Journal of Structural Biology 180 (2012) 271–279
Contents lists available at SciVerse ScienceDirect
Journal of Structural Biology
journal homepage: www.elsevier.com/locate/yjsbi
Structural insights into serine protease inhibition by a marine invertebrate BPTI
Kunitz-type inhibitor
Rossana García-Fernández a,b, Tirso Pons c, Markus Perbandt d,e, Pedro A. Valiente a, Ariel Talavera f,
Yamile González-González a,b, Dirk Rehders g, María A. Chávez a,b, Christian Betzel d, Lars Redecke g,⇑
a
Centro de Estudio de Proteínas, Facultad de Biología, Universidad de la Habana, Calle 25 No 411, 10400 Havana, Cuba
International Cooperation Network ‘‘Proteómica y Quimiogenómica de Inhibidores de Proteasas de Origen Natural con Potencial Terapéutico en Malaria’’ from the
Iberoamerican Programme of Science and Technology for Development, RED CYTED-PROMAL
c
Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre, C/Melchor Fernández Almagro 3, Madrid E-28029, Spain
d
Institute of Biochemistry and Molecular Biology, University of Hamburg, c/o DESY, Notkestr. 85, 22603 Hamburg, Germany
e
University Medical Center Hamburg-Eppendorf, Department of Medical Microbiology, Virology and Hygiene, Martinistr. 52, 20246 Hamburg, Germany
f
Center of Molecular Immunology, P.O. Box 16040, 11600 Havana, Cuba
g
Joint Laboratory for Structural Biology of Infection and Inflammation, Institute of Biochemistry and Molecular Biology, University of Hamburg, and Institute of
Biochemistry, University of Lübeck, c/o DESY, Notkestr. 85, 22603 Hamburg, Germany
b
a r t i c l e
i n f o
Article history:
Received 14 February 2012
Received in revised form 22 August 2012
Accepted 24 August 2012
Available online 5 September 2012
Keywords:
BPTI Kunitz-type inhibitor
Serine protease
Protein-inhibitor interaction
Crystal structure
Marine invertebrates
a b s t r a c t
Proteins isolated from marine invertebrates are frequently characterized by exceptional structural and
functional properties. ShPI-1, a BPTI Kunitz-type inhibitor from the Caribbean Sea anemone Stichodactyla
helianthus, displays activity not only against serine-, but also against cysteine-, and aspartate proteases.
As an initial step to evaluate the molecular basis of its activities, we describe the crystallographic structure of ShPI-1 in complex with the serine protease bovine pancreatic trypsin at 1.7 Å resolution. The overall structure and the important enzyme-inhibitor interactions of this first invertebrate BPTI-like
Kunitz-type inhibitor:trypsin complex remained largely conserved compared to mammalian BPTI-Kunitz
inhibitor complexes. However, a prominent stabilizing role within the interface was attributed to arginine at position P3. Binding free-energy calculations indicated a 10-fold decrease for the inhibitor affinity
against trypsin, if the P3 residue of ShPI-1 is mutated to alanine. Together with the increased role of Arg11
at P3 position, slightly reduced interactions at the prime side (Pn0 ) of the primary binding loop and at the
secondary binding loop of ShPI-1 were detected. In addition, the structure provides important information for site directed mutagenesis to further optimize the activity of rShPI-1A for biotechnological
applications.
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
Proteolytic enzymes are involved in the regulation of all physiological processes in living organisms. Deficiencies or alterations of
their activities can lead to abnormal development, disease, and
Abbreviations: APPI, inhibitor domain of human Alzheimer’s amyloid betaprotein precursor; BAPA, N-benzoyl-arginine-p-nitroanilide; BPTI, bovine pancreatic
trypsin inhibitor; BS, buried surface; DLS, dynamic laser light scattering; HPLC,
high-performance liquid chromatography; IF, intensity fading; Ki, equilibrium
dissociation constant; MALDI-TOF, matrix assisted laser desorption/ionization
time-of-flight; MS, mass spectrometry; MW, molecular weight; PDB, protein data
bank; RH, hydrodynamic radius; RMSD, root mean square deviation; RT, room
temperature; SDS–PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEC, size exclusion chromatography; ShPI-1, Stichodactyla helianthus protease
inhibitor 1; TFPI, tissue factor protease inhibitor; vdW, van der Waals.
⇑ Corresponding author. Fax: +49 40 8998 4739.
E-mail address: [email protected] (L. Redecke).
1047-8477/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jsb.2012.08.009
even death (Abbenante and Fairlie, 2005). Therefore, detailed
knowledge about the structure–function-relationship of enzymes
is of great interest to the pharmaceutical industry. Based on their
importance in human health and disease prevention, there is also
increased interest in applying protease inhibitors in therapeutic
and biotechnological applications (Abbenante and Fairlie, 2005).
Additionally, enzyme-inhibitor complexes are useful model systems to study protein–protein recognition (Laskowski et al.,
2000; Otlewski et al., 2001).
Canonical serine protease inhibitors have been the most extensively studied so far and 18 families are currently recognized
(Laskowski et al., 2000). Among them, the BPTI-Kunitz family
(PFAM: PF00014), which comprises extremely potent inhibitors
of serine proteases, is composed of more than 1000 different
sequences isolated from a variety of animals ranging from invertebrates to mammals (Delfín et al., 1996; Kunitz and Northrop,
1936). The three-dimensional (3D) structures of sixteen protein
272
R. García-Fernández et al. / Journal of Structural Biology 180 (2012) 271–279
inhibitors belonging to this family have been determined.
However, the majority of the reported structures belong to the bovine pancreatic trypsin inhibitor (BPTI), the prototypical member
of this family. Insights into the nature of inhibitory interactions
have been obtained by X-ray crystallographic studies investigating
serine proteases in complex mainly with mammalian BPTI-Kunitz
inhibitors (Burgering et al., 1997; Huber et al., 1974; Scheidig
et al., 1997).
BPTI-Kunitz inhibitors are small proteins (around 6 kDa) with a
compact structure composed of a hydrophobic core, containing a
central b-sheet and three disulfide bridges with conserved chiralities (Antuch et al., 1993; Czapinska et al., 2000; Deisenhofer and
Steigemann, 1975; Huber et al., 1974). This core is the scaffold that
supports the convex and exposed canonical binding loop at
positions P3–P30 , according to the Pn–Pn0 notation of Schechter
and Berger (1967). This loop is highly complementary to the
concave protease active site and is, thus, responsible for the
extreme stability of the interaction with the target enzyme in a
substrate-like manner (Laskowski et al., 2000). The dissociation
constants of enzyme:inhibitor complexes range from 1013 to
107 M, mainly depending on the nature of the residue at position
P1 and the number of contacts formed with the S1 site of the bound
protease (Czapinska et al., 2000). Typically, trypsin inhibitors
contain Arg/Lys at position P1, whereas chymotrypsin inhibitors
have Leu/Met at this position. However, other residues at the
primary binding site and within the secondary binding loop
(residues 34–39 in BPTI) are also suggested to have significant influence on the association energy (Scheidig et al., 1997; Czapinska
et al., 2000; Laskowski et al., 2000).
A molecule of 6.1 kDa sharing the basic structure of the BPTIKunitz family, named ShPI-1 (UniProt accession No. P31713),
was previously isolated by our group from the Caribbean Sea
anemone Stichodactyla helianthus (Antuch et al., 1993; Delfín
et al., 1996). In contrast to other BPTI-Kunitz inhibitors, ShPI-1
is able to inhibit not only serine proteases with Ki values in
the nanomolar range, but also cysteine and aspartic proteases
such as papain and pepsin (Delfín et al., 1996). In addition, its
reported activity against human neutrophil elastase (HNE) (Delfín
et al., 1996) is not usual for domains having a basic residue at
position P1. Therefore, the elucidation of the molecular basis
for the activity of this multifunctional inhibitor against
structurally unrelated proteases is of specific interest for further
site-directed mutagenesis experiments or increasing its potential
applications.
Considering its inhibitory efficiency, an active recombinant
variant of ShPI-1 (rShPI-1A) recently expressed in Pichia pastoris,
is successfully used in diagnostics and biotechnological processes
(Gil et al., 2011). Here we present the first crystal structure of
rShPI-1A in complex with the serine protease bovine pancreatic
trypsin at 1.7 Å resolution. Analyzing the molecular contacts by
comparison with trypsin complexes of mammalian BPTI-Kunitztype inhibitors indicated a prominent role not only for Lys13 at
position P1, but also for Arg11 at position P3 of rShPI-1A in the
stabilization of the complex that was so far not described for
BPTI-Kunitz-type inhibitors. However, less interaction than BPTI
at the primary and secondary binding loops of rShPI-1A reduced
the gain of stability attributed to Arg11, resulting in a decreased
trypsin-binding affinity compared to BPTI.
acids compared to rShPI-1A, Lys15 at position P1 of BPTI corresponds to Lys13 in rShPI-1A.
2.1. Purification of rShPI-1A and complex formation with bovine
trypsin
The recombinant inhibitor rShPI-1A was expressed and purified
as previously described (Gil et al., 2011). The homogeneity of the
purified protein was verified by SDS–PAGE, MALDI-TOF MS, and reverse phase HPLC on a C8 column (Grace-Vydac, USA). For complex
formation a 2-fold molar excess of rShPI-1A was incubated in the
presence of bovine pancreatic trypsin (E.C. 3.4.21.4, Sigma,
Germany) in 0.02 M Tris buffer (pH 8.0) supplemented with
0.15 M NaCl and 0.02 M CaCl2 for one hour at RT. The mixture
was applied onto a Superose 12HR gel filtration column
(1 30 cm, Biosciences, USA) previously equilibrated with the
same buffer. Separation of inhibitor excess was performed on an
ÄKTA purifier system (GE Healthcare, USA) by monitoring the
absorbance at 280 nm. A chromatographic run of free trypsin
was used as a control for complex formation. Residual trypsin
activity was tested in the isolated protein fractions using BAPA
(BACHEM, Germany) as a substrate (Erlanger et al., 1961). The
dissociation constant (Ki) of the rShPI-1A:trypsin complex was
calculated as described by Bieth (1974). Protein concentrations
were determined by absorbance at 280 nm using the extinction
coefficient reported for natural ShPI-1 (Delfín et al., 1996) and a
theoretical extinction coefficient (E280nm1%) of 13.8 calculated for
the rShPI-1A:trypsin complex based on both protein sequences,
using ProtParam program (Gasteiger et al., 2005). Protein solutions
were concentrated using 3 kDa and 10 kDa MW cut-off concentration devices (Centricon, Millipore, USA) for the free inhibitor and
the complex, respectively.
2.2. Intensity fading (IF) MALDI-TOF MS
A control mass spectrum was obtained after mixing 1.0 ll
(60 lM) of pure rShPI-1A and 2.0 ll of sinapinic acid solution
(10 mg/ml) in 30% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic
acid. Fading or disappearance of MS signal was investigated after
incubation of pure rShPI-1A with 2 ll of immobilized trypsin on
cyanogen bromide-activated SepharoseÒ4B for 3 min at RT. Further
washing and elution steps were performed as previously described
(Yanes et al., 2007).
2.3. Dynamic laser light scattering (DLS)
DLS measurements were performed using the spectroscatterer
201 (Molecular Dimensions, UK) with a He-Ne laser providing light
of 690 nm wavelength and an output power in the range of 10–
50 mW. All samples were filtered using 0.1 lm PVDF centrifugal
filters (Microcon, Millipore, USA) prior to measurements. The
samples (30 ll) were measured in a quartz cuvette at 20 °C using
an autopilot function accumulating 10 measurements per sample.
Associated molecular masses were estimated from the detected RH
using the ‘‘SpectroSize’’ software package (Molecular Dimensions,
UK). This estimation is based on the relation MW = NAqRH2.34/3p
where NA is the Avogadro constant and q is the density of the
protein (1.5 g/cm3) (Fischer et al., 2004).
2.4. Crystallization, X-ray data collection, and structure refinement
2. Materials and methods
Due to the cloning procedure, rShPI-1A contains an additional
Glu-Ala-Glu-Ala motif (numbered as 3 to 0) at the N-terminus
of the natural ShPI-1 sequence and two additional residues (LeuGly) at the C-terminus. Since BPTI contains two additional amino
A robot-assisted screening of crystallization conditions was performed using commercially available kits from Hampton Research
(USA) and Qiagen (Germany). The purified rShPI-1A:trypsin
complex was concentrated up to 13 mg/mL and mixed 1:1 (v/v)
with reservoir solutions. Using the sitting-drop vapor diffusion
273
R. García-Fernández et al. / Journal of Structural Biology 180 (2012) 271–279
technique at 20 °C, orthorhombic crystals measuring up to 0.8 mm
grew within one week in condition 83 of the Qiagen Classic Suite
Research screening kit (0.2 M (NH4)2SO4, 0.1 M NaAc pH 4.6, 25%
PEG 4000). Diffraction data were collected with synchrotron
radiation at the consortiums beamline X13 at HASYLAB/DESY
(Hamburg, Germany) equipped with a MARresearch CCD detector.
The crystal was mounted in a nylon loop and flash-cooled in a
nitrogen-gas stream at 100 K. Integration, scaling, and reduction
of the recorded reflections were performed using the programs
MOSFLM and SCALA (Collaborative Computational Project, Number
4, 1994). The structure was solved by molecular replacement
using the program MOLREP (Collaborative Computational Project,
Number 4, 1994) and the coordinates of a BPTI:trypsin complex
(PDB ID: 2FTL) (Hanson et al., 2007) as molecular probe. Within
cyclic steps of refinement using the programs Coot (Emsley and
Cowtan, 2004) and REFMAC (Murshudov, 1997), 526 water molecules and one phosphate ion have been added to the structure.
2.5. Structure analysis
The stereochemistry of the complex structure was evaluated
using the programs VERIFY-3D (Lüthy et al., 1992), WHATCHECK
(Vriend, 1990), and PROCHECK (Laskowski et al., 1993). Analysis
of atom–atom contacts and structural superposition of the trypsin-bound inhibitors rShPI-1A, BPTI (PDB ID: 2FTL) and APPI (PDB
ID: 1TAW) was performed using the program WHATIF (Vriend,
1990). Intermolecular contact maps were drawn based on an analysis of the program CMA (Sobolev et al., 2005) and the interface
properties of these complexes, which were evaluated using the
PROTORP (Reynolds et al., 2009) and the PDBePISA (Krissinel and
Henrick, 2007) servers.
2.6. Free energy calculations by Crooks Gaussian intersection (CGI)
method
The relative binding free energy difference (DDG) associated
with a R11A substitution in rShPI-1A was calculated according to
the thermodynamic cycle shown in Supplementary Fig. 1 using the
CGI method (Goette and Grubmüller, 2009). The effect of the R11A
substitution on the Ki value at 298 K was estimated considering that
mut
DDGðR11AÞ ¼ RT lnðKKi ðIðIwt ÞÞÞ: The mutation was automatically setup
i
with the PYMACS package (Seeliger and de Groot, 2010). All
Molecular Dynamics (MD) simulations were performed using the
GROMACS software package (Hess et al., 2008). For details of the
simulation setup of the equilibrium and fast growth thermodynamic
integration (FGTI) runs see Supplementary methods. Van der Waals
(vdW) and electrostatic interaction energies between Arg11 (Ala11)
and the contact residues in the trypsin S3-pocket (0.4 nm cut-off)
were calculated by rerunning the equilibrium ensemble using a
2.0 nm cut-off. All the other parameters were setup analogous to
the equilibrium runs (Supplementary methods). The average and
block averaging error estimate was calculated using the g_analyze
tool of the GROMACS software package (Hess et al., 2008).
2.7. Protein Data Bank accession number
The atomic coordinates and structure factors of the rShPI1A:trypsin complex have been deposited in the Protein Data Bank
with PDB accession number 3M7Q.
3. Results and discussion
3.1. Formation of the rShPI-1A:trypsin complex
Specific complex formation of rShPI-1A with bovine trypsin was
revealed by Intensity Fading MALDI-TOF MS and dynamic light
Table 1
Data collection and refinement statistics of the rShPI-1A:trypsin complex.
rShPI-1A:trypsin PDB 3M7Q
Space group
a (Å)
b (Å)
c (Å)
P212121
58.3
66.5
71.0
VM (Å3/Da)
Solvent content (%)
Completeness of data (%)
No. of total reflections
No. of unique reflections
Average I/sigma intensity
Resolution (Å)
Redundancy
Rmerge (%)
No. of reflections used in refinement
Rcrystal (%)
No. of reflections used in Rfree
Rfree (%)
Protein atoms
Solvent atoms
2.32
47.00
98.8 (97.8)
162,197
30,437
10.4 (6.1)
30.0–1.7
5.8 (5.8)
5.1 (12.0)
28,976
15.8 (18.9)
1540
18.8 (25.8)
2167
526
Average B-factor (Å2)
Main-chain atoms
Side chain atoms
Ligand molecules
Solvent molecules
Other atoms (PO43)
10.3
11.8
12.1
28.2
15.2
Root mean square deviation
Bonds (Å)
Bond angles (°)
0.007
0.955
Residues in regions of the Ramachandran plot (%)
Most favored
86.9
Allowed
13.1
Disallowed
–
Generally allowed
–
Numbers in parentheses refer to the highest resolution shell.
scattering (DLS). The MALDI mass spectrum of purified rShPI-1A
clearly showed that its m/z signal (6681) faded upon the addition
of trypsin (results not shown). Besides, incubation of a 1:1 molar
ratio of both proteins for 10 min at RT and further DLS measurements resulted in a monodisperse solution of molecules
characterized by an RH of 2.63 ± 0.07 nm and a calculated MW of
29.69 kDa. Both values are increased compared to that of free
trypsin measured under identical conditions (RH = 2.33 ± 0.24 nm;
MW = 22.56 kDa) and agree with the theoretical MW of the enzyme-inhibitor complex (29.95 kDa). Moreover, a shift in the SEC
elution volume combined with the absence of trypsin activity in
the associated SEC fractions supports the successful complex
formation. The DLS signal of the solution did not change for up
to 15 days incubation at 4 °C, confirming the stability of the
rShPI-1A:trypsin complex.
3.2. Crystallization, structure determination and refinement
The purified rShPI-1A:trypsin complex formed orthorhombic
crystals that diffracted up to a resolution of 1.7 Å. The asymmetric
unit contained one molecule of the complex. The structure was
determined by molecular replacement using the coordinates of a
BPTI:trypsin complex (PDB ID: 2FTL) (Hanson et al., 2007) as a
search model and refined to a final R-factor of 15.8% (Rfree = 18.8%).
The inhibitor and the enzyme chain fit well into the electron density map, including the entire N- and C-terminus of the proteins.
All residues are located within the most favored or allowed regions
of the Ramachandran plot, reflecting the quality of the rShPI1A:trypsin structure. Data collection and refinement statistics are
summarized in Table 1.
274
R. García-Fernández et al. / Journal of Structural Biology 180 (2012) 271–279
Fig. 1. X-ray structure of the rShPI-1A:trypsin complex compared to trypsin
complexes of mammalian BPTI-Kunitz inhibitors. (A) Cartoon representation of the
overall structure of rShPI-1A (blue), BPTI (red) (PDB ID: 2FTL), and APPI (grey) (PDB
ID: 1TAW) in complexes with bovine trypsin (green). Binding loops of rShPI-1A are
highlighted in cyan. The side chains of the catalytic triad residues (His57, Asp102, and
Ser195), and of Asp189 at the bottom of the catalytic pocket of trypsin (all highlighted
in orange), as well as the basic residues at P1 positions of the inhibitors are shown
in stick representation and labeled accordingly. The inset shows the interaction of
the P1 residue of the enzymes with trypsin residue Asp189 in detail. (B) Buried
surface (BS) area of the inhibitor residues at the interface represented as percentage
of the total surface area that is buried after complex formation. The corresponding
amino acid sequences of rShPI-1A (cyan), BPTI (red), and APPI (grey), are shown.
Secondary binding loop residues are numbered according to BPTI. (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of this article.)
3.3. rShPI-1A binding to trypsin and comparison with mammalian
inhibitor complexes
Structural superposition of free (PDB ID: 3OFW) and trypsinbound rShPI-1A revealed minor reorientations of flexible
side-chain atoms (RMSD 1.77 Å) as a consequence of complex
formation, while the backbone atoms remained almost unaffected
(RMSD 0.69 Å). Deviations in the main chain psi (6 20°) and
side-chain angles (P30°) were restricted to residues Arg11 (P3 site)
and Lys13 (P1 site). Residues located in the primary binding loop
(Lys7, Tyr15 and Phe16), in the b-hairpin (Ser23, Glu24, and Lys27),
and in the C-terminal a-helix (Glu44 to Arg50) of rShPI-1A showed
only small side chain rearrangements.
The overall fold of the invertebrate inhibitor complex rShPI1A:trypsin is highly similar to the homologous complexes of the
mammalian BPTI-Kunitz inhibitors BPTI (PDB ID: 2FTL) (Hanson
et al., 2007), which is characterized by the lowest dissociation
constant known so far for an inhibitor-protease interaction
(Ki = 6 1014 M, Fritz and Wunderer, 1983), and APPI (PDB ID:
1TAW) (Scheidig et al., 1997), which has a trypsin affinity similar to
that of ShPI-1 (Ki = 4.2 1010 M, van Nostrand et al., 1995)
(Fig. 1A). Residues P3–P30 adopt the canonical conformation observed
in all BPTI-Kunitz inhibitors with the side chain of the basic residue
at position P1 in the conserved down conformation (Scheidig
et al., 1997). The complex interface consistently extends up to
positions P5–P40 (9–17 in rShPI-1A). These residues are folded into
a hairpin-like structure, which forms together with residues 32–37
a two-stranded antiparallel b-sheet that is stabilized by the disulfide
bridge Cys12-Cys36 along with several hydrogen bonds. The interaction occurs via similar interface residues (Fig 1B) defined by total
buried surface (BS) areas ranging from 765.1 Å2 (18.2% of the total
accessible surface area) to 744.8 Å2 (18.9%) and 729.0 Å2 (19.8%)
for the rShPI-1A, BPTI, and APPI trypsin complexes, respectively.
As known for canonical BPTI-Kunitz-type inhibitors, the primary direct interactions within the rShPI-1A:trypsin interface are
provided by residues at positions P2, P1, and P10 (Helland et al.,
1998; Huber et al., 1974; Scheidig et al., 1997), which are completely buried after complex formation (Fig. 1B). Based on this
analysis, additional contributions to the rShPI-1A complex stability
are indicated by residues Arg11 at position P3 and Ile32, whereas
Tyr15 (P20 ), Phe16 (P30 ), Gly35, and Cys36 appear to have a reduced
impact compared to the corresponding residues in the BPTI and
APPI complexes. Arg39 is reported as an essential secondary interaction site of BPTI (Scheidig et al., 1997). However, this site is not
important in rShPI-1A, where the equivalent residue Gly37 loses
essentially no accessible surface area upon complexation, as
observed in APPI (Fig. 1B). The overall intermolecular interaction
pattern remained largely conserved between all three inhibitortrypsin complexes (Fig. 2). However, the number of direct
enzyme-inhibitor contacts shorter than 4 Å is slightly reduced for
the rShPI-1A:trypsin complex (116 hydrophobic interactions, 10
hydrogen bonds) compared to a total of 137 interactions observed
for the BPTI and APPI trypsin complexes, including 12 and 13
hydrogen bonds, respectively (Supplementary Table 1). The interface of the rShPI-1A:trypsin complex is further stabilized by interactions mediated by six water molecules (Supplementary Table 2).
Four of them (wat148, wat268, wat269 and wat381) are highly
conserved compared to the BPTI:trypsin complex (Helland et al.,
1998), connecting the P3 and P1 residues in the central core of
the interface as well as Gly35 at the secondary binding loop of
the inhibitor with the enzyme. In contrast, the APPI:trypsin
complex is significantly less stabilized by water-mediated contacts
(Supplementary Table 2).
3.3.1. Reactive residue (position P1)
Around 40% of the total interactions at the rShPI-1A:trypsin
interface are formed by Lys13 at position P1. This site is considered
to be the most important in terms of defining the specificity and
the strength of the inhibitory interaction (Helland et al., 1998;
Huber et al., 1974; Scheidig et al., 1997). The side chain of the P1
residue points directly into the catalytic pocket of the trypsin
molecule, interacting with Asp189 at the bottom of this pocket
(Fig. 3A). The interaction maps of the rShPI-1A:trypsin and
BPTI:trypsin complexes are almost identical at position P1
(Fig. 2). Depending on the analyzed BPTI-Kunitz:trypsin complex
and on the applied scoring criteria, the nature of the important
P1–S1 interaction has been variously described so far, including
R. García-Fernández et al. / Journal of Structural Biology 180 (2012) 271–279
275
Fig. 2. Protein–protein interaction maps of rShPI-1A:trypsin and homologous complexes. Inhibitor residues of the primary and secondary binding loops are located on the xaxis, while the trypsin residues involved in intermolecular interactions are presented on the y-axis. The maps were calculated based on the contacts provided by interaction
analysis using the WHATIF and PISA servers. (A) rShPI-1A:trypsin complex; (B) BPTI:trypsin complex (PDB ID: 2FTL); (C) APPI:trypsin complex (PDB ID: 1TAW). Squares
represent vdW interactions whereas cross and circles represent direct and indirect hydrogen bonds, respectively. For a detailed analysis of the associated hydrogen bonds see
Supplementary Tables 1 and 2.
no direct interaction (Huber et al., 1974) or a distance longer than
common for H-bonds (3.63 Å) (Helland et al., 1998). We clearly
observed a direct interaction between the NZ atom of Lys13 of
rShPI-1A and the OD1 atom of the S1 residue Asp189 in trypsin at
a distance of 3.35 Å (Fig. 3A), scored as a hydrogen bond consistent
with other BPTI-Kunitz:trypsin complexes (Helland et al., 1998;
Schmidt et al., 2005) (Supplementary Table 1).
The analysis of various mutants of BPTI and APPI revealed that
efficient trypsin inhibition strongly depend on a basic residue at
position P1 (Krowarsch et al., 2005; Otlewski et al., 2001). However,
the interaction is almost independent of the nature of the basic
residue at P1 position, since the vice versa substitutions Lys15Arg
in BPTI (pseudo-BPTI) and Arg15Lys in APPI did not significantly
affect the association energies with trypsin (Navaneetham et al.,
2010; Van Nostrand et al., 1995). This is further supported by the
comparable Ki values of rShPI-1A (Lys at P1 position) and APPI
(Arg at P1 position) for trypsin. Chymotrypsin and coagulation
factor IXa also displayed no preference for Arg or Lys at P1 when
inhibited by wild type or Arg15Lys substituted APPI (Van Nostrand
et al., 1995). In contrast, also highly residue specific inhibition of
serine proteases by BPTI-Kunitz inhibitors has been reported, e.g.
for kallikrein, which favors Arg over Lys (Fiedler, 1987; Grzesiak
et al., 2000), while plasmin inhibition is enhanced by Lys at P1
position (Van Nostrand et al., 1995). This agrees well with the
increased antiplasmin activity of ShPI-1 (1010 M) compared to
kallikrein (108 M) (Delfín et al., 1996).
3.3.2. Pn side of the primary binding loop
Most of the interactions at the N-terminal side of the scissile
bond of rShPI-1A are performed by residue Arg11 at position P3.
The backbone hydrogen bond between the carbonyl oxygen of
Arg11 and the nitrogen of Gly216 in trypsin is highly conserved in
all BPTI-Kunitz inhibitor-proteinase complexes described so far.
However, our crystal structure revealed that the arginine side
chain points directly into a pocket on the enzyme surface (S3 site)
that is formed by trypsin residues Ser96 to Ser99 as well as Gln175,
Ser214, and Trp215, establishing two additional H-bonds at the
rShPI-1A complex interface (Figs. 2 and 3B). Together with optimized hydrophobic contacts (Supplementary Table 1), further indirect polar interactions with trypsin residues Thr98/Gln175 as well as
Ser96/Asn97 are mediated by wat192 and wat540, respectively
(Fig. 3B, Supplementary Table 2). BPTI and APPI locate a proline
residue at this position that does not deeply enter the S3 pocket
of trypsin due to its cyclic nature, resulting in significantly less
side-chain interactions, as revealed by the corresponding trypsincomplex structures (Hanson et al., 2007; Scheidig et al., 1997)
(Fig. 3B). Thus, an important impact of arginine at position P3 is
indicated for the interface stabilization within the rShPI-1A:trypsin
complex. Other BPTI-Kunitz-type trypsin inhibitors, e.g. kalicludines from the sea anemone Anemonia sulcata and domain 1 of
hepatocyte growth factor activator inhibitor type I also contain
an equivalent Arg residue (Schweitz et al., 1995; Shimomura
et al., 1997), but its impact for complex stabilization was not structurally elucidated so far.
To support our suggestion, we calculated binding free energy
differences after in silico Arg11Ala mutation of rShPI-1A. Substitution by proline, which resembles the P3 position in BPTI and APPI,
is technically prevented, since the required destruction of covalent
bonds is conceptionally different from all other mutations. However, the methyl side chain of alanine approximates the hydrophobicity and the length of the proline side chain that sticks into the S3
pocket, allowing a rough estimation of the arginine-specific contribution to the stabilization of the rShPI-1A:trypsin complex.
According to the free energy cycle shown in Supplementary
Fig. 1, a positive DDG of 5.52 ± 1.90 kJ/mol was calculated,
accounting for a 10-fold increase in the theoretical Ki value against
trypsin as a result of the Arg11Ala mutation in rShPI-1A. Net positive electrostatic and vdW free energies were also obtained for the
individual binding free energy differences of each trypsin residue
within the S3 pocket as a consequence of the Arg11Ala mutation
(Table 2). All S3 residues suggested to be involved in hydrogen
bonds (Fig. 3B) provide a significant electrostatic contribution to
the complex interaction, while Leu99 and Trp215 form strong vdW
interactions with the hydrophobic carbon moiety of the arginine
side chain.
Next to Arg and Pro, a variety of further amino acids has been
observed at the P3 position of BPTI-Kunitz-type inhibitors
(Scheidig et al., 1997). However, the influence of the P3 residue
strongly depends on the individual enzyme-inhibitor complex.
While we show here that Arg strengthens the specific interaction
276
R. García-Fernández et al. / Journal of Structural Biology 180 (2012) 271–279
Fig. 3. Stereo views of the enzyme-inhibitor interface around the primary binding loop (positions P5 to P40 ) of rShPI-1A and BPTI. The active site at position P1 (A), the Nterminal (positions P5 to P2) (B) and C-terminal (positions P10 to P40 ) (C) sides of the primary binding loop of rShPI-1A (cyan) and BPTI (red) and the corresponding trypsin
residues (blue and light red, respectively) involved in direct or water-mediated interactions are shown using stick representation. Water molecules are shown as spheres and
H-bonds are depicted with dashed lines. Only the H-bond network around wat381 is conserved in BPTI and APPI complexes. For a detailed comparison of the H-bond pattern
at the trypsin complex interface of rShPI-1A, BPTI, and APPI, see Supplementary Tables 1 and 2. (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
of rShPI-1A with trypsin, a Pro/Arg but also a Pro/Ala substitution
at P3 significantly decreases the Ki value for pseudo-BPTI against
trypsin (Grzesiak et al., 2000; Otlewski et al., 2001). The transformation of the non-inhibitory collagen VI Kunitz-like module into
a specific trypsin inhibitor also required the introduction of Pro
at position P3 (Kohfeldt et al., 1996). Domain 1 of the protease
inhibitor nexin II, a secreted isoform of APPI, inhibited trypsin
independently of Pro or Ala at P3 position, whereas inhibition of
other proteases, e.g. kallikrein and plasmin, strongly depend
on the presence of a Pro residue (Navaneetham et al., 2010).
Interestingly its inhibitory activity against factor Xa was recovered
by introduction of Arg at P3 position (Navaneetham et al., 2005).
R. García-Fernández et al. / Journal of Structural Biology 180 (2012) 271–279
Table 2
Calculated binding free energy differences of trypsin S3 site residues as a result of
in silico Arg11Ala mutation in rShPI-1A.
Trypsin S3 residues
96
Ser
Asn97
Thr98
Leu99
Gln175
Trp215
Gly216
hDEv dW ia (kJ/mol)
hDEele ib (kJ/mol)
0.73 ± 0.07
1.82 ± 0.05
3.41 ± 0.08
8.22 ± 0.17
4.01 ± 0.20
11.28 ± 0.44
1.81 ± 0.47
20.68 ± 1.51
37.95 ± 1.13
22.98 ± 1.09
4.15 ± 0.40
19.90 ± 3.39
20.11 ± 0.31
2.75 ± 1.97
R11X
(a) hDEv dW ia ¼ hEA11X
v dW i hEv dW i.
A11X
(b) hDEele ib ¼ hEele
i hER11X
i.
ele
Differences in electrostatic and vdW energies were calculated considering equations (a) and (b), respectively. Consequently, positive values indicate that the
interactions are more favorable in the wild-type complex.
Consequently, a general trend for an optimal amino acid at position
P3 for efficient trypsin inhibition is not indicated.
Most of the interactions involving the residue at position P5 as
well as the conserved cysteine residue at position P2 are conserved
in the trypsin complexes of rShPI-1A, BPTI, and APPI. However, the
highly conserved H-bond between Cys14 at position P2 of APPI/
BPTI and Gln192 in trypsin as well as the hydrophobic contact between Gln192 (trypsin) and the highly conserved Gly10 at position
P4 is absent in the rShPI-1A complex (Supplementary Table 1).
Since the trypsin residue Gln192 is reported to have a stabilizing
impact on the enzyme-inhibitor complexes (Schmidt et al., 2005),
the absence of these interactions is suggested to attenuate the
stabilizing effect that was attributed to Arg13 at position P3 in
rShPI-1A.
3.3.3. Pn prime (Pn0 ) side of the primary binding loop
At the Pn’ side of the rShPI-1A:trypsin complex interface, the
number of hydrophobic as well as polar contacts is reduced compared to the trypsin complex of BPTI and APPI, due to sequences
alterations within this region (Fig. 2). A strong tendency for alanine, e.g. in BPTI and APPI, and glycine, e.g. in rShPI-1A, is observed
at position P10 within BPTI-Kunitz-type domains (Grzesiak et al.,
2000). However, the missing methyl-group of the Gly residue in
rShPI-1A resulted in reduced vdW interactions with Cys42 of trypsin, supported by a significant increase of the Ki value of BPTI
against trypsin after substitution of Ala16 by glycine (Krowarsch
et al., 2005; Otlewski et al., 2001). In addition, Arg17 at position
P20 of BPTI establishes an H-bond with His40 of trypsin, but
277
equivalent contacts of Tyr15 (rShPI-1A) and Met17 (APPI) are absent
(Fig. 3C). Another H-bond between the residue at position P40 and
Tyr39 of trypsin is only formed by Ile or Ser in BPTI and APPI
complexes, respectively, but not by Pro in the rShPI-1A complex.
Although the coordination of a water molecule (wat132) at position P40 (Pro17) in rShPI-1A is conserved compared to the trypsin
complexes of BPTI and APPI (Helland et al., 1998), water-mediated
interactions with the enzyme are only formed within the
BPTI:trypsin complex (Supplementary Table 2). These differences
within the interaction pattern at the prime site of the primary
binding loop in rShPI-1A are supposed to contribute to the
increased Ki value of rShPI-1A compared to BPTI. However, a
reduced impact on the overall complex stability is in general
supposed for the Pn0 side interactions compared to that at the Pn
side (Perona and Craik, 1997).
3.4. Secondary binding loop
The secondary binding loop, comprising residues 34 (P19’) to 39
(P24’) in BPTI, is considered to be an additional segment that influences the association energy of BPTI-Kunitz domains (Czapinska
et al., 2000; Chand et al., 2004; Dennis and Lazarus, 1994; Helland
et al., 1998). At the most divergent position P19’, a stabilizing
hydrophobic interaction with trypsin residue Tyr151 is only formed
by Ile32 of rShPI-1A, but not by equivalent residues of BPTI and
APPI. The importance of this position for specific inhibitory activity
is supported by previous mutagenesis studies of APPI (Dennis and
Lazarus, 1994) and of the non-inhibitory BPTI-Kunitz-like module
of collagen VI (Kohfeldt et al., 1996). At the positions P20’ to P23’
(Tyr33 to Cys36 according to rShPI-1A numbering), rShPI-1A, BPTI
and APPI locate identical amino acids. However, no vdW contacts
are mediated by P220 (Gly35) and P230 (Cys36) in the trypsin complex of rShPI-1A (Fig. 2), indicating a minor impact of these residues for complex stabilization in rShPI-1A. In BPTI, the
interactions formed by the polar residue Arg39 at position P24’
are suggested to significantly contribute to the exceptionally low
Ki value of this inhibitor (Scheidig et al., 1997). In addition to a
water-mediated interaction with trypsin residue Ser96, this is
largely attributed to a direct H-bond formed with trypsin residue
Asn 97 (Fig. 4) A similar interaction is reported to strongly
contribute to the protease specificity and complex stability of
bovine trypsin-bound domain 1 from TFPI-2 (Chand et al., 2004).
In rShPI-1A and APPI, comparable interactions are prevented by
the presence of a glycine residue at position P24’, consistent with
the significantly increased Ki value of both inhibitors for trypsin.
Fig. 4. Stereo view of the interaction of the secondary binding loop of rShPI-1 (cyan) and BPTI (red) with trypsin. The enzyme residues in the rShPI-1 and BPTI complexes are
colored in blue and light red, respectively. Positions 33–36 (rShPI-1A numbers) are conserved within the inhibitors. Dashed lines are used for direct and indirect H-bonds. For
a detailed comparison of the H-bond pattern at the trypsin complex interface of rShPI-1A, BPTI, and APPI, see Supplementary Tables 1 and 2. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)
278
R. García-Fernández et al. / Journal of Structural Biology 180 (2012) 271–279
4. Concluding remarks
Appendix A. Supplementary data
Structural information on proteins isolated from marine
invertebrates is rare to date, compared to the tremendous variety
of invertebrate species (Brusca and Brusca, 2003). Here we present
the first crystallographic structure of a BPTI-Kunitz-type inhibitor
from a marine invertebrate in complex with a serine protease, bovine trypsin. The common trypsin interaction pattern of canonical
BPTI-Kunitz-type inhibitors remained largely conserved throughout evolution from invertebrates to mammalian species,
significantly depending on the P2, P1, and P1’ residues of the
inhibitor (Helland et al., 1998; Huber et al., 1974; Scheidig et al.,
1997). In addition, we observed a prominent role of arginine at
position P3 in the stabilization of the rShPI-1A:trypsin complex,
partially compensating the absence of important contacts at other
positions within the primary and secondary binding loops.
Thus, the associated Ki value against trypsin is maintained at
1010 M (Delfín et al., 1996).
The structure presented here provides valuable information for
further design of ShPI-1 mutants, which will help to elucidate the
basis of its multifunctional activity, associated with an increased
knowledge about biomolecules from marine invertebrates. Due
to their general defensive role against prey and predators, protease
inhibitors from marine invertebrates have generally exceptional
structural and functional properties (González et al., 2007; Lenarčič
and Turk, 1999; Xue et al., 2009; Alonso del Rivero et al., 2012).
Although unusual within the BPTI-Kunitz family, ShPI-1 as well
as three other invertebrate domains of this family exhibit activity
against structurally unrelated proteases, but the mechanisms of
their cross-activity remain to be elucidated in detail (Delfín et al.,
1996; Sasaki et al., 2006; Alonso del Rivero et al., 2012; Minagawa
et al., 1997). ShPI-1 is supposed to inhibit other serine proteases,
e.g. plasmin, kallikrein, as well as chymotrypsin, due to the basic
residue at P1 position, as already reported for complexes of BPTI
and APPI (Scheidig et al., 1997). This also applies to the reported
inhibition of human neutrophil elastase (Delfín et al., 1996), which
tolerates a broad specificity of P1 residues due to a more flexible S1
pocket (McBride et al., 1999), including canonical inhibitors from
other families with basic P1 residues (González et al., 2007). These
preliminary considerations about the inhibitory mechanisms of
ShPI-1 against serine proteases have to be structurally validated
to understand more thoroughly the unusual multifunctional
activity of this inhibitor.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jsb.2012.08.009.
Acknowledgements
We are indebted to Prof. Dr. U. Hahn (University of Hamburg,
Germany) for his support to start this research. We are grateful
to Prof. Dr. J. Díaz, Dr. M. Mansur, and D. F. Gil for their contribution
to heterologous expression of ShPI-1. We thank D. Oberthuer (University of Hamburg, Germany) for his help during data processing
and refinement and Dr. G. Groenhof (Max Planck Institute for Biophysical Chemistry, Göttingen, Germany) for technical support
during free energy calculations. We also acknowledge Dr. C. Berry
(Cardiff University, Wales, UK) for critical reading of the manuscript. RGF, MCh, YGG, and PAV thank the International Foundation
for Science (IFS), Sweden and the German Academic Exchange Service (DAAD) for financial support. MP is member of the Hamburg
School for Structure and Dynamics in Infection (SDI) and thanks
the Hamburg Ministry of Science and Research and the JoachimHertz-Stiftung, as part of the Hamburg Initiative for Excellence in
Research, for financial support. LR and CB thank the German Federal Ministry of Education and Research (BMBF) for financial support [grants 01KX0806 and 01KX0807].
References
Abbenante, G., Fairlie, D.P., 2005. Protease inhibitors in the clinic. Med. Chem. 1, 71–
104.
Alonso del Rivero, M., Trejo, S.A., Reytor, M.L., Rodríguez de la Vega, M., Delfín, J.,
González, Y., Canals, F., Chávez, M.A., Avilés, F.X., 2012. A tri-domain
bifunctional inhibitor of metallocarboxypeptidases A and serine proteases
isolated from the marine annelid Sabellastarte magnifica. J. Biol. Chem. 287 (19),
15427–15438.
Antuch, W., Berndt, K.D., Chávez, M.A., Delfín, J., Wuethrich, K., 1993. The NMR
solution structure of a Kunitz-type proteinase inhibitor from the sea anemone
Stichodactyla helianthus. Eur. J. Biochem. 212, 675–684.
Bieth, J., 1974. Some kinetic consequences of the tight binding of protein-proteinase
inhibitor to proteolytic enzymes and their application to the determination of
dissociation constants. Bayer Symposium V ‘‘Proteinase Inhibitor’’. SpringerVerlag, Berlin, pp. 463–469.
Brusca, R.C., Brusca, G.J., 2003. Invertebrates, second ed. Sinauer Associates Inc,
Publishers Sunderland, Massachusetts.
Burgering, M.J., Orbons, L.P., van der Doelen, A., Mulders, J., Theunissen, H.J.,
Grootenhuis, P.D., Bode, W., Huber, R., Stubbs, M.T., 1997. The second Kunitz
domain of human tissue factor pathway inhibitor: cloning, structure
determination and interaction with factor Xa. J. Mol. Biol. 269, 395–407.
Chand, H.S., Schmidt, A.E., Bajaj, S.P., Kisiel, W., 2004. Structure-function analysis of
the reactive site in the first Kunitz-type domain of human tissue factor pathway
inhibitor-2. J. Biol. Chem. 279, 17500–17507.
Collaborative Computational Project, Number 4, 1994. The CCP4 suite: programs for
protein crystallography. Acta Crystallogr. D50, 760–763.
Czapinska, H., Otlewski, J., Krzywda, S., Sheldrick, G.M., Jaskólski, M., 2000. Highresolution structure of bovine pancreatic trypsin inhibitor with altered binding
loop sequence. J. Mol. Biol. 295, 1237–1249.
Deisenhofer, J., Steigemann, W., 1975. Crystallographic refinement of the structure
of bovine pancreatic trypsin inhibitor at l.5 Å resolution. Acta Crystallogr. B31,
238–250.
Delfín, J., Morera, V., González, Y., Díaz, J., Márquez, M., Larionova, N., Saroyán, A.,
Padrón, G., Chávez, M.A., 1996. Purification, characterization and
immobilization of proteinase inhibitors from Stichodactyla helianthus. Toxicon
34, 1367–1376.
Dennis, M.S., Lazarus, R.A., 1994. Kunitz domain inhibitors of tissue factor-factor
VIIa: potents and specific inhibitors by competitive phage selection. J. Biol.
Chem. 269, 22137–22144.
Emsley, P., Cowtan, K., 2004. Coot: model-building tools for molecular graphics.
Acta Crystallogr. D60, 2126–2132.
Erlanger, B.F., Kokowsky, N., Cohen, E., 1961. Preparation and properties of two new
chromogenic substrates of trypsin. Arch. Biochem. Biophys. 95, 271–278.
Fiedler, F., 1987. Effects of secondary interactions on the kinetics of peptide and
peptide ester hydrolysis by tissue kallikrein and trypsin. Eur. J. Biochem. 163,
303–312.
Fischer, H., Polikarpov, I., Craievich, A.F., 2004. Average protein density is a
molecular-weight-dependent function. Protein Sci. 13, 2825–2828.
Fritz, H., Wunderer, G., 1983. Biochemistry and applications of aprotinin, the
kallikrein inhibitor from bovine organs. Arzneimittelforschung 33, 479–494.
Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M.R., Appel, R.D.,
Bairoch, A., 2005. Protein Identification and Analysis Tools on the ExPASy
Server;. In: Walker, J.M. (Ed.), The Proteomics Protocols Handbook. Humana
Press, pp. 571–607.
Gil, D., García-Fernández, R., Alonso-del-Rivero, M., Lamazares, E., Pérez, M., Varas,
L., Díaz, J., Chávez, M.A., González-González, Y., Mansur, M., 2011. Recombinant
expression of ShPI-1A, a non-specific BPTI-Kunitz-type inhibitor, and its
protection effect on proteolytic degradation of recombinant human
miniproinsulin expressed in Pichia pastoris. FEMS Yeast Res. 7, 575–586.
Goette, M., Grubmüller, H., 2009. Accuracy and convergence of free energy
differences calculated from nonequilibrium switching processes. J. Comp.
Chem. 30, 447–456.
González, Y., Pons, T., Gil, J., Besada, V., Alonso-del-Rivero, M., Tanaka, A.S., Araujo,
M.S., Chávez, M.A., 2007. Characterization and comparative 3D modeling of
CmPI-II, a novel ‘non-classical’ Kazal-type inhibitor from the marine snail
Cenchritis muricatus (Mollusca). Biol. Chem. 388, 1183–1194.
Grzesiak, A., Krokoszynska, I., Krowarchsch, D., Dadlez, M., Otlewski, J., 2000.
Inhibition of six serine proteinases of the human coagulation system by
mutants of bovine pancreatic trypsin inhibitor. J. Biol. Chem. 321, 647–658.
Hanson, W.M., Domek, G.J., Horvath, M.P., Goldenberg, D.P., 2007. Rigidification of a
flexible protease inhibitor variant upon binding to trypsin. J. Mol. Biol. 366,
230–243.
Helland, R., Leiros, I., Berglund, G., Willassen, N.P., Smala, A., 1998. The crystal
structure of anionic salmon trypsin in complex with bovine pancreatic trypsin
inhibitor. Eur. J. Biochem. 256, 317–324.
Hess, B., Kutzner, C., Van der Spoel, D., Lindahl, E., 2008. GROMACS 4: algorithms for
highly efficient, load-balanced, and scalable molecular simulation. J. Chem.
Theory Comput. 4, 435–447.
R. García-Fernández et al. / Journal of Structural Biology 180 (2012) 271–279
Huber, R., Kukla, D., Bode, W., Schwager, P., Bartels, K., Deisenhofer, J., Steigemann,
W., 1974. Structure of the complex formed by bovine trypsin and bovine
pancreatic trypsin inhibitor. Crystallographic refinement at 1.9 Å resolution. J.
Mol. Biol. 89, 73–101.
Kohfeldt, E., Gohring, W., Mayer, U., Zweckstetter, M., Holak, T.A., Chu, M.L., Timpl,
R., 1996. Conversion of the Kunitz-type module of collagen VI into a highly
active trypsin inhibitor by site-directed mutagenesis. Eur. J. Biochem. 238, 333–
340.
Krissinel, E., Henrick, K., 2007. Inference of macromolecular assemblies from
crystalline state. J. Mol. Biol. 372, 774–797.
Krowarsch, D., Zakrzewska, M., Smalas, A.O., Otlewski, J., 2005. Structure-function
relationships in serine protease-bovine pancreatic trypin inhibitor interaction.
Protein Pept. Lett. 12, 403–407.
Kunitz, M., Northrop, J.H., 1936. Isolation from beef pancreas of crystalline
trypsinogen, trypsin, a trypsin inhibitor, and an inhibitor-trypsin compound.
J. Gen. Physiol. 19, 991–1007.
Laskowski Jr., M., Qasim, M.A., Lu, S.M., 2000. Oxford University Press, Oxford, pp.
228–279.
Laskowski, R.A., MacArthur, M.W., Moss, D.S., Thornton, J.M., 1993. PROCHECK - a
program to check the stereochemical quality of protein structures. J. App. Cryst.
26, 283–291.
Lenarčič, B., Turk, V., 1999. Thyroglobulin type-1 domains in equistatin inhibit both
papain-like cysteine proteinases and cathepsin D. J. Biol. Chem. 274, 563–566.
Lüthy, R., Bowie, J.U., Eisenberg, D., 1992. Assessment of protein models with threedimensional profiles. Nature 356, 83–85.
Navaneetham, D., Jin, L., Pandey, P., Strickler, J.E., Babine, R.E., Abdel-Meguid, S.S.,
Walsh, P.N., 2005. Structural and mutational analyses of the molecular
interactions between the catalytic domain of factor XIa and the Kunitz
protease inhibitor domain of protease nexin 2. J. Biol. Chem. 280, 36165–36175.
Navaneetham, D., Sinha, D., Walsh, P.N., 2010. Mechanisms and specificity of factor
XIa and trypsin inhibition by protease nexin 2 and basic pancreatic trypsin
inhibitor. J. Biochem. 148, 467–479.
McBride, J.D., Freeman, H.N.M., Leatherbarrow, R.J., 1999. Selection of human
elastase inhibitors from a conformationally constrained combinatorial peptide
library. Eur. J. Biochem. 266, 403–412.
Minagawa, S., Ishida, M., Shimakura, K., Nagashima, Y., Shiomi, K., 1997. Isolation
and amino acid sequences of two Kunitz-type protease inhibitors from the sea
anemone Anthopleura aff Xanthogrammica. Comp. Biochem. Physiol. 118B, 381–
386.
Murshudov, G.N., 1997. Refinement of macromolecular structures by the
maximum-likelihood method. Acta Crystallogr. D53, 240–255.
Otlewski, J., Jaskolski, M., Buseck, O., Cierpicki, T., Czapiñska, H., et al., 2001.
Structure-function relationship of serine protease-protein inhibitor interaction.
Acta Biochim. Pol. 38, 419–428.
279
Perona, J.J., Craik, C.S., 1997. Evolutionary divergence of substrate specificity within
the chymotrypsin-like serine protease fold. J. Biol. Chem. 272, 29987–29990.
Reynolds, C., Damerell, D., Jones, S., 2009. Protorp: a protein-protein interaction
analysis tool. Bioinformatics 25, 413–414.
Sasaki, S.D., Cotrin, S.S., Carmona, A.K., Tanaka, A.S., 2006. An unexpected inhibitory
activity of Kunitz-type serine proteinase inhibitor derived from Boophilus
microplus trypsin inhibitor of Cathepsin L. Biochem. Biophys. Res. Comm. 341,
266–272.
Schechter, I., Berger, A., 1967. On the size of the active site in proteases. Biochem.
Biophys. Res. Commun. 27, 157–162.
Scheidig, A.J., Hynes, T.R., Pelletier, L.A., Wells, J.A., Kossiakoff, A.A., 1997. Crystal
structures of bovine chymotrypsin and trypsin complexed to the inhibitor
domain of Alzheimer’s amyloid b-protein precursor (APPI) and basic trypsin
inhibitor (BPTI): engineering of inhibitors with altered specificities. Protein Sci.
6, 1806–1824.
Schmidt, A.E., Chand, H.S., Cascio, D., Kisiel, W., Bajaj, S.P., 2005. Crystal structure of
Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 in complex with
trypsin. J. Biol. Chem. 280, 27832–27838.
Schweitz, H., Bruhn, T., Guillemare, E., Moinier, D., Lancelin, J.M., Beress, L.,
Lazdunski, M., 1995. Kalicludines and kaliseptine. Two different classes of sea
anemone toxins for voltage sensitive K+ channels. J. Biol. Chem. 270, 25121–
25126.
Seeliger, D., de Groot, B.L., 2010. Protein thermostability calculations using
alchemical free energy simulations. Biophys. J. 98, 2309–2316.
Shimomura, T., Denda, K., Kitamura, A., Kawaguchi, T., Kito, M., Kondo, J., Kagaya, S.,
Qin, L., Takata, H., Miyazawa, K., Kitamura, N., 1997. Hepatocyte growth factor
activator inhibitor, a novel Kunitz-type serine protease inhibitor. J. Biol. Chem.
272, 6370–6376.
Sobolev, V., Eyal, E., Gerzon, S., Potapov, V., Babor, M., et al., 2005. SPACE: a suite of
tools for protein structure prediction and analysis based on complementarity
and environment. Nucleic Acids Res. 33, W39–W43.
Van Nostrand, W.E., Schmaier, A.H., Siegel, R.S., Wagner, S.L., Raschke, W.C., 1995.
enhanced plasmin inhibition by a reactive center lysine mutant of the Kunitztype protease inhibitor domain of the amyloid b-protein precursor. J. Biol.
Chem. 270, 22827–22830.
Vriend, G., 1990. WHAT IF, a molecular modeling and drug design program. J. Mol.
Graph. 8, 52–56.
Xue, Q., Itoh, N., Schey, K.L., Cooper, R.K., La Peyre, J.F., 2009. Evidence indicating the
existence of a novel family of serine protease inhibitors that may be involved in
marine invertebrate immunity. Fish Shellfish Immunol. 27, 250–259.
Yanes, O., Villanueva, J., Querol, E., Avilés, F.X., 2007. Detection of non-covalent
protein interactions by ‘intensity fading’ MALDI-TOF mass spectrometry:
applications to proteases and protease inhibitors. Nat. Protoc. 2, 119–130.