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Biochemistry 2001, 40, 7973-7983 7973 Solution Structure of the Squash Trypsin Inhibitor MCoTI-II A New Family for Cyclic Knottins†,‡ Annie Heitz,Đ Jean-Francá ois Hernandez,| Jean Gagnon,| Thai Trinh Hong, T Traˆn Chaˆu Pham,⊥ Tuyet Mai Nguyen,⊥ Dung Le-Nguyen,# and Laurent Chiche*,§ Centre de Biochimie Structurale, UMR5048 CNRS-UniVersite´ Montpellier I, UMR554 INSERM-UniVersite´ Montpellier I, Faculte´ de pharmacie, 15 aVenue Charles Flahault, 34060 Montpellier, France, Institut de Biologie Structurale Jean-Pierre Ebel (CEA-CNRS), 41, rue Jules Horowitz, 38027 Grenoble Cedex 1, France, Centre de Biotechnologie, UniVersite´ Nationale du Vietnam, 90, Nguyen Trai Street, Hanoă-Viet-Nam, and INSERM U376, CHU Arnaud-de-VilleneuVe, 371, rue du doyen Gaston Giraud, 34295 Montpellier-France ReceiVed April 2, 2001; ReVised Manuscript ReceiVed May 17, 2001 ABSTRACT: The “knottin” fold is a stable cysteine-rich scaffold, in which one disulfide crosses the macrocycle made by two other disulfides and the connecting backbone segments This scaffold is found in several protein families with no evolutionary relationships In the past few years, several homologous peptides from the Rubiaceae and Violaceae families were shown to define a new structural family based on macrocyclic knottin fold We recently isolated from Momordica Cochinchinensis seeds the first known macrocyclic squash trypsin inhibitors These compounds are the first members of a new family of cyclic knottins In this paper, we present NMR structural studies of one of them, MCoTI-II, and of a -Asp rearranged form, MCoTI-IIb Both compounds display similar and well-defined conformations These cyclic squash inhibitors share a similar conformation with noncyclic squash inhibitors such as CPTI-II, and it is postulated that the main effect of the cyclization is a reduced sensitivity to exo-proteases On the contrary, clear differences were detected with the three-dimensional structures of other known cyclic knottins, i.e., kalata B1 or circulin A The two-disulfide cystine-stabilized -sheet motif [Heitz et al (1999) Biochemistry 38, 10615-10625] is conserved in the two families, whereas in the C-to-N linker, one disulfide bridge and one loop are differently located The molecular surface of MCoTI-II is almost entirely charged in contrast to circulin A that displays a well-marked amphiphilic character These differences might explain why the isolated macrocyclic squash inhibitors from M cochinchinensis display no significant antibacterial activity, whereas circulins and kalata B1 A number of small, stable disulfide-rich proteins have been found in plants and animals The corresponding scaffolds have been largely used by nature to achieve a variety of tasks (inhibition, toxicity, defense, regulation, etc.) and thus represent very interesting starting frameworks for building new active molecules, i.e., by grafting active sites or recognition fragments on them (1-4) However, only few different structural motifs are found in proteins with very diverse origins and functions and with no apparent evolutionary relationship (5) Although this is consistent with the fact that possible protein folds are limited in number and that similar folds can be observed in proteins with essentially no sequence identity (6-8), it appears necessary to accumulate information on new structural motifs and on as many of their variants as possible in order to rationalize the sequence structure-function relationship One such small and stable motif with three disulfide bridges is found in the squash trypsin inhibitors (9-16) These small disulfide-rich proteins (28-32 amino acids, cysteines) are composed of a small antiparallel triple-stranded -sheet, one and a half-turn of a 310 helix, two -turns and the inhibitory loop These secondary structural elements are organized around the three disulfide bridges that largely participate in stabilizing the protein core It was observed that one disulfide bridge crosses the macrocycle formed by the two other disulfide bridges and the interconnecting backbone, hence the terms “knottins”, “cystine-knot”, or “inhibitor cystine-knot” (17-19) The knottin scaffold is based on the elementary cystine stabilized -sheet (CSB)1 motif (20), and opens new interesting perspectives for the engineering of small stable proteins with various novel activities (21, 22) † This work was supported by the collaboration program between CNRS (France) and CNST (Vietnam) ‡ The coordinates for the 30 refined conformers of MCoTI-II have been deposited in the Brookhaven Protein Data Bank (entry 1HA9) * To whom correspondence should be addressed Phone: +33 [0]4 67 04 34 32 Fax: +33 [0]4 67 52 96 23 E-mail: chiche@ cbs.univ-montp1.fr § Centre de Biochimie Structurale | Institut de Biologie Structurale Jean-Pierre Ebel (CEA-CNRS) ⊥ Centre de Biotechnologie # INSERM U376 Abbreviations: 1D, one-dimensional; 2D, two-dimensional; 3D, three-dimensional; CSB, cystine-stabilized -sheet; COSY, correlated spectroscopy; CPTI-II, Cucurbita pepo trypsin inhibitor II; CSI, chemical shift index; EETI II, Ecballium elaterium trypsin inhibitor II; H-bond, hydrogen bond; HSQC, heteronuclear single quantum coherence spectroscopy; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; PCI, potato carboxypeptidase inhibitor; RMS, root-meansquare; TI, trypsin inhibitor; TOCSY, total correlated spectroscopy; TSP, 3-(trimethylsilyl)-propionate, sodium salt 10.1021/bi0106639 CCC: $20.00 © 2001 American Chemical Society Published on Web 06/13/2001 7974 Biochemistry, Vol 40, No 27, 2001 Heitz et al clization and by the disulfide bonds, render these molecules very attractive In this paper, we report NMR solution structure studies on the most abundant cyclic squash trypsin inhibitor, MCoTIII, and on the rearranged form containing a -Asp residue, MCoTI-IIb The peptide segment that was absent in noncylic squash inhibitors but present in cyclic MCoTI-II and -IIb has been termed the C-to-N linker since it links residues that used to be the C-terminus and the N-terminus in previously known squash inhibitors The well-defined conformation calculated for MCoTI-II is compared with both the conformation of noncyclic squash inhibitors and the conformation of nonsquash cyclic knottins Structural similarities and/or differences are detailed and their possible impact on functional aspects are discussed MATERIALS AND METHODS FIGURE 1: (A) Sequence alignment of members of the squash inhibitors family Sequences were taken from (28) and from the Swiss-Prot, TrEMBL, and PIR databases, except for SATI-I, -II, and -III (56) The alignment and sequence order is that given by the CLUSTALW program (57) The precursor sequences were truncated at the first corresponding residue in the MCoTI-II sequence Three sequences are reported for MCTI-II and are followed by the corresponding PDB ID or the indication sw (SwissProt) in parentheses The bottom line indicates fully conserved residues (*) or physicochemical properties (:) (B) Structural alignment between MCoTI-II and cyclic knottins kalata B1, circulin A, and cycloviolacin O1 The alignment was done manually The conserved triple-stranded -sheet is shown as arrows and only structurally conserved residues are aligned In the past few years, macrocyclic peptides kalata B1 (23), cyclopsychotride A (24), circulin A and B (25), cycloviolacin O1 (26), and cycloviolins A-D (27) were shown to share this structural motif, thus defining a new structural family of macrocyclic knottins All these peptides are homologous and were named plant cyclotides They were grouped into two subfamilies following sequence comparisons (26) However, shortly after, we identified new members of the cyclic knottin structural family from seeds of Momordica cochinchinensis, a common cucurbitaceae in Vietnam (28) Two major cyclic trypsin inhibitors named MCoTI-I and -II were isolated, along with rearranged forms containing a -Asp residue (MCoTI-Ib and MCoTI-IIb) All these compounds share large sequence identity with other squash inhibitors but only very low sequence identity, if any, with previously known cyclic knottins (Figure 1) The small size and yet very high stability afforded both by the macrocy- Materials The proteins were isolated from M cochinchinensis seeds as described previously (28) Natural MCoTI-II and MCoTI-IIb were obtained in quantities sufficient for structural studies Microbial Strains Escherichia coli D31 was from H G Boman (Department of microbiology, University of Stockholm, Stockholm, Sweden) Micrococcus luteus A270 was from the Pasteur Institute Neurospora crassa (CBS 32754) was a gift from W F Broekaert (Jansens Laboratory of Genetic, Catholic University of Leuven, Heverlee, Belgium) Antimicrobial Assays Antibacterial activities were measured using a liquid-growth inhibition assay as described previously (29) Briefly, 10 µL from 2-fold serial dilutions of the peptides (100-0.2 µM, final concentrations) were incubated in 96-wells microtiter plates with a starting OD600 of 0.001 After a 24 h incubation at 25 °C, the antibacterial activity was monitored by measuring the culture absorbance at 595 nm using a microplate reader The antifungal activity against N crassa used liquid-growth inhibition assay (29) Briefly, fungal spores (final concentration of 10-4 spores/ mL) were suspended in a growth medium containing Potato Dextrose Broth [DIFCO, in half-strength, supplemented with tetracycline (10µg/mL) and cefotaxim (100µg/mL)], dispensed by aliquots of 90 µL into wells of a microplate containing 10 µL of the serial dilution of the peptides, and incubated for 48 h at 25 °C in the dark Growth of fungi was evaluated as above Positive controls were obtained using insect antimicrobial peptides (thanatin and androctonin) NMR Spectroscopy Samples were prepared by dissolving peptides in either 90% H2O/10% 2H2O (v/v) or 100% 2H2O to a concentration of approximately 2.5 mM with the pH adjusted to 3.4 by addition of dilute HCl or NaOH All 1H NMR spectra were recorded on a Bruker AMX-600 spectrometer Data were acquired at 12 and 27 °C, and TSP-d4 was used as an internal reference All 2D experiments, COSY, TOCSY, and NOESY, were performed according to standard procedures (30) using quadrature detection in both dimensions with spectral widths of 6849.3 Hz in both dimensions The carrier frequency was centered on the water signal, and the solvent was suppressed by continuous low power irradiation during the relaxation delay and during the mixing time for NOESY spectra The 2D spectra were obtained using 2048 or 4096 points for each t1 value, and 512 t1 experiments were acquired for COSY, TOCSY, and Solution Structure of MCoTI-II NOESY experiments TOCSY spectra were recorded with spin lock times of 30 and 60 ms The mixing time was 150 and 300 ms in NOESY spectra Spectra were processed using XWINNMR (Bruker) The t1 dimension was zero filled to 1024 points and π/8- and π/4-shifted sine bell functions were applied in t1 and t2 domains, respectively, prior to Fourier transform 3JNH-HR coupling constants were measured on 1D spectra The exchange of amide protons with deuterium was studied at 12 °C on 2.5 mM samples lyophilized from H2O at pH 3.4 and dissolved in 2H2O A series of 1D, TOCSY, and NOESY spectra were acquired over a 72-h period 1H13 C HSQC spectra (31, 32) were recorded on the samples in 2H O Spectral widths were 6849.3 and 25 000 Hz in the 1H and 13C dimensions, respectively A total of 2048 data points was acquired with 512 t1 increments Structure Calculations All calculations were performed on a Silicon Graphics Origin 200 workstation The structures were displayed and analyzed using either INSIGHT II (MSI, San Diego) on a Silicon Graphics O2 workstation or MOLMOL 2K.1 (33) on a Linux box The NOE intensities were classified as strong, medium, and weak, and converted into distance constraints of 2.5, 3, and Å, respectively If the connectivity involved side-chain protons, 3.0, 4.0, and 5.0 Å upper bounds were used instead to account for higher mobility For sequential dRN and dNN connectivities, we used bounds of 2.5, 3.0, and 3.5 Å and 2.8, 3.3, and 4.0 Å, respectively When necessary, the distance constraints were corrected for pseudoatoms (34) φ angles of residues with small or large 3JHN-HR coupling constants (8.5 Hz) were constrained in the -90° to -40° or -160° to -80° ranges angles of residues for which stereospecific attribution of the -protons could be achieved were constrained in the corresponding range Disulfide bridges were imposed through distance constraints of 2.0-2.1 Å, 3.03.1 Å, and 3.75-3.95 Å on Si-Sj, Si-C j, Sj-C i, and C i-C j distances, respectively No H-bond was imposed 3D structures were obtained from the distance and angle restraints using the torsion angle molecular dynamics method available in the DYANA program (35) Preliminary DYANA runs and analyses with the GLOMSA routine (36) were used to perform stereospecific assignment whenever possible Gly2, Gly6, and Gly34 R-protons could be unambiguously assigned with this method One thousand structures were then calculated with the standard simulated annealing protocol The thirty structures with the lowest violation of the target function were selected for further refinement They were submitted to molecular mechanics energy refinement with the SANDER module of the AMBER program (37), using the parm94 force field (38) and the GB/SA implicit solvation system (39) During the molecular dynamics runs, the covalent bond lengths were kept constant by applying the SHAKE algorithm (40) allowing a 1.5 fs time step to be used The nonbonded pair list was updated every 20 steps, and the temperature was regulated by coupling the system to a heat bath with a coupling constant of 0.2 ps Pseudoenergy terms taking into account the NMR interproton distance restraints were defined as follows via four threshold distance values: r1, r2, r3, and r4 In all cases, r1 and r2 were set to 1.3 and 1.8 Å, respectively r3 was taken as the upper boundary used in the DYANA calculations and r4 was chosen as r3 + 0.5 Å For an observed distance lying between r2 and r3, no restraint was applied Between r1 and r2 or between Biochemistry, Vol 40, No 27, 2001 7975 r3 and r4, parabolic restraints were applied Outside the r1 to r4 range, the restraints were linear with slopes identical at parabolic slopes at points r1 and r4 A similar strategy was used for dihedral restraints When no stereospecific assignment could be achieved for methyl or methylene protons, an 〈r-6〉-1/6 averaging scheme was used instead of pseudoatoms Five thousand cycles of restrained energy minimization were first carried out followed by a 30-ps long simulated annealing procedure in which the temperature was raised to 900 K for 20 ps then gradually lowered to 300 K During this stage, the force constant for the NMR distance and dihedral constraints were gradually increased from 3.2 to 32 kcal mol-1 Å-2 and from 0.5 to 50 kcal mol-1 rad-2, respectively Color Figures 8, 9, and 10 were produced with the MOLMOL (33) and POV-Ray (http://www.povray.org) programs RESULTS AND DISCUSSION In the late 1980s, we determined the first 3D structure of EETI-II, a squash trypsin inhibitor This compound, with only 28 amino acids but three disulfide bridges arranged in a pseudo-knotted topology, displayed a particularly high degree of stability along with protease resistance (9, 17) Since then, more than 20 different small disulfide rich protein families were shown to share the same “knottin” topology More recently, nearly 40 homologous peptides from plants of the Rubiaceae and Violaceae families have been reported to belong to a new cyclic knottins structural family (23, 2527, 41) We reported recently the first known macrocyclic trypsin inhibitors (TI) from the squash family, MCoTI-I and MCoTI-II, and rearranged -Asp isoforms that belong to the cyclic knottin family but display no significant sequence similarity with cyclic knottins of the Rubiaceae and Violaceae families We have determined the solution structure of MCoTI-II and MCoTI-IIb to analyze structural differences with homologous noncyclic squash TIs and with nonhomologous cyclic knottins Solution Structure of MCoTI-II (1) NMR Assignments The assignment of all the 1H and 13C resonances present in the spectrum was achieved using well-established techniques (30) and part of the sequential assignment is presented in Figure The lists of 1H and 13C chemical shifts are available as Supporting Information (2) Secondary Structure Figure summarized the sequential and medium range NOEs, 3JHN-HR coupling constants, slowly exchanging amide protons and the CR chemical shift index (CSI) (42, 43) The observation of two small 3J 18 19 HN-HR coupling constants for residues Asp and Ser , and the dNN(i,i+2), dRN(i,i+2), and dRN(i,i+3) NOEs in the region 16-21 shows the presence of a short 310 helix which is generally detected between the second and the third cysteine of the TIs of the squash family The NMR parameters measured in the region 22-25 are in agreement with a -turn [dRN(i,i+2) NOE and slowly exchanging amide proton of residue 25] It is now well-established that squash TIs share a common structural motif with others cysteine-rich peptides This motif made of an antiparallel triple-stranded -sheet is well-defined in MCoTI-II The three regions of the sequence involved in this motif are 13-15, 26-28 and 32-34 Large 3J HN-HR coupling constants and slowly exchanging amide 7976 Biochemistry, Vol 40, No 27, 2001 Heitz et al FIGURE 4: Distribution of the number of experimental constraints deduced from medium (hatched bars) and long range (filled bars) NOEs as a function of the sequence of MCoTI-II Each constraint is counted twice, once for each proton involved Table Constraint Violations and Structural Statisticsa FIGURE 2: Fingerprint region of the 600 MHz NOESY spectrum of MCoTI-II at 12 °C and pH 3.4 in 90% H2O/10% 2H2O The following sequences are traced: 5-8, 10-13, 16-20, 23-26, and 28-3 spatial constraints distancesb short medium long-range dihedralsc φ constraint violationsd distances number > 0.2 Å number > 0.1 Å sum maximum dihedral number > 5° number > 2° FIGURE 3: NMR data summary of the sequential and medium range NOE connectivities, 3JHN-HR coupling constants and slowly exchanging amide protons observed for MCoTI-II The asterisk indicate the sequential HR-Hδ(i-1) connectivities for proline residues The height of the bar correspond to the strength of the NOE The values of the 3JHN-HR coupling constants are indicated by V (8.5 Hz) Open and filled squares indicate backbone amide protons that were still observed after and 24 h, respectively, in 2H2O The chemical shift index (CSI) derived from the CR chemical shifts of MCoTI-II is plotted at the bottom of the figure protons measured in these parts of the sequence are in agreement with this structure In addition, all the characteristic inter-strand NOEs were detected Region 26-34 is a -hairpin with a -turn involving residues 28-31 that was ascertained by the presence of dNN(i,i+2) and dRN(i,i+2) NOEs Concerning the C-to-N linker, weak dNN(i,i+2) and dRN(i,i+2) NOEs were observed in the region 3-6, but all the amide protons of the residues constituting this loop are rapidly exchanging This part of the sequence thus appears as rather flexible and poorly structured, in accordance with the CSI (3) Structure Calculations The three-dimensional structure of the cyclic compound MCoTI-II was determined from NMR data using the same strategy previously used for structural studies of native squash inhibitor EETI II and of analogues (9, 20, 44-47) The NMR study led to 86 AMBER energies (kcal mol-1) bond angle + dihedral van der Waals generalized Born surface based total AMBER constraint PROCHECK statistics residues in most favored regions (A,B,L) residues in additional allowed regions (a,b,l,p) deviations from ideal geometry bond angle 86 31 140 8,11,15,16,18,19,21,26,32 8,15,18,20,21,25,27,30,32,33 2.97 (0.81) 1.17 (0.83) 11.46 (1.71) 0.55 (0.17) 2.49 (0.26) 0.11 (0.02) 0.35 (0.07) 0.67 (0.48) 0.77 (0.77) 1.37 (0.85) 20.1 (0.47) 189.9 (44.2) -113.7 (3.54) -523.3 (145.9) 11.7 (0.28) -1279.3 (43.96) 2.51 (0.52) 85% 14% 0.012 (10-4) 2.15 (0.38) a Values in parentheses indicate standard deviations b Number of constraints c Residue numbers d Values are for refined structures and for DYANA structures in italics sequential and 171 medium and long-range NOEs The distribution of the medium and long-range NOEs along the sequence is displayed in Figure Nine φ angles were determined from the 3JHN-HR coupling constants and stereospecific assignment of the H protons was achieved for 10 residues (Figure and Table 1) Although not experimentally determined, the disulfide bridge pattern was assumed to be the same as that derived from the three-dimensional structures of closely related noncyclic homologues (Figure 1) (28) The Solution Structure of MCoTI-II Biochemistry, Vol 40, No 27, 2001 7977 FIGURE 5: Stereoview of the refined solution structures of MCoTI-II Thirty structures were superimposed for backbone atoms of residues 13-33 Backbone atoms (N, CA, C, O) and disulfide bridges (atoms CB and SG) are shown, and cysteine residue numbers are displayed NMR data were converted into distance and angle constraints as usual The list of constraints used is available as Supporting Information, and the statistics for constraint violations and for molecular mechanics energies are shown in Table The program DYANA (35) was used to compute 1000 3D conformations compatible with the constraints using the torsion angle dynamics method The large number of calculation was to avoid convergence problems due to the highly constrained knotted topology of the molecule Using AMBER 6.0 (37), the 30 best resulting models were further refined using a molecular dynamics simulated annealing protocol including a 20-ps long heating period (900 K) to better search the conformational space Molecular dynamics refinements in previous studies used either a cpu-intensive explicit water treatment (10, 44) or a simple distance dependent dielectric function coupled to reduced charges on charged side chains (20) In this study, the more accurate GB/SA implicit solvation model (39), made available in AMBER 6.0, was used instead Structure Analysis and Comparison with Noncyclic Squash Inhibitors The calculated structures satisfy the NMR data very well with no distance and dihedral violation equal or larger than 0.2 Å or 5°, respectively (Table 1) Statistical analyses using the PROCHECK-NMR software (48) show that the overall stereochemistry of the MCoTI-II solution structures is very good with 99% of nonglycine and nonproline residues lying in the most favored and additional allowed regions of the Ramachandran map (Table 1) As expected, the refined models also display large negative molecular mechanics AMBER energies The stereochemical quality of the calculated structures, is supported by the very good similarity with X-ray structures of homologous proteins (see below) These results constitute a good validation of the refinement protocol using the implicit GB/SA solvation model (39) Since it has been shown that the quality of solution structures may be correlated to the refinement protocols or softwares (49), then the AMBER-GB/SA combination does appear well suited to the refinement of solution structures Table Global RMS Deviationsa (Å) residues MCoTI-II solution structures trace backbone heavy atoms MCoTI-II vs CPTI-II trace backbone a 1-34 7-34 13-33 1.53 (0.47) 1.44 (0.45) 2.18 (0.40) 0.78 (0.30) 0.74 (0.28) 1.72 (0.29) 0.33 (0.09) 0.29 (0.08) 1.57 (0.33) 0.86 0.82 0.62 0.57 Values in parentheses indicate standard deviations FIGURE 6: Average per residue RMS deviation between calculated structures of MCoTI-II (plain line) and per residue RMS deviation between MCoTI-II and CPTI-II (dashed line) MCoTI-II structures were superimposed pairwise for the backbone atoms of residues 13-33 The solution structure closest to the average conformation of MCoTI-II was superimposed onto the X-ray structure of CPTIII for backbone atoms of residues 13-33 (MCoTI-II numbering) Grey boxes at the top indicate different segments Lnk: C-to-N linker; Inh: inhibitory loop; CSB: cystine stabilized -sheet motif The structure of MCoTI-II is particularly well resolved with a very low global backbone RMS deviation of 0.29 ( 0.08 Å for superimposition of backbone atoms of core residues 13-33 (Table and Figures and 6) Even the inhibitory loop displays rather low (