Journal of Structural Biology 180 (2012) 271–279

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Journal of Structural Biology

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Structural insights into serine protease inhibition by a marine invertebrate BPTIKunitz-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, Cubab International Cooperation Network ‘‘Proteómica y Quimiogenómica de Inhibidores de Proteasas de Origen Natural con Potencial Terapéutico en Malaria’’ from theIberoamerican Programme of Science and Technology for Development, RED CYTED-PROMALc Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre, C/Melchor Fernández Almagro 3, Madrid E-28029, Spaind Institute of Biochemistry and Molecular Biology, University of Hamburg, c/o DESY, Notkestr. 85, 22603 Hamburg, Germanye University Medical Center Hamburg-Eppendorf, Department of Medical Microbiology, Virology and Hygiene, Martinistr. 52, 20246 Hamburg, Germanyf Center of Molecular Immunology, P.O. Box 16040, 11600 Havana, Cubag Joint Laboratory for Structural Biology of Infection and Inflammation, Institute of Biochemistry and Molecular Biology, University of Hamburg, and Institute ofBiochemistry, University of Lübeck, c/o DESY, Notkestr. 85, 22603 Hamburg, Germany

a r t i c l e i n f o

Article history:Received 14 February 2012Received in revised form 22 August 2012Accepted 24 August 2012Available online 5 September 2012

Keywords:BPTI Kunitz-type inhibitorSerine proteaseProtein-inhibitor interactionCrystal structureMarine invertebrates

1047-8477/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.jsb.2012.08.009

Abbreviations: APPI, inhibitor domain of human Atein precursor; BAPA, N-benzoyl-arginine-p-nitroanitrypsin inhibitor; BS, buried surface; DLS, dynamichigh-performance liquid chromatography; IF, intendissociation constant; MALDI-TOF, matrix assistedtime-of-flight; MS, mass spectrometry; MW, moleculbank; RH, hydrodynamic radius; RMSD, root meantemperature; SDS–PAGE, sodium dodecyl sulfate-polysis; SEC, size exclusion chromatography; ShPI-1, Sticinhibitor 1; TFPI, tissue factor protease inhibitor; vdW⇑ Corresponding author. Fax: +49 40 8998 4739.

E-mail address: redecke@biochem.uni-luebeck.de

a b s t r a c t

Proteins isolated from marine invertebrates are frequently characterized by exceptional structural andfunctional properties. ShPI-1, a BPTI Kunitz-type inhibitor from the Caribbean Sea anemone Stichodactylahelianthus, 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 struc-ture of ShPI-1 in complex with the serine protease bovine pancreatic trypsin at 1.7 Å resolution. The over-all structure and the important enzyme-inhibitor interactions of this first invertebrate BPTI-likeKunitz-type inhibitor:trypsin complex remained largely conserved compared to mammalian BPTI-Kunitzinhibitor complexes. However, a prominent stabilizing role within the interface was attributed to argi-nine at position P3. Binding free-energy calculations indicated a 10-fold decrease for the inhibitor affinityagainst 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 thesecondary binding loop of ShPI-1 were detected. In addition, the structure provides important informa-tion for site directed mutagenesis to further optimize the activity of rShPI-1A for biotechnologicalapplications.

� 2012 Elsevier Inc. All rights reserved.

1. Introduction

Proteolytic enzymes are involved in the regulation of all physi-ological processes in living organisms. Deficiencies or alterations oftheir activities can lead to abnormal development, disease, and

ll rights reserved.

lzheimer’s amyloid betapro-lide; BPTI, bovine pancreaticlaser light scattering; HPLC,sity fading; Ki, equilibriumlaser desorption/ionization

ar weight; PDB, protein datasquare deviation; RT, roomacrylamide gel electrophore-hodactyla helianthus protease

, van der Waals.

(L. Redecke).

even death (Abbenante and Fairlie, 2005). Therefore, detailedknowledge about the structure–function-relationship of enzymesis of great interest to the pharmaceutical industry. Based on theirimportance in human health and disease prevention, there is alsoincreased interest in applying protease inhibitors in therapeuticand biotechnological applications (Abbenante and Fairlie, 2005).Additionally, enzyme-inhibitor complexes are useful model sys-tems to study protein–protein recognition (Laskowski et al.,2000; Otlewski et al., 2001).

Canonical serine protease inhibitors have been the most exten-sively 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 inhibitorsof serine proteases, is composed of more than 1000 differentsequences isolated from a variety of animals ranging from inverte-brates 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 bo-vine pancreatic trypsin inhibitor (BPTI), the prototypical memberof this family. Insights into the nature of inhibitory interactionshave been obtained by X-ray crystallographic studies investigatingserine proteases in complex mainly with mammalian BPTI-Kunitzinhibitors (Burgering et al., 1997; Huber et al., 1974; Scheidiget al., 1997).

BPTI-Kunitz inhibitors are small proteins (around 6 kDa) with acompact structure composed of a hydrophobic core, containing acentral b-sheet and three disulfide bridges with conserved chirali-ties (Antuch et al., 1993; Czapinska et al., 2000; Deisenhofer andSteigemann, 1975; Huber et al., 1974). This core is the scaffold thatsupports the convex and exposed canonical binding loop atpositions P3–P30, according to the Pn–Pn0 notation of Schechterand Berger (1967). This loop is highly complementary to theconcave protease active site and is, thus, responsible for theextreme stability of the interaction with the target enzyme in asubstrate-like manner (Laskowski et al., 2000). The dissociationconstants of enzyme:inhibitor complexes range from 10�13 to10�7 M, mainly depending on the nature of the residue at positionP1 and the number of contacts formed with the S1 site of the boundprotease (Czapinska et al., 2000). Typically, trypsin inhibitorscontain Arg/Lys at position P1, whereas chymotrypsin inhibitorshave Leu/Met at this position. However, other residues at theprimary binding site and within the secondary binding loop(residues 34–39 in BPTI) are also suggested to have significant influ-ence on the association energy (Scheidig et al., 1997; Czapinskaet al., 2000; Laskowski et al., 2000).

A molecule of 6.1 kDa sharing the basic structure of the BPTI-Kunitz family, named ShPI-1 (UniProt accession No. P31713),was previously isolated by our group from the Caribbean Seaanemone Stichodactyla helianthus (Antuch et al., 1993; Delfínet al., 1996). In contrast to other BPTI-Kunitz inhibitors, ShPI-1is able to inhibit not only serine proteases with Ki values inthe nanomolar range, but also cysteine and aspartic proteasessuch as papain and pepsin (Delfín et al., 1996). In addition, itsreported activity against human neutrophil elastase (HNE) (Delfínet al., 1996) is not usual for domains having a basic residue atposition P1. Therefore, the elucidation of the molecular basisfor the activity of this multifunctional inhibitor againststructurally unrelated proteases is of specific interest for furthersite-directed mutagenesis experiments or increasing its potentialapplications.

Considering its inhibitory efficiency, an active recombinantvariant 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 ofrShPI-1A in complex with the serine protease bovine pancreatictrypsin at 1.7 Å resolution. Analyzing the molecular contacts bycomparison with trypsin complexes of mammalian BPTI-Kunitz-type inhibitors indicated a prominent role not only for Lys13 atposition P1, but also for Arg11 at position P3 of rShPI-1A in thestabilization of the complex that was so far not described forBPTI-Kunitz-type inhibitors. However, less interaction than BPTIat the primary and secondary binding loops of rShPI-1A reducedthe gain of stability attributed to Arg11, resulting in a decreasedtrypsin-binding affinity compared to BPTI.

2. Materials and methods

Due to the cloning procedure, rShPI-1A contains an additionalGlu-Ala-Glu-Ala motif (numbered as �3 to 0) at the N-terminusof the natural ShPI-1 sequence and two additional residues (Leu-Gly) at the C-terminus. Since BPTI contains two additional amino

acids compared to rShPI-1A, Lys15 at position P1 of BPTI corre-sponds to Lys13 in rShPI-1A.

2.1. Purification of rShPI-1A and complex formation with bovinetrypsin

The recombinant inhibitor rShPI-1A was expressed and purifiedas previously described (Gil et al., 2011). The homogeneity of thepurified protein was verified by SDS–PAGE, MALDI-TOF MS, and re-verse phase HPLC on a C8 column (Grace-Vydac, USA). For complexformation a 2-fold molar excess of rShPI-1A was incubated in thepresence of bovine pancreatic trypsin (E.C. 3.4.21.4, Sigma,Germany) in 0.02 M Tris buffer (pH 8.0) supplemented with0.15 M NaCl and 0.02 M CaCl2 for one hour at RT. The mixturewas applied onto a Superose 12HR gel filtration column(1 � 30 cm, Biosciences, USA) previously equilibrated with thesame buffer. Separation of inhibitor excess was performed on anÄKTA purifier system (GE Healthcare, USA) by monitoring theabsorbance at 280 nm. A chromatographic run of free trypsinwas used as a control for complex formation. Residual trypsinactivity was tested in the isolated protein fractions using BAPA(BACHEM, Germany) as a substrate (Erlanger et al., 1961). Thedissociation constant (Ki) of the rShPI-1A:trypsin complex wascalculated as described by Bieth (1974). Protein concentrationswere determined by absorbance at 280 nm using the extinctioncoefficient reported for natural ShPI-1 (Delfín et al., 1996) and atheoretical extinction coefficient (E280nm

1%) of 13.8 calculated forthe rShPI-1A:trypsin complex based on both protein sequences,using ProtParam program (Gasteiger et al., 2005). Protein solutionswere concentrated using 3 kDa and 10 kDa MW cut-off concentra-tion devices (Centricon, Millipore, USA) for the free inhibitor andthe 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) trifluoroaceticacid. Fading or disappearance of MS signal was investigated afterincubation of pure rShPI-1A with 2 ll of immobilized trypsin oncyanogen bromide-activated Sepharose�4B for 3 min at RT. Furtherwashing 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 spectroscatterer201 (Molecular Dimensions, UK) with a He-Ne laser providing lightof 690 nm wavelength and an output power in the range of 10–50 mW. All samples were filtered using 0.1 lm PVDF centrifugalfilters (Microcon, Millipore, USA) prior to measurements. Thesamples (30 ll) were measured in a quartz cuvette at 20 �C usingan 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 = NA�q�RH

2.3�4/3pwhere NA is the Avogadro constant and q is the density of theprotein (1.5 g/cm3) (Fischer et al., 2004).

2.4. Crystallization, X-ray data collection, and structure refinement

A robot-assisted screening of crystallization conditions was per-formed using commercially available kits from Hampton Research(USA) and Qiagen (Germany). The purified rShPI-1A:trypsincomplex was concentrated up to 13 mg/mL and mixed 1:1 (v/v)with reservoir solutions. Using the sitting-drop vapor diffusion

Table 1Data collection and refinement statistics of the rShPI-1A:trypsin complex.

rShPI-1A:trypsin PDB 3M7Q

Space group P212121

a (Å) 58.3b (Å) 66.5c (Å) 71.0

VM (Å3/Da) 2.32Solvent content (%) 47.00Completeness of data (%) 98.8 (97.8)No. of total reflections 162,197No. of unique reflections 30,437Average I/sigma intensity 10.4 (6.1)Resolution (Å) 30.0–1.7Redundancy 5.8 (5.8)Rmerge (%) 5.1 (12.0)No. of reflections used in refinement 28,976Rcrystal (%) 15.8 (18.9)No. of reflections used in Rfree 1540Rfree (%) 18.8 (25.8)Protein atoms 2167Solvent atoms 526

Average B-factor (Å2)Main-chain atoms 10.3Side chain atoms 11.8Ligand molecules 12.1Solvent molecules 28.2Other atoms (PO4

3�) 15.2

Root mean square deviationBonds (Å) 0.007Bond angles (�) 0.955

Residues in regions of the Ramachandran plot (%)Most favored 86.9Allowed 13.1Disallowed –Generally allowed –

Numbers in parentheses refer to the highest resolution shell.

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technique at 20 �C, orthorhombic crystals measuring up to 0.8 mmgrew within one week in condition 83 of the Qiagen Classic SuiteResearch screening kit (0.2 M (NH4)2SO4, 0.1 M NaAc pH 4.6, 25%PEG 4000). Diffraction data were collected with synchrotronradiation 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 anitrogen-gas stream at 100 K. Integration, scaling, and reductionof the recorded reflections were performed using the programsMOSFLM and SCALA (Collaborative Computational Project, Number4, 1994). The structure was solved by molecular replacementusing 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. Withincyclic steps of refinement using the programs Coot (Emsley andCowtan, 2004) and REFMAC (Murshudov, 1997), 526 water mole-cules and one phosphate ion have been added to the structure.

2.5. Structure analysis

The stereochemistry of the complex structure was evaluatedusing the programs VERIFY-3D (Lüthy et al., 1992), WHATCHECK(Vriend, 1990), and PROCHECK (Laskowski et al., 1993). Analysisof atom–atom contacts and structural superposition of the tryp-sin-bound inhibitors rShPI-1A, BPTI (PDB ID: 2FTL) and APPI (PDBID: 1TAW) was performed using the program WHATIF (Vriend,1990). Intermolecular contact maps were drawn based on an anal-ysis of the program CMA (Sobolev et al., 2005) and the interfaceproperties of these complexes, which were evaluated using thePROTORP (Reynolds et al., 2009) and the PDBePISA (Krissinel andHenrick, 2007) servers.

2.6. Free energy calculations by Crooks Gaussian intersection (CGI)method

The relative binding free energy difference (DDG) associatedwith a R11A substitution in rShPI-1A was calculated according tothe thermodynamic cycle shown in Supplementary Fig. 1 using theCGI method (Goette and Grubmüller, 2009). The effect of the R11Asubstitution on the Ki value at 298 K was estimated considering thatDDGðR11AÞ ¼ RT � lnðKiðImutÞ

KiðIwtÞ Þ:The mutation was automatically setupwith the PYMACS package (Seeliger and de Groot, 2010). AllMolecular Dynamics (MD) simulations were performed using theGROMACS software package (Hess et al., 2008). For details of thesimulation setup of the equilibrium and fast growth thermodynamicintegration (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 a2.0 nm cut-off. All the other parameters were setup analogous tothe equilibrium runs (Supplementary methods). The average andblock averaging error estimate was calculated using the g_analyzetool of the GROMACS software package (Hess et al., 2008).

2.7. Protein Data Bank accession number

The atomic coordinates and structure factors of the rShPI-1A:trypsin complex have been deposited in the Protein Data Bankwith 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 wasrevealed by Intensity Fading MALDI-TOF MS and dynamic light

scattering (DLS). The MALDI mass spectrum of purified rShPI-1Aclearly showed that its m/z signal (6681) faded upon the additionof trypsin (results not shown). Besides, incubation of a 1:1 molarratio of both proteins for 10 min at RT and further DLS measure-ments resulted in a monodisperse solution of moleculescharacterized by an RH of 2.63 ± 0.07 nm and a calculated MW of29.69 kDa. Both values are increased compared to that of freetrypsin measured under identical conditions (RH = 2.33 ± 0.24 nm;MW = 22.56 kDa) and agree with the theoretical MW of the en-zyme-inhibitor complex (29.95 kDa). Moreover, a shift in the SECelution volume combined with the absence of trypsin activity inthe associated SEC fractions supports the successful complexformation. The DLS signal of the solution did not change for upto 15 days incubation at 4 �C, confirming the stability of therShPI-1A:trypsin complex.

3.2. Crystallization, structure determination and refinement

The purified rShPI-1A:trypsin complex formed orthorhombiccrystals that diffracted up to a resolution of 1.7 Å. The asymmetricunit contained one molecule of the complex. The structure wasdetermined by molecular replacement using the coordinates of aBPTI:trypsin complex (PDB ID: 2FTL) (Hanson et al., 2007) as asearch 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 den-sity map, including the entire N- and C-terminus of the proteins.All residues are located within the most favored or allowed regionsof the Ramachandran plot, reflecting the quality of the rShPI-1A:trypsin structure. Data collection and refinement statistics aresummarized in Table 1.

Fig. 1. X-ray structure of the rShPI-1A:trypsin complex compared to trypsincomplexes of mammalian BPTI-Kunitz inhibitors. (A) Cartoon representation of theoverall structure of rShPI-1A (blue), BPTI (red) (PDB ID: 2FTL), and APPI (grey) (PDBID: 1TAW) in complexes with bovine trypsin (green). Binding loops of rShPI-1A arehighlighted in cyan. The side chains of the catalytic triad residues (His57, Asp102, andSer195), and of Asp189 at the bottom of the catalytic pocket of trypsin (all highlightedin orange), as well as the basic residues at P1 positions of the inhibitors are shownin stick representation and labeled accordingly. The inset shows the interaction ofthe P1 residue of the enzymes with trypsin residue Asp189 in detail. (B) Buriedsurface (BS) area of the inhibitor residues at the interface represented as percentageof the total surface area that is buried after complex formation. The correspondingamino acid sequences of rShPI-1A (cyan), BPTI (red), and APPI (grey), are shown.Secondary binding loop residues are numbered according to BPTI. (For interpreta-tion of the references to color in this figure legend, the reader is referred to the webversion of this article.)

274 R. García-Fernández et al. / Journal of Structural Biology 180 (2012) 271–279

3.3. rShPI-1A binding to trypsin and comparison with mammalianinhibitor complexes

Structural superposition of free (PDB ID: 3OFW) and trypsin-bound rShPI-1A revealed minor reorientations of flexibleside-chain atoms (RMSD 1.77 Å) as a consequence of complexformation, while the backbone atoms remained almost unaffected(RMSD 0.69 Å). Deviations in the main chain psi (6 20�) andside-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 showedonly small side chain rearrangements.

The overall fold of the invertebrate inhibitor complex rShPI-1A:trypsin is highly similar to the homologous complexes of themammalian BPTI-Kunitz inhibitors BPTI (PDB ID: 2FTL) (Hansonet al., 2007), which is characterized by the lowest dissociationconstant known so far for an inhibitor-protease interaction(Ki = 6 � 10�14 M, Fritz and Wunderer, 1983), and APPI (PDB ID:1TAW) (Scheidig et al., 1997), which has a trypsin affinity similar tothat of ShPI-1 (Ki = 4.2� 10�10 M, van Nostrand et al., 1995)(Fig. 1A). Residues P3–P30 adopt the canonical conformation observedin all BPTI-Kunitz inhibitors with the side chain of the basic residueat position P1 in the conserved down conformation (Scheidiget al., 1997). The complex interface consistently extends up topositions P5–P40 (9–17 in rShPI-1A). These residues are folded intoa hairpin-like structure, which forms together with residues 32–37a two-stranded antiparallel b-sheet that is stabilized by the disulfidebridge Cys12-Cys36 along with several hydrogen bonds. The interac-tion occurs via similar interface residues (Fig 1B) defined by totalburied surface (BS) areas ranging from 765.1 Å2 (18.2% of the totalaccessible 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 pri-mary direct interactions within the rShPI-1A:trypsin interface areprovided by residues at positions P2, P1, and P10 (Helland et al.,1998; Huber et al., 1974; Scheidig et al., 1997), which are com-pletely buried after complex formation (Fig. 1B). Based on thisanalysis, additional contributions to the rShPI-1A complex stabilityare indicated by residues Arg11 at position P3 and Ile32, whereasTyr15 (P20), Phe16 (P30), Gly35, and Cys36 appear to have a reducedimpact compared to the corresponding residues in the BPTI andAPPI complexes. Arg39 is reported as an essential secondary inter-action site of BPTI (Scheidig et al., 1997). However, this site is notimportant in rShPI-1A, where the equivalent residue Gly37 losesessentially no accessible surface area upon complexation, asobserved in APPI (Fig. 1B). The overall intermolecular interactionpattern remained largely conserved between all three inhibitor-trypsin complexes (Fig. 2). However, the number of directenzyme-inhibitor contacts shorter than 4 Å is slightly reduced forthe rShPI-1A:trypsin complex (116 hydrophobic interactions, 10hydrogen bonds) compared to a total of 137 interactions observedfor the BPTI and APPI trypsin complexes, including 12 and 13hydrogen bonds, respectively (Supplementary Table 1). The inter-face of the rShPI-1A:trypsin complex is further stabilized by inter-actions mediated by six water molecules (Supplementary Table 2).Four of them (wat148, wat268, wat269 and wat381) are highlyconserved compared to the BPTI:trypsin complex (Helland et al.,1998), connecting the P3 and P1 residues in the central core ofthe interface as well as Gly35 at the secondary binding loop ofthe inhibitor with the enzyme. In contrast, the APPI:trypsincomplex 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 consideredto be the most important in terms of defining the specificity andthe strength of the inhibitory interaction (Helland et al., 1998;Huber et al., 1974; Scheidig et al., 1997). The side chain of the P1residue points directly into the catalytic pocket of the trypsinmolecule, interacting with Asp189 at the bottom of this pocket(Fig. 3A). The interaction maps of the rShPI-1A:trypsin andBPTI:trypsin complexes are almost identical at position P1(Fig. 2). Depending on the analyzed BPTI-Kunitz:trypsin complexand on the applied scoring criteria, the nature of the importantP1–S1 interaction has been variously described so far, including

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 x-axis, while the trypsin residues involved in intermolecular interactions are presented on the y-axis. The maps were calculated based on the contacts provided by interactionanalysis 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). Squaresrepresent vdW interactions whereas cross and circles represent direct and indirect hydrogen bonds, respectively. For a detailed analysis of the associated hydrogen bonds seeSupplementary Tables 1 and 2.

R. García-Fernández et al. / Journal of Structural Biology 180 (2012) 271–279 275

no direct interaction (Huber et al., 1974) or a distance longer thancommon for H-bonds (3.63 Å) (Helland et al., 1998). We clearlyobserved a direct interaction between the NZ atom of Lys13 ofrShPI-1A and the OD1 atom of the S1 residue Asp189 in trypsin ata distance of 3.35 Å (Fig. 3A), scored as a hydrogen bond consistentwith 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 thatefficient trypsin inhibition strongly depend on a basic residue atposition P1 (Krowarsch et al., 2005; Otlewski et al., 2001). However,the interaction is almost independent of the nature of the basicresidue at P1 position, since the vice versa substitutions Lys15Argin BPTI (pseudo-BPTI) and Arg15Lys in APPI did not significantlyaffect the association energies with trypsin (Navaneetham et al.,2010; Van Nostrand et al., 1995). This is further supported by thecomparable Ki values of rShPI-1A (Lys at P1 position) and APPI(Arg at P1 position) for trypsin. Chymotrypsin and coagulationfactor IXa also displayed no preference for Arg or Lys at P1 wheninhibited by wild type or Arg15Lys substituted APPI (Van Nostrandet al., 1995). In contrast, also highly residue specific inhibition ofserine proteases by BPTI-Kunitz inhibitors has been reported, e.g.for kallikrein, which favors Arg over Lys (Fiedler, 1987; Grzesiaket al., 2000), while plasmin inhibition is enhanced by Lys at P1position (Van Nostrand et al., 1995). This agrees well with theincreased antiplasmin activity of ShPI-1 (�10�10 M) compared tokallikrein (10�8 M) (Delfín et al., 1996).

3.3.2. Pn side of the primary binding loopMost 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 ofArg11 and the nitrogen of Gly216 in trypsin is highly conserved inall BPTI-Kunitz inhibitor-proteinase complexes described so far.However, our crystal structure revealed that the arginine sidechain 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 therShPI-1A complex interface (Figs. 2 and 3B). Together with opti-mized hydrophobic contacts (Supplementary Table 1), further indi-rect 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 prolineresidue at this position that does not deeply enter the S3 pocketof trypsin due to its cyclic nature, resulting in significantly lessside-chain interactions, as revealed by the corresponding trypsin-complex structures (Hanson et al., 2007; Scheidig et al., 1997)(Fig. 3B). Thus, an important impact of arginine at position P3 isindicated for the interface stabilization within the rShPI-1A:trypsincomplex. Other BPTI-Kunitz-type trypsin inhibitors, e.g. kaliclu-dines from the sea anemone Anemonia sulcata and domain 1 ofhepatocyte growth factor activator inhibitor type I also containan equivalent Arg residue (Schweitz et al., 1995; Shimomuraet al., 1997), but its impact for complex stabilization was not struc-turally elucidated so far.

To support our suggestion, we calculated binding free energydifferences after in silico Arg11Ala mutation of rShPI-1A. Substitu-tion by proline, which resembles the P3 position in BPTI and APPI,is technically prevented, since the required destruction of covalentbonds is conceptionally different from all other mutations. How-ever, the methyl side chain of alanine approximates the hydropho-bicity and the length of the proline side chain that sticks into the S3pocket, allowing a rough estimation of the arginine-specific contri-bution to the stabilization of the rShPI-1A:trypsin complex.According to the free energy cycle shown in SupplementaryFig. 1, a positive DDG of 5.52 ± 1.90 kJ/mol was calculated,accounting for a 10-fold increase in the theoretical Ki value againsttrypsin as a result of the Arg11Ala mutation in rShPI-1A. Net posi-tive electrostatic and vdW free energies were also obtained for theindividual binding free energy differences of each trypsin residuewithin the S3 pocket as a consequence of the Arg11Ala mutation(Table 2). All S3 residues suggested to be involved in hydrogenbonds (Fig. 3B) provide a significant electrostatic contribution tothe complex interaction, while Leu99 and Trp215 form strong vdWinteractions with the hydrophobic carbon moiety of the arginineside chain.

Next to Arg and Pro, a variety of further amino acids has beenobserved at the P3 position of BPTI-Kunitz-type inhibitors(Scheidig et al., 1997). However, the influence of the P3 residuestrongly depends on the individual enzyme-inhibitor complex.While we show here that Arg strengthens the specific interaction

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 N-terminal (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 trypsinresidues (blue and light red, respectively) involved in direct or water-mediated interactions are shown using stick representation. Water molecules are shown as spheres andH-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 patternat 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 isreferred to the web version of this article.)

276 R. García-Fernández et al. / Journal of Structural Biology 180 (2012) 271–279

of rShPI-1A with trypsin, a Pro/Arg but also a Pro/Ala substitutionat P3 significantly decreases the Ki value for pseudo-BPTI againsttrypsin (Grzesiak et al., 2000; Otlewski et al., 2001). The transfor-mation of the non-inhibitory collagen VI Kunitz-like module intoa specific trypsin inhibitor also required the introduction of Proat position P3 (Kohfeldt et al., 1996). Domain 1 of the protease

inhibitor nexin II, a secreted isoform of APPI, inhibited trypsinindependently of Pro or Ala at P3 position, whereas inhibition ofother proteases, e.g. kallikrein and plasmin, strongly dependon the presence of a Pro residue (Navaneetham et al., 2010).Interestingly its inhibitory activity against factor Xa was recoveredby introduction of Arg at P3 position (Navaneetham et al., 2005).

Table 2Calculated binding free energy differences of trypsin S3 site residues as a result ofin silico Arg11Ala mutation in rShPI-1A.

Trypsin S3 residues hDEvdW ia (kJ/mol) hDEelei

b (kJ/mol)

Ser96 0.73 ± 0.07 20.68 ± 1.51Asn97 1.82 ± 0.05 37.95 ± 1.13Thr98 3.41 ± 0.08 22.98 ± 1.09Leu99 8.22 ± 0.17 4.15 ± 0.40Gln175 4.01 ± 0.20 19.90 ± 3.39Trp215 11.28 ± 0.44 20.11 ± 0.31Gly216 1.81 ± 0.47 2.75 ± 1.97

(a) hDEvdW ia ¼ hEA11�X

vdW i � hER11�XvdW i.

(b) hDEeleib ¼ hEA11�X

ele i � hER11�Xele i.

Differences in electrostatic and vdW energies were calculated considering equa-tions (a) and (b), respectively. Consequently, positive values indicate that theinteractions are more favorable in the wild-type complex.

R. García-Fernández et al. / Journal of Structural Biology 180 (2012) 271–279 277

Consequently, a general trend for an optimal amino acid at positionP3 for efficient trypsin inhibition is not indicated.

Most of the interactions involving the residue at position P5 aswell as the conserved cysteine residue at position P2 are conservedin the trypsin complexes of rShPI-1A, BPTI, and APPI. However, thehighly conserved H-bond between Cys14 at position P2 of APPI/BPTI and Gln192 in trypsin as well as the hydrophobic contact be-tween Gln192 (trypsin) and the highly conserved Gly10 at positionP4 is absent in the rShPI-1A complex (Supplementary Table 1).Since the trypsin residue Gln192 is reported to have a stabilizingimpact on the enzyme-inhibitor complexes (Schmidt et al., 2005),the absence of these interactions is suggested to attenuate thestabilizing effect that was attributed to Arg13 at position P3 inrShPI-1A.

3.3.3. Pn prime (Pn0) side of the primary binding loopAt the Pn’ side of the rShPI-1A:trypsin complex interface, the

number of hydrophobic as well as polar contacts is reduced com-pared to the trypsin complex of BPTI and APPI, due to sequencesalterations within this region (Fig. 2). A strong tendency for ala-nine, e.g. in BPTI and APPI, and glycine, e.g. in rShPI-1A, is observedat position P10 within BPTI-Kunitz-type domains (Grzesiak et al.,2000). However, the missing methyl-group of the Gly residue inrShPI-1A resulted in reduced vdW interactions with Cys42 of tryp-sin, supported by a significant increase of the Ki value of BPTIagainst trypsin after substitution of Ala16 by glycine (Krowarschet al., 2005; Otlewski et al., 2001). In addition, Arg17 at positionP20 of BPTI establishes an H-bond with His40 of trypsin, but

Fig. 4. Stereo view of the interaction of the secondary binding loop of rShPI-1 (cyan) andcolored in blue and light red, respectively. Positions 33–36 (rShPI-1A numbers) are consea detailed comparison of the H-bond pattern at the trypsin complex interface of rShPIreferences to color in this figure legend, the reader is referred to the web version of thi

equivalent contacts of Tyr15 (rShPI-1A) and Met17 (APPI) are absent(Fig. 3C). Another H-bond between the residue at position P40 andTyr39 of trypsin is only formed by Ile or Ser in BPTI and APPIcomplexes, respectively, but not by Pro in the rShPI-1A complex.Although the coordination of a water molecule (wat132) at posi-tion P40 (Pro17) in rShPI-1A is conserved compared to the trypsincomplexes of BPTI and APPI (Helland et al., 1998), water-mediatedinteractions with the enzyme are only formed within theBPTI:trypsin complex (Supplementary Table 2). These differenceswithin the interaction pattern at the prime site of the primarybinding loop in rShPI-1A are supposed to contribute to theincreased Ki value of rShPI-1A compared to BPTI. However, areduced impact on the overall complex stability is in generalsupposed for the Pn0 side interactions compared to that at the Pnside (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 influ-ences the association energy of BPTI-Kunitz domains (Czapinskaet al., 2000; Chand et al., 2004; Dennis and Lazarus, 1994; Hellandet al., 1998). At the most divergent position P19’, a stabilizinghydrophobic interaction with trypsin residue Tyr151 is only formedby Ile32 of rShPI-1A, but not by equivalent residues of BPTI andAPPI. The importance of this position for specific inhibitory activityis supported by previous mutagenesis studies of APPI (Dennis andLazarus, 1994) and of the non-inhibitory BPTI-Kunitz-like moduleof collagen VI (Kohfeldt et al., 1996). At the positions P20’ to P23’(Tyr33 to Cys36 according to rShPI-1A numbering), rShPI-1A, BPTIand APPI locate identical amino acids. However, no vdW contactsare mediated by P220 (Gly35) and P230 (Cys36) in the trypsin com-plex of rShPI-1A (Fig. 2), indicating a minor impact of these resi-dues for complex stabilization in rShPI-1A. In BPTI, theinteractions formed by the polar residue Arg39 at position P24’are suggested to significantly contribute to the exceptionally lowKi value of this inhibitor (Scheidig et al., 1997). In addition to awater-mediated interaction with trypsin residue Ser96, this islargely attributed to a direct H-bond formed with trypsin residueAsn97 (Fig. 4) A similar interaction is reported to stronglycontribute to the protease specificity and complex stability ofbovine trypsin-bound domain 1 from TFPI-2 (Chand et al., 2004).In rShPI-1A and APPI, comparable interactions are prevented bythe presence of a glycine residue at position P24’, consistent withthe significantly increased Ki value of both inhibitors for trypsin.

BPTI (red) with trypsin. The enzyme residues in the rShPI-1 and BPTI complexes arerved within the inhibitors. Dashed lines are used for direct and indirect H-bonds. For-1A, BPTI, and APPI, see Supplementary Tables 1 and 2. (For interpretation of thes article.)

278 R. García-Fernández et al. / Journal of Structural Biology 180 (2012) 271–279

4. Concluding remarks

Structural information on proteins isolated from marineinvertebrates is rare to date, compared to the tremendous varietyof invertebrate species (Brusca and Brusca, 2003). Here we presentthe first crystallographic structure of a BPTI-Kunitz-type inhibitorfrom a marine invertebrate in complex with a serine protease, bo-vine trypsin. The common trypsin interaction pattern of canonicalBPTI-Kunitz-type inhibitors remained largely conserved through-out evolution from invertebrates to mammalian species,significantly depending on the P2, P1, and P1’ residues of theinhibitor (Helland et al., 1998; Huber et al., 1974; Scheidig et al.,1997). In addition, we observed a prominent role of arginine atposition P3 in the stabilization of the rShPI-1A:trypsin complex,partially compensating the absence of important contacts at otherpositions within the primary and secondary binding loops.Thus, the associated Ki value against trypsin is maintained at10�10 M (Delfín et al., 1996).

The structure presented here provides valuable information forfurther design of ShPI-1 mutants, which will help to elucidate thebasis of its multifunctional activity, associated with an increasedknowledge about biomolecules from marine invertebrates. Dueto their general defensive role against prey and predators, proteaseinhibitors from marine invertebrates have generally exceptionalstructural and functional properties (González et al., 2007; Lenarcicand Turk, 1999; Xue et al., 2009; Alonso del Rivero et al., 2012).Although unusual within the BPTI-Kunitz family, ShPI-1 as wellas three other invertebrate domains of this family exhibit activityagainst structurally unrelated proteases, but the mechanisms oftheir cross-activity remain to be elucidated in detail (Delfín et al.,1996; Sasaki et al., 2006; Alonso del Rivero et al., 2012; Minagawaet al., 1997). ShPI-1 is supposed to inhibit other serine proteases,e.g. plasmin, kallikrein, as well as chymotrypsin, due to the basicresidue at P1 position, as already reported for complexes of BPTIand APPI (Scheidig et al., 1997). This also applies to the reportedinhibition of human neutrophil elastase (Delfín et al., 1996), whichtolerates a broad specificity of P1 residues due to a more flexible S1pocket (McBride et al., 1999), including canonical inhibitors fromother families with basic P1 residues (González et al., 2007). Thesepreliminary considerations about the inhibitory mechanisms ofShPI-1 against serine proteases have to be structurally validatedto understand more thoroughly the unusual multifunctionalactivity of this inhibitor.

Acknowledgements

We are indebted to Prof. Dr. U. Hahn (University of Hamburg,Germany) for his support to start this research. We are gratefulto Prof. Dr. J. Díaz, Dr. M. Mansur, and D. F. Gil for their contributionto heterologous expression of ShPI-1. We thank D. Oberthuer (Uni-versity of Hamburg, Germany) for his help during data processingand refinement and Dr. G. Groenhof (Max Planck Institute for Bio-physical Chemistry, Göttingen, Germany) for technical supportduring free energy calculations. We also acknowledge Dr. C. Berry(Cardiff University, Wales, UK) for critical reading of the manu-script. RGF, MCh, YGG, and PAV thank the International Foundationfor Science (IFS), Sweden and the German Academic Exchange Ser-vice (DAAD) for financial support. MP is member of the HamburgSchool for Structure and Dynamics in Infection (SDI) and thanksthe Hamburg Ministry of Science and Research and the Joachim-Hertz-Stiftung, as part of the Hamburg Initiative for Excellence inResearch, for financial support. LR and CB thank the German Fed-eral Ministry of Education and Research (BMBF) for financial sup-port [grants 01KX0806 and 01KX0807].

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.jsb.2012.08.009.

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