Protein crystallography
1. Tracking the structural dynamics of proteins in solution
using time-resolved wide-angle X-ray scattering
Cammarata M, Levantino M, Schotte F, Anfinrud PA, Ewald F, Choi J, Cupane A, Wulff M, Ihee H.
Nat Methods. 2008 Oct;5(10):881-6. Epub 2008 Sep 21.
We demonstrate tracking of protein structural changes with time-resolved wide-angle X-ray
scattering (TR-WAXS) with nanosecond time resolution. We investigated the tertiary and
quaternary conformational changes of human hemoglobin under nearly physiological
conditions triggered by laser-induced ligand photolysis. We also report data on optically
induced tertiary relaxations of myoglobin and refolding of cytochrome c to illustrate the wide
applicability of the technique. By providing insights into the structural dynamics of proteins
functioning in their natural environment, TR-WAXS complements and extends results obtained
with time-resolved optical spectroscopy and X-ray crystallography.
2. Light-induced structural changes in a photosynthetic reaction
center caught by Laue diffraction
Wöhri AB, Katona G, Johansson LC, Fritz E, Malmerberg E, Andersson M, Vincent
J, Eklund M, Cammarata M, Wulff M, Davidsson J, Groenhof G, Neutze R.
Science. 2010 Apr 30;328(5978):630-3.
Photosynthetic reaction centers convert the energy content of light into a transmembrane
potential difference and so provide the major pathway for energy input into the biosphere. We
applied time-resolved Laue diffraction to study light-induced conformational changes in the
photosynthetic reaction center complex of Blastochloris viridis. The side chain of TyrL162,
which lies adjacent to the special pair of bacteriochlorophyll molecules that are photooxidized
in the primary light conversion event of photosynthesis, was observed to move 1.3 angstroms
closer to the special pair after photoactivation. Free energy calculations suggest that this
movement results from the deprotonation of this conserved tyrosine residue and provides a
mechanism for stabilizing the primary charge separation reactions of photosynthesis.
3. Hydrogen location in stages of an enzyme-catalyzed reaction:
time-of-flight neutron structure of D-xylose isomerase with
bound D-xylulose
Kovalevsky AY, Katz AK, Carrell HL, Hanson L, Mustyakimov M, Fisher SZ, Coates L, Schoenborn
BP, Bunick GJ, Glusker JP, Langan P.
Biochemistry. 2008 Jul 22;47(29):7595-7. Epub 2008 Jun 26.
The time-of-flight neutron Laue technique has been used to determine the location of hydrogen
atoms in the enzyme d-xylose isomerase (XI). The neutron structure of crystalline XI with bound
product, d-xylulose, shows, unexpectedly, that O5 of d-xylulose is not protonated but is
hydrogen-bonded to doubly protonated His54. Also, Lys289, which is neutral in native XI, is
protonated (positively charged), while the catalytic water in native XI has become activated to a
hydroxyl anion which is in the proximity of C1 and C2, the molecular site of isomerization of
xylose. These findings impact our understanding of the reaction mechanism.
4. Macromolecular crystal data phased by negative-stained
electron-microscopy reconstructions
Trapani S, Schoehn G, Navaza J, Abergel C.
Acta Crystallogr D Biol Crystallogr. 2010 May;66(Pt 5):514-21. Epub 2010 Apr 21.
The combination of transmission electron microscopy with X-ray diffraction data is usually
limited to relatively large particles. Here, the approach is continued one step further by utilizing
negative staining, a technique that is of wider applicability than cryo-electron microscopy, to
produce models of medium-size proteins suitable for molecular replacement. The technique
was used to solve the crystal structure of the dodecameric type II dehydroquinase enzyme from
Candida albicans (approximately 190 kDa) and that of the orthologous Streptomyces coelicolor
protein.
5. With phases: how two wrongs can sometimes make a right
Roversi P, Johnson S, Lea SM.
Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):420-5. Epub 2010 Mar 24.
In isolation, both weak isomorphous/anomalous difference signals from heavy-atom
derivatization and phases from partial molecular-replacement solutions for a subset of the
asymmetric unit often fall short of producing interpretable electron-density maps. Phases
generated from very partial molecular-replacement models (if generated carefully) can be used
to reliably locate heavy-atom sites, even if the signal is not sufficiently strong to allow robust
finding of the sites using Patterson interpretation or direct methods. Additional advantages are
that using molecular-replacement phases to define the heavy-atom substructure avoids the
need for subsequent hand determination and/or origin-choice reconciliation and that the
partial model can be used to aid the mask determination during solvent flattening. Two case
studies are presented in which it was only by combining experimental and molecularreplacement phasing approaches that the crystal structures could be determined.
6. Experimental phasing with SHELXC/D/E: combining chain
tracing with density modification.
Sheldrick GM.
Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt4):479-85. Epub 2010 Mar 24.
The programs SHELXC, SHELXD and SHELXE are designed to provide simple, robust and efficient
experimental phasing of macromolecules by the SAD, MAD, SIR, SIRAS and RIP methods and are
particularly suitable for use in automated structure-solution pipelines. This paper gives a
general account of experimental phasing using these programs and describes the extension of
iterative density modification in SHELXE by the inclusion of automated protein main-chain
tracing. This gives a good indication as to whether the structure has been solved and enables
interpretable maps to be obtained from poorer starting phases. The autotracing algorithm
starts with the location of possible seven-residue alpha-helices and common tripeptides. After
extension of these fragments in both directions, various criteria are used to decide whether to
accept or reject the resulting poly-Ala traces. Noncrystallographic symmetry (NCS) is applied to
the traced fragments, not to the density. Further features are the use of a 'no-go' map to
prevent the traces from passing through heavy atoms or symmetry elements and a splicing
technique to combine the best parts of traces (including those generated by NCS) that partly
overlap.
7. How to get the magic triangle and the MAD triangle into your
protein crystal
Beck T, da Cunha CE, Sheldrick GM.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2009 Oct 1;65(Pt 10):1068-70. Epub 2009
Sep 25.
The magic triangle 5-amino-2,4,6-triiodoisophthalic acid (I3C) and the MAD triangle 5-amino2,4,6-tribromoisophthalic acid (B3C) are two representatives of a novel class of compounds that
combine heavy atoms for experimental phasing with functional groups for protein interactions.
These compounds are readily available and provide easy access to experimental phasing. The
preparation of stock solutions and the incorporation of the compounds into protein crystals are
discussed. As an example of incorporation via cocrystallization, the incorporation of B3C into
bovine trypsin, resulting in a single site with high occupancy, is described.
NMR
1. Structure of the p53 Transactivation Domain in Complex with
the Nuclear Receptor Coactivator Binding Domain of CREB
Binding Protein.
Lee CW, Martinez-Yamout MA, Dyson HJ, Wright PE
Biochemistry. 2010 Nov 23;49(46):9964-71. Epub 2010 Oct 29.
The activity and stability of the tumor suppressor p53 are regulated by interactions with key
cellular proteins such as MDM2 and CBP/p300. The transactivation domain (TAD) of p53
contains two subdomains (AD1 and AD2) and interacts directly with the N-terminal domain of
MDM2 and with several domains of CBP/p300. Here we report the NMR structure of the fulllength p53 TAD in complex with the nuclear coactivator binding domain (NCBD) of CBP. Both
the p53 TAD and NCBD are intrinsically disordered and fold synergistically upon binding, as
evidenced by the observed increase in helicity and increased level of dispersion of the amide
proton resonances. The p53 TAD folds to form a pair of helices (denoted Pα1 and Pα2), which
extend from Phe19 to Leu25 and from Pro47 to Trp53, respectively. In the complex, the NCBD
forms a bundle of three helices (Cα1, residues 2066-2075; Cα2, residues 2081-2092; and Cα3,
residues 2095-2105) with a hydrophobic groove into which p53 helices Pα1 and Pα2 dock. The
polypeptide chain between the p53 helices remains flexible and makes no detectable
intermolecular contacts with the NCBD. Complex formation is driven largely by hydrophobic
contacts that form a stable intermolecular hydrophobic core. A salt bridge between D49 of p53
and R2105 of NCBD may contribute to the binding specificity. The structure provides the first
insights into simultaneous binding of the AD1 and AD2 motifs to a target protein.
2. Cooperative interaction of transcription termination factors
with the RNA polymerase II C-terminal domain
Lunde BM, Reichow SL, Kim M, Suh H, Leeper TC, Yang F, Mutschler H, Buratowski
S, Meinhart A, Varani G
Nat Struct Mol Biol. 2010 Oct;17(10):1195-201. Epub 2010 Sep 5.
Phosphorylation of the C-terminal domain (CTD) of RNA polymerase II controls the cotranscriptional assembly of RNA processing and transcription factors. Recruitment relies on
conserved CTD-interacting domains (CIDs) that recognize different CTD phosphoisoforms during
the transcription cycle, but the molecular basis for their specificity remains unclear. We show
that the CIDs of two transcription termination factors, Rtt103 and Pcf11, achieve high affinity
and specificity both by specifically recognizing the phosphorylated CTD and by cooperatively
binding to neighboring CTD repeats. Single-residue mutations at the protein-protein interface
abolish cooperativity and affect recruitment at the 3' end processing site in vivo. We suggest
that this cooperativity provides a signal-response mechanism to ensure that its action is
confined only to proper polyadenylation sites where Ser2 phosphorylation density is highest.
3. A split active site couples cap recognition by Dcp2 to
activation
Floor SN, Jones BN, Hernandez GA, Gross JD
Nat Struct Mol Biol. 2010 Sep;17(9):1096-101. Epub 2010 Aug 15.
Decapping by Dcp2 is an essential step in 5'-to-3' mRNA decay. In yeast, decapping requires an
open-to-closed transition in Dcp2, though the link between closure and catalysis remains
elusive. Here we show using NMR that cap binds conserved residues on both the catalytic and
regulatory domains of Dcp2. Lesions in the cap-binding site on the regulatory domain reduce
the catalytic step by two orders of magnitude and block the formation of the closed state,
whereas Dcp1 enhances the catalytic step by a factor of 10 and promotes closure. We conclude
that closure occurs during the rate-limiting catalytic step of decapping, juxtaposing the capbinding region of each domain to form a composite active site. This work suggests a model for
regulation of decapping where coactivators trigger decapping by stabilizing a labile composite
active site.
4. Structure of the s5a:k48-linked diubiquitin complex and its
interactions with rpn13
Zhang N, Wang Q, Ehlinger A, Randles L, Lary JW, Kang Y, Haririnia A, Storaska AJ, Cole JL,
Fushman D, Walters KJ.
Mol Cell. 2009 Aug 14;35(3):280-90.
Degradation by the proteasome typically requires substrate ubiquitination. Two ubiquitin
receptors exist in the proteasome, S5a/Rpn10 and Rpn13. Whereas Rpn13 has only one
ubiquitin-binding surface, S5a binds ubiquitin with two independent ubiquitin-interacting
motifs (UIMs). Here, we use nuclear magnetic resonance (NMR) and analytical
ultracentrifugation to define at atomic level resolution how S5a binds K48-linked diubiquitin, in
which K48 of one ubiquitin subunit (the "proximal" one) is covalently bonded to G76 of the
other (the "distal" subunit). We demonstrate that S5a's UIMs bind the two subunits
simultaneously with a preference for UIM2 binding to the proximal subunit while UIM1 binds to
the distal one. In addition, NMR experiments reveal that Rpn13 and S5a bind K48-linked
diubiquitin simultaneously with subunit specificity, and a model structure of S5a and Rpn13
bound to K48-linked polyubiquitin is provided. Altogether, our data demonstrate that S5a is
highly adaptive and cooperative toward binding ubiquitin chains.
5. Structurally conserved five nucleotide bulge determines the
overall topology of the core domain of human telomerase RNA.
Zhang Q, Kim NK, Peterson RD, Wang Z, Feigon J.
Proc Natl Acad Sci U S A. 2010 Nov 2;107(44):18761-8. Epub 2010 Oct 21.
Telomerase is a unique ribonucleoprotein complex that catalyzes the addition of telomeric DNA
repeats onto the 3' ends of linear chromosomes. All vertebrate telomerase RNAs contain a
catalytically essential core domain that includes the template and a pseudoknot with extended
helical subdomains. Within these helical regions is an asymmetric 5-nt internal bulge loop
(J2a/b) flanked by helices (P2a and P2b) that is highly conserved in its location but not
sequence. NMR structure determination reveals that J2a/b forms a defined S-shape and creates
an ∼90 ° bend with a surprisingly low twist (∼10 °) between the flanking helices. A search of
RNA structures revealed only one other example of a 5-nt bulge, from hepatitis C virus internal
ribosome entry site, with a different sequence but the same structure. J2a/b is intrinsically
flexible but the interhelical motions across the loop are remarkably restricted. Nucleotide
substitutions in J2a/b that affect the bend angle, direction, and interhelical dynamics are
correlated with telomerase activity. Based on the structures of P2ab (J2a/b and flanking
helices), the conserved region of the pseudoknot (P2b/P3, previously determined) and the
remaining helical segment (P2a.1-J2a.1 refined using residual dipolar couplings and the
modeling program MC-Sym) we have calculated an NMR-based model of the full-length
pseudoknot. The model and dynamics analysis show that J2a/b serves as a dominant structural
and dynamical element in defining the overall topology of the core domain, and suggest that
interhelical motions in P2ab facilitate nucleotide addition along the template and template
translocation.
6. NMR structure of the let-7 miRNA interacting with the site
LCS1 of lin-41 mRNA from Caenorhabditis elegans.
Cevec M, Thibaudeau C, Plavec J.
Nucleic Acids Res. 2010 Jul 26.
We have determined the 3D structure of a 34-nt RNA construct, herein named LCS1co, which
mimics the interaction of let-7 microRNA (miRNA) to one of its complementary binding sites,
LCS1, in the 3'-untranslated region of lin-41 mRNA by solution-state NMR spectroscopy. let-7
miRNAs control the timing of development of the nematode Caenorhabditis elegans and are
highly conserved in mammals. The sequence and structure of the two conserved let-7
complementary sites, LCS1 and LCS2, in the 3'-untranslated region of lin-41 mRNA are
important for a proper downregulation of lin-41. The high-resolution NMR structure reveals
details of the binding of let-7 miRNA to lin-41 mRNA which involves formation of a complex
with non-canonical structural elements within the seed region. LCS1co exhibits a stem-loop
structure with two stems, an asymmetric internal loop and an adenine bulge. Comparison with
the NMR solution-state structure of the let-7:lin-41 complex involving the LCS2-binding site
shows that conformational freedom of the asymmetric internal loop of LCS1co correlates with a
smaller bend between the upper and lower stems in comparison to the well-defined
asymmetric loop of LCS2co.
7. Structure of a Conserved Retroviral RNA Packaging Element
by NMR Spectroscopy and Cryo-Electron Tomography.
Miyazaki Y, Irobalieva RN, Tolbert BS, Smalls-Mantey A, Iyalla K, Loeliger K, D'Souza V, Khant H,
Schmid MF, Garcia EL, Telesnitsky A, Chiu W, Summers MF.
J Mol Biol. 2010 Oct 8.
The 5'-untranslated regions of all gammaretroviruses contain a conserved "double-hairpin
motif" (Ψ(CD)) that is required for genome packaging. Both hairpins (SL-C and SL-D) contain
GACG tetraloops that, in isolated RNAs, are capable of forming "kissing" interactions stabilized
by two intermolecular G-C base pairs. We have determined the three-dimensional structure of
the double hairpin from the Moloney murine leukemia virus (*Ψ(CD)+(2), 132 nt, 42.8 kDa) using
a (2)H-edited NMR-spectroscopy-based approach. This approach enabled the detection of (1)H(1)H dipolar interactions that were not observed in previous studies of isolated SL-C and SL-D
hairpin RNAs using traditional (1)H-(1)H correlated and (1)H-(13)C-edited NMR methods. The
hairpins participate in intermolecular cross-kissing interactions (SL-C to SL-D' and SLC' to SL-D)
and stack in an end-to-end manner (SL-C to SL-D and SL-C' to SL-D') that gives rise to an
elongated overall shape (ca 95 Å×45 Å×25 Å). The global structure was confirmed by cryoelectron tomography (cryo-ET), making *Ψ(CD)+(2) simultaneously the smallest RNA to be
structurally characterized to date by cryo-ET and among the largest to be determined by NMR.
Our findings suggest that, in addition to promoting dimerization, *Ψ(CD)+(2) functions as a
scaffold that helps initiate virus assembly by exposing a cluster of conserved UCUG elements for
binding to the cognate nucleocapsid domains of assembling viral Gag proteins.
Molecular Dynamics
1. Binding of ADP in the mitochondrial ADP/ATP carrier is
driven by an electrostatic funnel
Dehez F, Pebay-Peyroula E, Chipot C.
J Am Chem Soc. 2008 Sep 24;130(38):12725-33. Epub 2008 Aug 26.
The ADP/ATP carrier (AAC) is a membrane protein of paramount importance for the energyfueling function of the mitochondria, transporting ADP from the intermembrane space to the
matrix and ATP in the opposite direction. On the basis of the high-resolution, 2.2-A structure of
the bovine carrier, a total of 0.53 micros of classical molecular dynamics simulations were
conducted in a realistic membrane environment to decipher the early events of ADP (3-)
translocation across the inner membrane of the mitochondria. Examination of apo-AAC
underscores the impermeable nature of the carrier, impeding passive transport of permeants
toward the matrix. The electrostatic funnel illuminated from three-dimensional mapping of the
electrostatic potential forms a privileged passageway anticipated to drive the diphosphate
nucleotide rapidly toward the bottom of the internal cavity. This conjecture is verified in the
light of repeated, independent numerical experiments, whereby the permeant is dropped near
the mouth of the mitochondrial carrier. Systematic association of ADP (3-) to the crevice of the
AAC, an early event in its transport across the inner membrane, is accompanied by the
formation of an intricate network of noncovalent bonds. Simulations relying on the use of an
adaptive biasing force reveal for the first time that the proposed binding site corresponds to a
minimum of the free energy landscape delineating the translocation of ADP (3-) in the carrier.
The present work paves the way to the design of novel nucleotides and new experiments aimed
at unveiling key structural features in the chronology of ADP/ATP transport across the
mitochondrial membrane.
2. Diffusion of glycerol
aquaglyceroporin GlpF
through
Escherichia
coli
Hénin J, Tajkhorshid E, Schulten K, Chipot C.
Biophys J. 2008 Feb 1;94(3):832-9. Epub 2007 Oct 5.
The glycerol uptake facilitator, GlpF, a major intrinsic protein found in Escherichia coli,
selectively conducts water and glycerol across the inner membrane. The free energy landscape
characterizing the assisted transport of glycerol by this homotetrameric aquaglyceroporin has
been explored by means of equilibrium molecular dynamics over a timescale spanning 0.12
micros. To overcome the free energy barriers of the conduction pathway, an adaptive biasing
force is applied to the glycerol molecule confined in each of the four channels. The results
illuminate the critical role played by intramolecular relaxation on the diffusion properties of the
permeant. These free energy calculations reveal that glycerol tumbles and isomerizes on a
timescale comparable to that spanned by its adaptive-biasing-force-assisted conduction in GlpF.
As a result, reorientation and conformational equilibrium of glycerol in GlpF constitute a
bottleneck in the molecular simulations of the permeation event. A profile characterizing the
position-dependent diffusion of the permeant has been determined, allowing reaction rate
theory to be applied for investigating conduction kinetics based on the measured free energy
landscape.