Interestingly, the native mass spectrometry data suggested that the oxidized SAP30L
retains a folded structure in solution, which is further supported by the observed affinity
toward the phospholipids. This may have important implications in relation to its
function in the Sin3A complex. In the absence of a three-dimensional structure for the
oxidized SAP30L, we modeled the disulfide bonds to the NMR structure of holoSAP30L. The disulfide-bonded structure was generated by removing the zinc ion and
rotating the torsional angles of the cysteine residues to be able to make the two disulfide
bonds, followed by an energy-minimization of the resulting structure. The modeled
structure shows that the formation of these two disulfide bonds is plausible without any
major conformational changes in the overall structure (the backbone atom RMSD was
0.68 Å between the two structures). The peptide bond in the resulting eight-membered
ring of the vicinal Cys29-Cys30 adopts a clearly distorted trans-conformation, consistent
with the other reported vicinal disulfide bonds.
Therefore, the zinc center in SAP30L forms a redox switch, which, upon oxidative stress,
releases the coordinated zinc ion with a concomitant formation of the two specific
disulfide bonds, a vicinal Cys29-Cys30 and Cys38-Cys74. Figure 18 deciphers the
schematic of the redox-active zinc center in SAP30L and the three-dimensional
structures of the zinc centers of the holo and oxidized proteins.
38
The oxidized SAP30L was found to bind all the lipids with markedly higher affinity than
the holoprotein, and these differences suggest a conformational change upon the
oxidation of the protein. The binding of the lipids with the oxidized SAP30L further
supports that it remains folded in solution, likely having a functional role. The zinc
coordination is not required for the binding of the lipids and the oxidation might enable
conformational freedom not available for the holoprotein. The increased lipid binding
affinity of the oxidized SAP30L might further affect its release from the DNA and
therefore the repression activity of the protein complex.
Figure 18. Schematic representation of the redox switch in SAP30L N-terminal ZnF motif. Upon
oxidative stress, the two specific disulfide bonds, a vicinal Cys29-Cys30 and Cys38-Cys74, are
formed. In the holoprotein, the zinc coordination is perfectly tetrahedral. In the oxidized form,
the vicinal Cys29-Cys30 forms an eight-membered ring with the ring peptide bond adopting a
highly distorted trans-conformation.
39
4.5.
DESIGNED ZINC FINGER PEPTIDES III
4.5.1.
GENERAL
Small zinc finger (ZnF) motifs are promising molecular scaffolds for protein design,
owing to their structural robustness and versatility. Moreover, their characterization
provides important insights into protein folding in general. ZnF motifs usually possess
an exceptional specificity and high affinity towards the Zn(II) ion to drive folding. A
recently discovered non-native CHANCE (Cys/His peptide exhibiting a nonexpected
conformational ensemble) motif, serendipitously found during the analysis of the first
Cys/His-rich domain (CH1) of the transcriptional regulator CREB binding protein (CBP;
see Figure 1), has shown that ZnF-like designer peptides can mimic well their natural
counterparts.24,28,125 Based on the CHANCE motif, Sharpe et al. designed a number of
“minimal mutant” (MM) peptides, where several amino acid residues were mutated to
alanines, and a set of “designed functional finger” (DFF) peptides, where the surface
residues were further mutated to mimic some common ZnF motifs (Figure 19).24,28 Their
objective was to find minimal sequence features, which could retain the original fold,
and to test a possibility of grafting the surface of a designer peptide with a specific
(DNA or protein binding) function. Although the latter objective appeared challenging,28
it was demonstrated that the CHANCE motif retains a stable fold upon multiple alanine
mutations, suggesting its potential as a versatile molecular scaffold for protein design.
Based on the NMR structure of MM1, the zinc ion is coordinated by Cys5, Cys10, His19,
and Cys23 residues (Figure 19). In addition, there is an additional histidine residue
(His22) in close vicinity (ca. 3.7 Å), which could participate in the transient metal ion
binding upon folding, or act as an important second-shell ligand.125
Figure 19. NMR structure of MM1 zinc finger motif (PDB 1WO3).28 Underneath are the amino
acid sequences of the peptides used in this study. The zinc binding residues are marked with a
blue background and changes from the CHANCE motif are marked with red.
40
In this study, high-resolution FT-ICR mass spectrometry was used for characterization
of the metal ion specificity and affinity, as well as the primary coordination sphere
robustness of several small ZnF motifs. The earlier studies demonstrated the structural
robustness of the CHANCE motif, where the most stable MM peptide, named MM1,
was chosen as the starting point.24,27,28 For comparison, the designer mutant named
DFF2, which mimics the N-terminal ZnF motif of the HIV-1 nucleocapsid protein 7
(NCp7), was characterized and compared to the native 18-residue peptide (NC), which
represents one of the smallest known stable ZnF motifs.29 Moreover, several Cys/Histo-Ala mutant peptides of MM1 were synthesized to study the importance of the primary
and some secondary amino acid ligands toward the zinc ion binding.
4.5.2.
METAL ION BINDING OF ZINC FINGER PEPTIDES
Initial measurements in the denaturing solution conditions showed that the peptides were
synthesized correctly and no other impurities were present in the samples (see Table S1
in III). In these conditions, the peptides were found to be unfolded and no metal ion
binding could be observed (Figure 20a and Figure S2 in III). The analyses also indicated
that the MM1 peptide was partly oxidized, by forming an internal disulfide bond, which
was determined by CID experiments to be between Cys5 and Cys10. Similarly, the DFF2
peptide was also found to be partially oxidized forming the same disulfide bond. The
NC peptide was found to exist as a disulfide-linked dimer. The oxidation of NCp7 and
similar zinc finger motifs in the absence of zinc has been previously observed.126–129 The
formed disulfide could easily be reduced with DTT, an ESI-MS compatible reducing
agent.67 However, during prolonged storage times the peptides were found to reoxidize
even in the presence of DTT. However, as DTT can also chelate zinc ions, the use of
excessive DTT was avoided in further experiments.
To characterize zinc ion binding of the peptides, the experiments were first attempted in
water. However, peptide re-oxidation occurred rapidly in the absence of zinc. Therefore,
a mixture of water and MeCN was tested as an alternative solvent mixture and a solvent
ratio of 50:50 (v/v) was found optimal for further experiments, as it minimized the
peptide oxidation in the absence, while it preserved the folding of the peptides in the
presence of zinc.
A mass spectrum measured for MM1 in the presence of 10-fold molar excess of Zn(II)
ions showed a marked change in the CSD (2+ to 4+; average charge zav ≈ 3.0), as
compared to the apo-peptide (Figure 20 a,b) and the mass increased by ∼61.9111 Da,
consistent with the binding of one zinc ion. Only a small amount of the apo-peptide was
observed in this sample. The solvent accessible surface area for MM1 as calculated from
the NMR structure (PDB ID: 1WO3) by using the PDBePISA server130 is 2032 Å2. This
value translates into zav ≈ 2.9, based on the empirical charge–surface area correlation
proposed earlier,131 which is essentially the same as observed experimentally. Therefore,
the observed shift in the CSD, and a saturative binding of one specific zinc ion indicate
the Zn(II) ion-induced folding of MM1 into a well-defined three-dimensional structure.
41
Figure 20. ESI FT-ICR mass spectra of a) MM1 peptide without zinc and b) with 10-fold molar
excess of Zn2+ ions in solution. The peptide concentration was 2.5 µM in both. c) Titration curve
of zinc binding to MM1 peptide.
For better estimation of the zinc binding affinity, a zinc titration was also performed with
MM1. The fractional saturation of the peptide versus free zinc ion concentration is
shown in Figure 21c. The dissociation constant (Kd) determined from the curve fitting is
(112 ± 9) × 10–9 M. The obtained sub-micromolar affinity is approximately 7–8 orders
of magnitude weaker than that observed in native zinc fingers. Similar results were
obtained with the DFF2 and NC peptides (Figure S2 in III). However, the zinc ion
concentration requirements were markedly different for these peptides. The DFF2
peptide required ca. 15-fold zinc excess to reach saturation, suggesting a somewhat
lower affinity than MM1. In contrast, the NC peptide was fully saturated at a 3-fold zinc
concentration, implying very high affinity. The dissociation constant between NC and
zinc has been estimated to be around ~10–15 M.132 When acetic acid (pH 3.2) was added
to the peptide solutions, only the apo-peptides were observed due to the acid-induced
unfolding.
The specificity of MM1 to bind zinc ions was assessed by using a variety of other alkali
and transition metals. Of the metals tested, MM1 was able to bind Mn2+ and Ca2+, but
with very low affinity. In addition, up to three Hg2+ ions were seen to bind the peptide,
suggesting mostly non-specific binding. Surprisingly, only weak binding with Co2+ was
observed, although cobalt is used as a probe to study the coordination spheres of zinc
fingers. In fact, Sharpe et al. reported the use of Co2+ to probe the coordination geometry
of MM1 by absorption spectroscopy measurements.28 Only gold (Au+) was able to form
an abundant complex with the MM1 peptide (Figure 22). However, the binding of the
gold ion to the peptide did not shift the CSD, indicating that it does not induce similar
folding of the peptide as compared to zinc. Gold ions usually have a two-dentate binding
mode in ZnFs by two cysteine residues. A further increase of the gold ion concentration
caused the peptide to oxidize rapidly, forming a disulfide bond. However, the gold ion
did not bind to the oxidized peptide, indicating a requirement for free thiols for the
complex formation.
42
Figure 21. ESI FT-ICR mass spectra of MM1 peptide with 12-fold molar excess of a) Mn2+, b)
Ca2+, c) Co2+, d) Hg2+ and e) Au+ ions. The peptide concentration was 2.5 µM in each.
4.5.3.
ZINC COORDINATION SPHERE ROBUSTNESS OF MM1
Several Cys/His-to-Ala mutants of MM1 were synthesized in order to study the
importance of the residues in the primary coordination sphere for efficient zinc binding.
A robust and stable primary coordination sphere would be essential for the design of
novel ZnF based protein scaffolds.
The preliminary experiments showed that the affinity of the mutants was slightly lower
than the MM1 peptide. Therefore, the zinc concentration was raised to 36-fold (90 µM)
while keeping the peptide concentration constant. Surprisingly, all of the mutant
peptides, except C10A, were able to bind zinc ions (Figure 22), suggesting that Cys10 is
the most critical residue for the zinc ion binding in MM1 and cannot be replaced or
substituted by any other residue. In contrast, the C23A mutant was fully saturated in
these conditions, suggesting that the absence of this residue can be compensated for by
other residues, such as the non-coordinating His22. Also, the H19C mutant was almost
fully saturated in these conditions, however the affinity was lower than that of the MM1
peptide. An increase in the amount of cysteine residues in the coordination sphere
generally correlates with a higher entropy contribution in the binding free energy,
resulting in a higher sensitivity to the factors such as pH, temperature or ionic strength.133
The C5A and H19A mutants had similar affinities to MM1, but lower in comparison to
the C23A and H19C mutants.
43
Figure 22. Mass spectra of zinc binding to Cys/His-to-Ala mutants of MM1 peptide. a) C5A, b)
C10A, c) C23A, d) H19A and e) H19C. Zinc ion concentration was 36-fold in each case.
Second-shell interactions also play an important role in the stabilization of structural zinc
sites. MM1 contains a non-coordinating His22 residue, which is only ~3.7 Å away from
the coordinated zinc ion (Figure 19), having a plausible role as a second-shell ligand.
Previously, H22Y mutation was shown to prevent complete folding of the peptide.28 In
contrast, the H22A mutant was capable of binding zinc, although the affinity was lower
when compared to the MM1 peptide (data not shown). As tyrosine rather than alanine
was used in the previous study, the direct comparison is difficult. In summary, it seems
that His22 can act as a coordinating residue in MM1, especially in the case of C23A
mutation. In order to rationalize the results, the structural models for the studied MM1
mutants were obtained through molecular mechanics calculations by using the solution
NMR structure of MM1 as a template. The energy-minimized model for MM1 well
retained the overall fold with a backbone root-mean-square deviation (RMSD) of only
0.780 Å. In addition, all mutant peptide models also retained the overall fold (backbone
RMSDs between 0.187 and 0.846 Å) with their zinc coordination sites, showing a
tetrahedral coordination geometry (see Figure 5 in III). These results also suggest that
His22 could act as a substitutional zinc ligand. Interestingly, the obtained model for the
C10A mutant also showed nearly perfect zinc binding geometry. However, the absence
of Cys10 residue residing in the long loop connecting the two helical regions (V2–A4 and
V16–M20), supposedly results in destabilization since there are no other polar contacts
(e.g. hydrogen bonds) present in that region. A more detailed structural analysis would
require molecular dynamics simulations or heteronuclear NMR analysis to probe
structural changes and possible unfolding of this mutant. These results suggest that small
designed zinc finger motifs, such as MM1 and the like, may be more robust structurally
than their natural counterparts in larger proteins or enzymes.22,134–136
44
5.
CONCLUSIONS
Zinc proteins are of utmost importance in many physiological processes. Thus, their
analysis provides a way to understand these processes at the molecular level, which may
have important implications in finding targeted therapies for diseases or, for example,
designing artificial nanocatalysts, such as zinc finger nucleases or specific biosensors for
a variety of purposes.22–25 In this study, the structure and functions of several different
zinc proteins were studied by using three-dimensional protein structures available in the
structure database (PDB) and by experimental methods, especially high-resolution mass
spectrometry.
The large-scale analysis of zinc protein structures deposited in the PDB revealed that the
coordination spheres are very diverse and exhibit large variations in the metal-to-ligand
bond distances. Previous database surveys have been either limited in scale or have used
improper analysis methods or statistical rules, which have led to severe
misinterpretations of the true coordination chemistry of zinc ions.80–82 For example, diand tricoordinated structures have been reported, which are very unlikely for any
transition metal in biological environments. A wealth of new information regarding the
reasons behind the shortcomings of the previous database surveys were pointed out in
this study, emphasizing the need for establishing better analysis and validation protocols
for structural characterization of metalloproteins in general. Although zinc ions are
redox-inert and unique in their coordination environments, the principles used here can
be applied to any other biological metal, where the oxidation state and the electron
configuration may play an even more significant role.
Zinc ions themselves cannot participate in the redox-regulation in cellular conditions;
however, the coordination to cysteine residues in proteins provides the necessary redoxactivity for the structural and enzymatically active zinc sites.9,137 As zinc finger motifs
have several cysteine residues which coordinate to the zinc ion, they are sensitive
towards oxidative stress. This may result in cysteine oxidation, which releases the zinc
ion and alters the protein conformation. In this work, redox-regulation of the SAP30L
corepressor protein was studied. The N-terminal zinc finger motif of SAP30L was shown
to undergo in vitro oxidation, which directly affects the binding affinity toward nuclear
phospholipids. As shown in the previous studies, the presence of hydrogen peroxide led
to the detachment of SAP30L from the chromatin and its relocalization to the cytoplasm,
although this was associated with the increased amount of phospholipids upon oxidative
stress.102,109 In this study, the molecular basis for the redox-regulation of SAP30L by the
formation of the two specific disulfide bonds and the release of the coordinated zinc ion
was demonstrated as having a plausible role in the repression activity of the Sin3A corepressor complex.
The design of ZnF based molecular scaffolds, whether based on a known or a novel
amino acid sequence, has gained considerable interest in recent years.22–25,28 The designer
zinc finger peptides have shown that the primary structures of these peptides can be
tolerant to several amino acid mutations without marked difference in their folding or
45
metal ion binding characteristics. This makes them very robust and versatile molecular
templates for further design of ZnF based protein scaffolds for a variety of purposes.
Previously, a non-native CHANCE motif was shown to be exceptionally tolerant for
replacement of most of its amino acid residues with alanine without a marked decrease
in its ability to adopt a fully folded structure. One of the resulting “minimal mutant”
structures, named MM1, was further characterized in this study. As a continuation of the
previous studies, the robustness of the structure of MM1 was evaluated in terms of its
primary zinc coordination sphere. It was demonstrated that MM1 retains its zinc
specificity and binding affinity and remains folded upon selective Cys/His-to-Ala
mutations of its primary zinc coordinating residues. This suggests that small designer
peptides may have access to a wider conformational space required for efficient metal
ion binding, as compared to similar structural motifs in natural proteins. Threedimensional structures for the MM1 mutants would offer further structural insights into
the robustness of the zinc center in MM1, in particular the roles of some secondary amino
acid residues and the conformations adopted by the mutant structures.
Biochemical studies of zinc-binding proteins are challenging due to the limited number
of applicable spectroscopic methods for their characterization. This study showed that
high-resolution mass spectrometry serves as an excellent analytical tool for
characterizing various structural aspects in small and large zinc metalloproteins,
including metal ion specificity and affinity, ligand binding, redox chemistry and metal
dependent folding. The high sensitivity, specificity, and direct information regarding the
binding stoichiometry and are the key figures of merit, which make mass spectrometry
a competitive technique over the more traditional techniques.
46
ACKNOWLEDGEMENTS
This work was carried out at the Department of Chemistry, University of Eastern Finland
between 2008-2016. Grants from the Department of Chemistry at the beginning of these
studies, the financial support and travel grants from the Graduate School of Organic
Chemistry and Chemical Biology (GSOCCB), and funding from the TEKES project
“Biomedical product concept development based on big data gathered from
immunogenomics and –proteomics” are gratefully acknowledged.
I express my deepest gratitude to my supervisor, Professor Janne Jänis for the
opportunity to work with mass spectrometry and zinc proteins. Your guidance and
support over the years have been invaluable for the completion of these studies.
I would also like to thank Ph.D. Jarkko Valjakka for his guidance during these studies,
critical reading of the manuscripts and especially for asking the important questions.
Special thanks are also due to our former laboratory technician, Ritva Romppanen, for
her assistance in operating the mass spectrometry instruments during the research and
being able to locate the necessary laboratory equipment and chemicals.
The referees of this dissertation, Prof. Risto Kostiainen and Assoc. Prof. Vesa Hytönen,
are also owed special recognition and thanks for their critical reading and suggested
improvements. Docent Greg Watson was responsible for the language revision of this
manuscript.
The former and current members of the mass spectrometry and the protein group, as well
as the entire staff and student body of the Department of Chemistry also deserve warm
acknowledgement for having created a pleasant atmosphere to work within.
Finally, I would like to thank my family for their constant support throughout my life.
Without your support and care during these years the completion of this work would not
have been possible.
Joensuu, November 2016
Mikko Laitaoja
47
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Mikko Laitaoja: Structure-Function Studies of Zinc Proteins
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118/2013TIMONENJuri:Synthesis,characterizationandanti-inflammatoryeffectsofsubstitutedcoumarin
derivatives
119/2013 TAKKUNEN Laura: Three-dimensional roughness analysis for multiscale textured surfaces:
Quantitative characterization and simulation of micro- and nanoscale structures
120/2014 STENBERG Henna: Studies of self-organizing layered coatings
121/2014 KEKÄLÄINEN Timo: Characterization of petroleum and bio-oil samples by ultrahigh-resolution
Fourier transform ion cyclotron resonance mass spectrometry
122/2014 BAZHENOV Andrey: Towards deeper atomic-level understanding of the structure of magnesium
dichloride and its performance as a support in the Ziegler-Natta catalytic system
123/2014 PIRINEN Sami: Studies on MgCl2/ether supports in Ziegler–Natta catalysts for ethylene
polymerization
124/2014 KORPELA Tarmo: Friction and wear of micro-structured polymer surfaces
125/2014 HUOVINEN Eero: Fabrication of hierarchically structured polymer surfaces
126/2014 EROLA Markus: Synthesis of colloidal gold and polymer particles and use of the particles in
preparation of hierarchical structures with self-assembly
127/2015 KOSKINEN Laura: Structural and computational studies on the coordinative nature of halogen
bonding
128/2015 TUIKKA Matti: Crystal engineering studies of barium bisphosphonates, iodine bridged ruthenium
complexes, and copper chlorides
129/2015JIANGYu:Modificationandapplicationsofmicro-structuredpolymersurfaces
130/2015 TABERMAN Helena: Structure and function of carbohydrate-modifying enzymes
131/2015KUKLINMikhailS.:Towardsoptimizationofmetaloceneolefinpolymerizationcatalystsvia structuralmodifications:acomputationalapproach
132/2015SALSTELAJanne:Influenceofsurfacestructuringonphysicalandmechanicalpropertiesof polymer-cellulosefibercompositesandmetal-polymercompositejoints
133/2015 CHAUDRI Adil Maqsood: Tribological behavior of the polymers used in drug delivery devices
134/2015 HILLI Yulia: The structure-activity relationship of Pd-Ni three-way catalysts for H2S suppression
135/2016 SUN Linlin: The effects of structural and environmental factors on the swelling behavior of
Montmorillonite-Beidellite smectics: a molecular dynamics approach
136/2016 OFORI Albert: Inter- and intramolecular interactions in the stabilization and coordination of
palladium and silver complexes: DFT and QTAIM studies
137/2016 LAVIKAINEN Lasse: The structure and surfaces of 2:1 phyllosilicate clay minerals
138/2016 MYLLER Antti T.: The effect of a coupling agent on the formation of area-selective monolayers of
iron a-octabutoxy phthalocyanine on a nano-patterned titanium dioxide carrier
139/2016KIRVESLAHTIAnna:Polymerwettabilityproperties:theirmodificationandinfluencesupon water movement
Dissertations
Department of Chemistry
University of Eastern Finland
No. 140 (2016)
Mikko Laitaoja
Structure-Function Studies
of Zinc Proteins