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The Structure of Leishmania mexicana ICP Provides Evidence for Convergent Evolution of Cysteine Peptidase Inhibitors

2005; Elsevier BV; Volume: 281; Issue: 9 Linguagem: Inglês

10.1074/jbc.m510868200

1083-351X

Brian O. Smith, Nichola Picken, Gareth D. Westrop, Krystyna Bromek, Jeremy C. Mottram, Graham H. Coombs,

Enzyme Production and Characterization

Clan CA, family C1 cysteine peptidases (CPs) are important virulence factors and drug targets in parasites that cause neglected diseases. Natural CP inhibitors of the I42 family, known as ICP, occur in some protozoa and bacterial pathogens but are absent from metazoa. They are active against both parasite and mammalian CPs, despite having no sequence similarity with other classes of CP inhibitor. Recent data suggest that Leishmania mexicana ICP plays an important role in host-parasite interactions. We have now solved the structure of ICP from L. mexicana by NMR and shown that it adopts a type of immunoglobulin-like fold not previously reported in lower eukaryotes or bacteria. The structure places three loops containing highly conserved residues at one end of the molecule, one loop being highly mobile. Interaction studies with CPs confirm the importance of these loops for the interaction between ICP and CPs and suggest the mechanism of inhibition. Structure-guided mutagenesis of ICP has revealed that residues in the mobile loop are critical for CP inhibition. Data-driven docking models support the importance of the loops in the ICP-CP interaction. This study provides structural evidence for the convergent evolution from an immunoglobulin fold of CP inhibitors with a cystatin-like mechanism. Clan CA, family C1 cysteine peptidases (CPs) are important virulence factors and drug targets in parasites that cause neglected diseases. Natural CP inhibitors of the I42 family, known as ICP, occur in some protozoa and bacterial pathogens but are absent from metazoa. They are active against both parasite and mammalian CPs, despite having no sequence similarity with other classes of CP inhibitor. Recent data suggest that Leishmania mexicana ICP plays an important role in host-parasite interactions. We have now solved the structure of ICP from L. mexicana by NMR and shown that it adopts a type of immunoglobulin-like fold not previously reported in lower eukaryotes or bacteria. The structure places three loops containing highly conserved residues at one end of the molecule, one loop being highly mobile. Interaction studies with CPs confirm the importance of these loops for the interaction between ICP and CPs and suggest the mechanism of inhibition. Structure-guided mutagenesis of ICP has revealed that residues in the mobile loop are critical for CP inhibition. Data-driven docking models support the importance of the loops in the ICP-CP interaction. This study provides structural evidence for the convergent evolution from an immunoglobulin fold of CP inhibitors with a cystatin-like mechanism. Characterization of the structure and mechanisms of action of natural inhibitors of cysteine peptidases (CPs) 2The abbreviations used are: CP, cysteine peptidase; ICP, inhibitor of cysteine peptidase; HSQC, heteronuclear single quantum correlation; NOESY, nuclear Overhauser enhancement spectroscopy; NOE, nuclear Overhauser effect; RMSD, root mean square deviation; NRMSD, normalized RMSD. 2The abbreviations used are: CP, cysteine peptidase; ICP, inhibitor of cysteine peptidase; HSQC, heteronuclear single quantum correlation; NOESY, nuclear Overhauser enhancement spectroscopy; NOE, nuclear Overhauser effect; RMSD, root mean square deviation; NRMSD, normalized RMSD. has provided important insights into the functional roles of the inhibitors themselves and also those of the target CPs. CP inhibitors occur widely in nature, and several distinct groups with unrelated primary structures are recognized (1Rawlings N.D. Tolle D.P. Barrett A.J. Biochem. J. 2004; 378: 705-716Crossref PubMed Scopus (464) Google Scholar). Members of the cystatin superfamily (clan IH, family I25 (2Rawlings N.D. Tolle D.P. Barrett A.J. Nucleic Acids Res. 2004; 32: 160-164Crossref PubMed Google Scholar)) have been extensively characterized at the molecular level (3Bode W. Engh R. Musil D. Thiele U. Huber R. Karshikov A. Brzin J. Kos J. Turk V. EMBO J. 1988; 7: 2593-2599Crossref PubMed Scopus (542) Google Scholar, 4Stubbs M.T. Laber B. Bode W. Huber R. Jerala R. Lenarcic B. Turk V. EMBO J. 1990; 9: 1939-1947Crossref PubMed Scopus (463) Google Scholar, 5Jenko S. Dolenc I. Guncar G. Dobersek A. Podobnik M. Turk D. J. Mol. Biol. 2003; 326: 875-885Crossref PubMed Scopus (96) Google Scholar). A second group of natural CP inhibitors, assigned to clan IX, family I31, are similar to cystatin in inhibiting CPs of clan CA, family C1, such as papain, but are distinguished by the presence of a thyroglobulin type I domain (6Guncar G. Pungercic G. Klemencic I. Turk V. Turk D. EMBO J. 1999; 18: 793-803Crossref PubMed Scopus (174) Google Scholar). The recently discovered chagasin family of CP inhibitors, designated ICP for inhibitor of cysteine peptidases (clan I-, family I42), also inhibit papain-like CPs and yet have no significant sequence identity with the other groups of CP inhibitors (7Monteiro A.C.S. Abrahamson M. Lima A.P.C.A. Vannier-Santos M.A. Scharfstein J. J. Cell Sci. 2001; 114: 3933-3942Crossref PubMed Google Scholar). This raises the intriguing question: have the different groups of CP inhibitors evolved related tertiary structures to interact with the same target CPs in a similar fashion, as predicted from in silico analysis by Rigden et al. (8Rigden D.J. Mosolov V.V. Galperin M.Y. Protein Sci. 2002; 11: 1971-1977Crossref PubMed Scopus (52) Google Scholar), or does ICP act in a different way? ICP family members appear to occur in species from a very limited phylogenetic range (some parasitic protozoa, bacteria (including Pseudomonas aeruginosa), and Archaea, but no metazoa), suggesting that the genes have been acquired by horizontal gene transfer or retained for some special function. Although there is good evidence that the in vivo target for ICPs are clan CA, family C1 CPs, it remains uncertain whether the main function of the inhibitors is to modulate the activity of enzymes of the parasite itself (as is suggested for protozoan parasite Trypanosoma cruzi (9Santos C.C. Sant'Anna C. Terres A. Cunha-e-Silva N. Scharfstein J. de Lima A. J. Cell Sci. 2005; 118: 901-915Crossref PubMed Scopus (81) Google Scholar)) or the host (as suggested for the related parasite Leishmania (10Besteiro S. Coombs G.H. Mottram J.C. Mol. Microbiol. 2004; 54: 1224-1236Crossref PubMed Scopus (54) Google Scholar)). Interestingly, no clan CA, family C1 CPs appear to be present in the P. aeruginosa genome, which provides further support for the suggestion that one role of ICPs in pathogens might be to regulate host CP activity and so facilitate infection. This has been investigated with Leishmania by gene targeting to create parasite lines that either lack or overexpress the ICP gene (10Besteiro S. Coombs G.H. Mottram J.C. Mol. Microbiol. 2004; 54: 1224-1236Crossref PubMed Scopus (54) Google Scholar). ICP null mutants grow normally axenically in vitro and are as infective to macrophages in vitro as wild type parasites. However, they have reduced infectivity to mice. Lines that overexpress ICP also show markedly reduced virulence in vivo. Thus, ICP may be important after it is released in the interaction of the parasite with its mammalian host or as a mechanism of preventing damage from host CPs taken up by the parasite through endocytosis (e.g. from the parasitophorous vacuole, which contains host lysosomal enzymes). The ICP proteins are small (∼13 kDa) and very divergent, with L. mexicana ICP having only 31% identity with ICP of T. cruzi and 24% identity with ICP of P. aeruginosa (11Sanderson S.J. Westrop G.D. Scharfstein J. Mottram J.C. Coombs G.H. FEBS Lett. 2003; 542: 12-16Crossref PubMed Scopus (65) Google Scholar). Nevertheless, there are highly conserved motifs that suggest important functional regions. This has facilitated the identification of predicted ICPs from genome data and recombinant ICPs have been produced from the L. mexicana, T. brucei, Entamoeba histolytica, and P. aeruginosa genes and confirmed to have potent inhibitory activity toward CPs, notably cathepsin L homologues (7Monteiro A.C.S. Abrahamson M. Lima A.P.C.A. Vannier-Santos M.A. Scharfstein J. J. Cell Sci. 2001; 114: 3933-3942Crossref PubMed Google Scholar, 11Sanderson S.J. Westrop G.D. Scharfstein J. Mottram J.C. Coombs G.H. FEBS Lett. 2003; 542: 12-16Crossref PubMed Scopus (65) Google Scholar, 12Riekenberg S. Witjes B. Saric M. Bruchhaus I. Scholze H. FEBS Lett. 2005; 579: 1573-1578Crossref PubMed Scopus (43) Google Scholar) To date, the structural basis of the inhibitory activity of ICP is unknown. Previous threading studies have suggested that the binding site of T. cruzi ICP may be located on the loops between β-strands in a fold that resembles immunoglobulin light chain variable domains (8Rigden D.J. Mosolov V.V. Galperin M.Y. Protein Sci. 2002; 11: 1971-1977Crossref PubMed Scopus (52) Google Scholar, 13Rigden D.J. Monteiro A.C.S. De Sá M.F.G. FEBS Lett. 2001; 504: 41-44Crossref PubMed Scopus (32) Google Scholar). Another study drew parallels between the sequence conservation in predicted loops of the ICP family and the peptidase-binding regions of the cystatin family (12Riekenberg S. Witjes B. Saric M. Bruchhaus I. Scholze H. FEBS Lett. 2005; 579: 1573-1578Crossref PubMed Scopus (43) Google Scholar). We have now determined the structure of L. mexicana ICP in solution by NMR spectroscopy, confirmed residues key for its inhibitory activity using site-directed mutagenesis, and investigated how the key residues may bind to the model clan CA, family C1 peptidase papain, and an important L. mexicana CP, known as CPB (14Sanderson S.J. Pollock K.G. Hilley J.D. Meldal M. St. Hilaire P.M. Juliano M.A. Juliano L. Mottram J.C. Coombs G.H. Biochem. J. 2000; 347: 383-388Crossref PubMed Scopus (72) Google Scholar). Protein Production—Recombinant L. mexicana ICP was expressed from a pET28 (Novagen)-derived plasmid in Escherichia coli BL21 (DE3) cells as described previously (11Sanderson S.J. Westrop G.D. Scharfstein J. Mottram J.C. Coombs G.H. FEBS Lett. 2003; 542: 12-16Crossref PubMed Scopus (65) Google Scholar). 15N,13C-labeled protein was produced by growing the cells in M9 medium using 15NH4Cl and [13C]glucose (Spectra Stable Isotopes) as the sole nitrogen and carbon sources. The fusion protein was purified by nickel chelate chromatography and digested with thrombin (Novagen). The cleaved histidine tag and thrombin were removed by nickel chelate and benzamidine-Sepharose (Sigma) affinity chromatography. The protein comprising the complete native sequence (Q868H1 3UniProt number. ;CAD68975) with the addition of three residues (GSH) at the N terminus (designated ICP-(-2-113)) was buffer-exchanged into 25 mm sodium phosphate, pH 4.5, 50 mm NaCl, 0.001% NaN3 by extensive diafiltration using a 5,000 MWCO centrifugal concentrator (Vivascience) and concentrated to ∼1 mm. D2O was added to a final concentration of 10% (v/v). NMR samples of L. mexicana ICP-(-2-113) underwent proteolysis over 2-3 days under NMR sample conditions to produce an N-terminally truncated protein starting at residue serine 6 (ICP-(6-113)) as confirmed by mass spectrometry, which then remained stable. No difference in Ki for L. mexicana CPB could be detected between ICP-(-2-113) and ICP-(6-113). Interaction studies were carried out using papain from Papaya latex (Sigma) and L. mexicana CPB2.8Δ CTE, produced as described previously (14Sanderson S.J. Pollock K.G. Hilley J.D. Meldal M. St. Hilaire P.M. Juliano M.A. Juliano L. Mottram J.C. Coombs G.H. Biochem. J. 2000; 347: 383-388Crossref PubMed Scopus (72) Google Scholar). In each case, peptidase was mixed with an excess of 15N-labeled ICP in NMR sample buffer, and the complex was isolated by gel filtration on a Superdex 75 HR10/30 column (APBiotech) and then concentrated using a 10,000 MWCO centrifugal concentrator. NMR Spectroscopy and Data Analysis—Resonance assignments were determined using standard triple resonance NMR techniques and have been deposited as described (15Smith B.O. Westrop G.D. Mottram J.C. Coombs G.H. J. Biomol. NMR. 2005; (in press)Google Scholar). Distance restraints for structure calculation were derived from three-dimensional 15N and 13C HSQC-NOESY spectra recorded with 100 ms mixing times recorded on an 800 MHz Bruker Avance spectrometer. Slowly exchanging amide protons were identified by redissolving a lyophilized sample in D2O and recording a series of 15N HSQC spectra. Spectra were processed with AZARA 4Available at www.bio.cam.ac.uk/azara. and analyzed using CCPN analysis (16Vranken W.F. Boucher W. Stevens T.J. Fogh R.H. Pajon A. Llinas P. Ulrich E.L. Markley J.L. Ionides J. Laue E.D. Proteins Struct. Funct. Bioinf. 2005; 59: 687-696Crossref PubMed Scopus (2216) Google Scholar). Structure Calculation—Assigned, partially assigned, and ambiguous NOESY cross-peaks were used to generate distance constraints within CCPN analysis that were exported directly to CNS/XPLOR format and used as input for structure calculations using CNS v1.1 (17Brunger A.T. Adams P.D. Clore G.M. Delano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D. Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16919) Google Scholar) using a modified version of the PARALLHDG 5.3 forcefield (18Linge J.P. Williams M.A. Spronk C.A.E.M. Bonvin A.M.J.J. Nilges M. Proteins Struct. Funct. Genet. 2003; 50: 496-506Crossref PubMed Scopus (538) Google Scholar) with IUPAC-recommended nomenclature (19Markley J.L. Bax A. Arata Y. Hilbers C.W. Kaptein R. Sykes B.D. Wright P.E. Wuthrich K. Pure Appl. Chem. 1998; 70: 117-142Crossref Scopus (265) Google Scholar). Structures were generated from random atomic coordinates following the scheme of the rand, dgsa, and refine scripts from the XPLOR 3.1 manual (20Brunger A.T. X-PLOR 3.1. Yale University Press, New Haven, CT1992Google Scholar) reimplemented in CNS and modified to incorporate floating chirality at prochiral centers (21Folmer R.H.A. Hilbers C.W. Konings R.N.H. Nilges M. J. Biomol. NMR. 1997; 9: 245-258Crossref PubMed Scopus (110) Google Scholar) using a metropolis acceptance criterion. The tools provided by the ARIA (22Nilges M. Macias M.J. ODonoghue S.I. Oschkinat H. J. Mol. Biol. 1997; 269: 408-422Crossref PubMed Scopus (387) Google Scholar) module within CNS were used to identify consistently violated restraints for checking, to reduce the ambiguity of ambiguous distance restraints based on the ensemble of structures calculated, to remove duplicate restraints, and to recalibrate the distance-intensity mapping. Distance restraints representing hydrogen bonds were incorporated for slowly exchanging amides where corroborating NOEs existed and an acceptor atom could be unambiguously identified. Atomic coordinates have been deposited at the PDB (PDB code 2c34), and structural statistics are summarized in Table 1.TABLE 1NMR structural statistics for L. mexicana ICPNMR distance constraintsDistance constraintsTotal NOE2370Ambigous812Unambiguous1558Intra-residue869Inter-residueSequential (|i - j| = 1)302Medium-range (|i - j| < 4)83Long-range (|i - j| > 5)304Hydrogen bonds22Structure StatisticsViolations (mean and S.D.)Distance constraints (Å)0.052 ± 0.002Distance constraint violations > 0.5 Å1.3 ± 0.98Deviations from idealized geometryBond lengths (Å)2.54e-3 ± 1.00e-4Bond angles (°)0.382 ± 1.69e-2Impropers (°)0.354 ± 1.71e-2R.m.s.d. to the unbiased mean structureaRoot mean square deviation to the unbiased mean structure was calculated over residues 14-60 and 73-112 among 38 refined structures. (Å)Heavy0.854 ± 0.189Backbone0.586 ± 0.181Ramachandran plot (% residues in)Most favoured regions52.5Additionally allowed regions44.4Generously allowed2.7Disallowed regions0.5a Root mean square deviation to the unbiased mean structure was calculated over residues 14-60 and 73-112 among 38 refined structures. Open table in a new tab 15N Relaxation Measurements—Protein dynamics were probed through 15N relaxation measurements of the backbone amide groups. 15N T1, T2, and 1H, 15N-heteronuclear NOE experiments were recorded at 600.13MHz (1H), and the data were analyzed according to the Lipari-Szabo model-free formalism using the programs curvefit and modelfree (23Mandel A.M. Akke M. Palmer A.G. J. Mol. Biol. 1995; 246: 144-163Crossref PubMed Scopus (896) Google Scholar). Docking—Data-driven molecular docking was carried out using HADDOCK v1.3 (24Dominguez C. Boelens R. Bonvin A.M.J.J. J. Am. Chem. Soc. 2003; 125: 1731-1737Crossref PubMed Scopus (2087) Google Scholar) using the default parameters and using chemical shift perturbation data as input. In the HADDOCK terminology, the “active” ICP residues are those whose backbone or side-chain amide chemical shifts are significantly perturbed in the complex with papain and which are surface-exposed. The “passive” ICP residues are their immediate, surface-exposed, neighbors. In the absence of chemical shift perturbation data for the peptidase, and given the superficial similarity between the ICP and stefin B structures, the “active” papain residues were defined as those residues in close contact with stefin B in the crystal structure of the stefin B·papain complex. The passive papain residues are their immediate, surface-exposed neighbors. “Semiflexible” regions are defined to be two residues either side of the active and passive residues in the primary sequences of both proteins. The ICP D-E loop was made “fully flexible” to reflect its high mobility in unbound ICP. Mutations—Mutations of L. mexicana ICP were incorporated into the expression vector using the QuikChange site-directed mutagenesis kit (Stratagene) and the following pairs of complementary primers (mutated sites in lowercase): NT300, CCCGACCACTGGAgcCATGTGGACGCGC and NT301, GCGCGTCCACATGgcTCCAGTGGTCGGG to generate pBP191 (encoding Y34A); NT282, CATCCTCGACTCCTgacGacGGAGaTGGTGGCATCTAC and NT283, GTAGATGCCACCAtCTCCgtCgtcAGGAGTCGAGGATG to generate pBP193 (encoding M64D, V68D, V70D); NT298, CCTCGACTCCTATGGTGccAGTTccTcccATCTACGTTGTGCTCG and NT299, CGAGCACAACGTAGATgggAggAACTggCACCATAGGAGTCGAGG to generate pBP194, (encoding G69P, G71P, G72P); NT284, CTGGTCTACACGgcCCCCgcCGAGGGCATCAAGC and NT285, GCTTGATGCCCTCGgcGGGGgcCGTGTAGACCAG to generate pBP197, (encoding R94A, F96A); NT280, GAAGGGCAACCCGggCggTGGATACATGTGGACG and NT281, CGTCCACATGTATCCAccGccCGGGTTGCCCTTC to generate pBP199 (encoding T31,T32G). The mutated plasmids were verified by nucleotide sequencing. Ki Determinations—Kis for L. mexicana ICP and its mutants against L. mexicana CPB2.8Δ CTE were determined essentially as described previously (11Sanderson S.J. Westrop G.D. Scharfstein J. Mottram J.C. Coombs G.H. FEBS Lett. 2003; 542: 12-16Crossref PubMed Scopus (65) Google Scholar). In summary, concentrations of ICP stock solutions were determined by titration against a known concentration of CPB2.8Δ CTE in a colorimetric assay using N-benzoyl-PFR-p-nitroanilide hydrochloride (Sigma) as a substrate under pseudoirreversible conditions. For mutants with a detectable inhibitor activity, IC50s were determined in a fluorometric assay using 10 μm benzyloxycarbonyl-FR-7-amino-4-methylcoumarin hydrochloride (ZFR-AMC, Sigma) as a substrate and Kis calculated using the relationship Ki = IC50(1/(1 + [S]/Km) where Km = 0.7 μm (25Alves L.C. Judice W.A.S. St. Hilaire P.M. Meldal M. Sanderson S.J. Mottram J.C. Coombs G.H. Juliano L. Juliano M.A. Mol. Biochem. Parasitol. 2001; 116: 1-9Crossref PubMed Scopus (23) Google Scholar). Circular Dichroism—Near (250-320 nm) and far (190-240 nm) UV CD analyses were performed with 0.6 mg/ml protein in 0.5 and 0.02 cm path length quartz cells, respectively, using a JASCO J-810 spectropolarimeter. Eight scans were collected for each protein using a bandwidth of 1.0 nm, a scanning speed of 50 nm min-1 and a response time of 0.25 s. Data were corrected for cell path length and protein concentration and smoothed using the Savitsky-Golay algorithm (convolution width 9.0). The secondary structure analyses of wild type and mutant proteins were obtained using the CDSSTR method (26Manavalan P. Johnson W.C. Anal. Biochem. 1987; 167: 76-85Crossref PubMed Scopus (659) Google Scholar) available from the Dichroweb website, at the University of Birkbeck. All three proteins gave similar secondary structure estimates with low NRMSD values (∼0.025). ICP Adopts an Immunoglobulin Fold—In solution, ICP adopts an immunoglobulin-like (Ig) fold with seven β-strands (Fig. 1). One β-sheet is formed by anti-parallel strands B, E, and D, whereas the other is formed by anti-parallel strands G, F, and C with strand A parallel to strand G. A search for structural homologues using the DALI server (27Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3545) Google Scholar) identified the N-terminal Ig domain from α-dystroglycan (PDB:1u2c (28Bozic D. Sciandra F. Lamba D. Brancaccio A. J. Biol. Chem. 2004; 279: 44812-44816Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar)) as the closest match with a Z-score of 5.1 and an RMSD of 3.2 Å over 82 residues. The SCOP data base (29Murzin A.G. Brenner S.E. Hubbard T. Chothia C. J. Mol. Biol. 1995; 247: 536-540Crossref PubMed Scopus (5542) Google Scholar) classifies this as a cadherin-like superfamily fold, which also closely resembles the I-set immunoglobulin-like fold (30Harpaz Y. Chothia C. J. Mol. Biol. 1994; 238: 528-539Crossref PubMed Scopus (374) Google Scholar, 31Halaby D.M. Poupon A. Mornon J.P. Protein Eng. 1999; 12: 563-571Crossref PubMed Scopus (190) Google Scholar). The β-sheets enclose a well packed core of principally hydrophobic residues that correspond to the alternating pattern of conserved residues along the strands (see Fig. 2). The C-D loop of ICP folds back on strand C with a number of hydrophobic side-chains making contacts that are not part of the main core of the molecule. These interactions involving residues Met-35, Thr-37, Val-39, Val-42, Val-91, Thr-93, Pro-95, and Ile-99 serve to pin the F-G loop back to the outer face of one β-sheet. The prediction of the ICP family structure based on threading (13Rigden D.J. Monteiro A.C.S. De Sá M.F.G. FEBS Lett. 2001; 504: 41-44Crossref PubMed Scopus (32) Google Scholar) correctly identified the Ig-like fold, but the details of the predicted strands and loops were out of register after the C-D loop, possibly because of the misidentification of the hydrophobic residues in the C-D loop as representative of an extended C strand. The three highly conserved groups of residues previously noted (8Rigden D.J. Mosolov V.V. Galperin M.Y. Protein Sci. 2002; 11: 1971-1977Crossref PubMed Scopus (52) Google Scholar, 11Sanderson S.J. Westrop G.D. Scharfstein J. Mottram J.C. Coombs G.H. FEBS Lett. 2003; 542: 12-16Crossref PubMed Scopus (65) Google Scholar, 12Riekenberg S. Witjes B. Saric M. Bruchhaus I. Scholze H. FEBS Lett. 2005; 579: 1573-1578Crossref PubMed Scopus (43) Google Scholar) are all located in loops at one end of the molecule (Fig. 1B). The GNPTTGY motif lies in the B-C loop, the GXGG motif lies in the highly mobile D-E loop and the RPW/F motif in the F-G loop. The co-location of all three motifs, and the finding that none of the residues appear to have roles that are key to the integrity of the fold of the protein, suggests that they together form the CP-binding site. Residues 30-32 in the B-C loop form a turn of 310-helix projecting the side chains of Asn-29, Thr-31, and Thr-32 toward the solvent. Gly-28 and Gly-33 allow the loop to be accommodated between the B and C strands by adopting conformations with positive φ dihedral angles. The conserved Tyr-34, which caps the main hydrophobic core of the protein, interacts with residues in the F-G loop, and contributes to the solvent-exposed surface at this end of the protein. Pinned back to the GFC β-sheet, the 11-amino acid F-G loop (residues 93-103) forms a broad saddle with residues 96-101 running roughly perpendicular to the direction of the β-strands. The side chain of conserved Arg-94 interacts with Tyr-34 and, along with Tyr-96, projects out of this end of the protein accompanied by Glu-97, Lys-100, and Glu-102. The D-E loop (residues 61-72) projects from this end of the protein on the other side of the B-C loop and is poorly defined by the experimental data. The D-E Loop Is Flexible—15N relaxation measurements were used to probe the backbone dynamics of ICP-(6-113). 15N NOE, 15N T1, and 15N T2 were measured at 60.8 MHz (15N), the derived order parameters and correlation times are shown in Fig. 2. The majority of the residues display uniform relaxation rates close to the average values typical of a compactly folded monomeric protein of this size, and their behavior could be modeled using either a single order parameter (S2) or S2 and an internal correlation time. The exceptions are the N terminus up to residue 13 and residues 61-72 in the D-E loop, which display depressed NOE values and lengthened T2 values. These residues were best modeled with two order parameters representing internal motion on distinct timescales and an internal correlation time of the order of a nanosecond. In addition, residue Asn-29, which could not be well fitted by any model, has a significantly shortened T2 value indicative of millisecond timescale chemical exchange, which may reflect backbone flexibility, or given the proximity of Tyr-92 may simply be because of the combination of local motion and the ring current shift from this aromatic side chain. The Conserved Motifs in the Loops Are Involved in CP Binding—Chemical shift perturbation mapping was used to identify the CP-binding site on ICP. Complexes of 15N-labeled ICP with L. mexicana CPB and with P. latex papain were purified, and their 15N HSQC spectra were compared with free ICP. A similar subset of backbone HN and side chain cross-peaks are perturbed from their free ICP chemical shifts in both complexes, and the most distinctive are shown in Fig. 3 for the papain complex. It is clear that all the most perturbed residues fall in the ranges 28-37 (covering the B-C loop), 59-74 (covering the D-E loop), and 94-104 (covering the F-G loop). Chemical shifts are sensitive to changes in chemical environment caused either by proximity to a ligand or by propagated structural changes. The co-location of all the significant changes in the vicinity of the three conserved loops strongly suggests that this is because of their involvement in binding to target CPs. Residues in the D-E Loop Are Critical for CP Binding—Based on our L. mexicana ICP structure and on sequence similarity between ICP family members, we made a limited number of mutant ICPs to test their effect on the inhibitory activity of ICP. In the highly conserved B-C loop, we mutated the pair of threonines to glycine (T31G,T32G) and mutated the conserved tyrosine at the base of the loop to alanine (Y34A). In the flexible D-E loop, we made two triple mutants, one to replace the semiconserved hydrophobic residues with charged residues (M67D,V68D,V70D) and the other to replace the glycines by more conformationally restricted residues (G69P,G71P,G72P). In the F-G loop, we made a double mutant of the conserved arginine and aromatic residues (R94A,F96A). The D-E loop mutants lacked inhibitory activity against L. mexicana CPB. To ensure that these mutants had retained their three-dimensional fold we analyzed them using CD spectroscopy. Wild type ICP and mutant M67D,V68D,V70D possess superimposable CD spectra in both the near and far UV regions indicating very similar, if not identical, secondary and tertiary structures. Mutant G69P,G71P,G72P gave rise to a slightly different far UV spectrum, while retaining similar spectral features in the near UV region, and CDSSTR analysis placed the regular secondary structure estimates within a few percent of the wild type ICP. Of the other mutants, the B-C loop mutations resulted in somewhat lower Kis, whereas the Ki of the F-G loop mutant was ∼3-fold higher (see Table 2).TABLE 2Ki values for L. mexicana ICP mutants against L. mexicana CPB ND, not determined.LoopKipML. mexicana ICP132.8 ± 21.1T31G, T32GB-C53.0 ± 8.3Y34AB-C46.4 ± 6.5M67D, V68D, V70DD-END, no inhibition observedG69P, G71P, G72PD-END, no inhibition observedR94A, F96AF-G428.5 ± 86.5 Open table in a new tab A Model for the Interaction between ICP and CPs—The identification of the three conserved loops as the CP-binding site prompted a comparison of the ICP structure with other CP inhibitors. A slight, but suggestive, structural similarity was detected with the cystatin, stefin B (see below), where the CP-binding site is formed by a short central loop flanked by a flexible N-terminal peptide and a longer flattened loop that incorporates a proline (see Fig. 4). We speculated that ICP might bind target CPs in a similar fashion with the flexible D-E loop binding in place of the N-terminal peptide of stefin B. To produce a model for the interaction between ICP and a CP, we used the chemical shift perturbation data for ICP in complex with papain, together with information from the crystal structure of the complex between papain and stefin B, to drive a docking simulation using HADDOCK (24Dominguez C. Boelens R. Bonvin A.M.J.J. J. Am. Chem. Soc. 2003; 125: 1731-1737Crossref PubMed Scopus (2087) Google Scholar). The input data dictated that ICP should bind into the active site of papain but did not, a priori, dictate that ICP should bind the peptidase in a stefin-like orientation. Of the 200 lowest energy structures from the HADDOCK run, all adopted a stefin-like mode of binding (Fig. 4) with residues 67-70 from the D-E loop filling the “unprimed” sites of the active site cleft, whereas the B-C loop approaches the active site cysteine-histidine pair, and side chains from residues in the F-G lo