Protein Engineering vol 5 no.6 pp.473-477. 1992
The solution structure of echistatin: evidence for disulphide bond rearrangement in homologous snake toxins
Robert M.Cooke, Brian G.Carter, Peter Murray-Rust, Michael J.Hartshorn1, Pawel Herzyk1 and Roderick E.Hubbard1 Glaxo Group Research, Greenford Road, Greenford, Middlesex UB6 OHE and 'Department of Chemistry, University of York, Heslington, York YO1 5DD, UK
Introduction Considerable pharmaceutical interest was aroused by the discovery that the 49-residue snake toxin echistatin is a potent fibrinogen antagonist (Gan etal., 1988). This was attributed to the presence of an arginine—glycine —aspartic acid (RGD) sequence, a characteristic receptor-binding motif in many extracellular proteins including fibronectin, collagen and fibrinogen (Ruoslahti and Pierschbacher, 1987). The RGD sequence enables echistatin to compete with fibrinogen in binding to the Gp IIB/IIIA receptor on the surface of platelets and thus inhibit the blood clotting process (Gan et al., 1988). Amino acid sequencing of echistatin (Gan etal., 1988) revealed a high proportion of charged residues but showed that cysteine was the most common component (Figure 1). There is no overall sequence homology between echistatin and fibrinogen, but there is extensive homology between echistatin and other anti-coagulant snake toxins (Figure 1). Considerable efforts have been directed towards producing fibrinogen antagonists from smaller peptides containing an RGD sequence. We have studied the solution conformation of echistatin by nuclear magnetic resonance spectroscopy (NMR). The 'H NMR spectrum of echistatin has been assigned and the secondary structure of the protein deduced (Cooke et al., 1991). We have now determined the tertiary structure of echistatin by a simulated annealing analysis of the NMR data (Wuthrich, 1986; Nilges et al., 1988). In addition, the pattern of disulphide bridges, which could not be determined by classical methods of digestion and mass spectrometic analysis, has been predicted. This pattern is distinct from that in the closely related protein kistrin (Adler et al., 1991), contradicting conventional beliefs that homologous proteins possess, where possible, the same topology of disulphide bridging (Creighton, 1984). © Oxford University Press
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The solution structure of the fibrinogen antagonist, echistatin, has been determined by a combination of NMR and simulated annealing methods. While the structure of the disulphidelinked core is well-defined by the NMR data, the N- and Ctermini and the loop bearing the RGD sequence (which is responsible for the fibrinogen antagonist properties) are poorly defined. The pattern of disulphide bridges, which could not be determined by classical methods, was predicted by a statistical analysis of the simulated annealing structures. This pattern is distinct from that for the homologous protein kistrin, leading to the novel suggestion that homologous proteins possess non-conserved patterns of disulphide bridges. Key words: disulphide bonds/fibrinogen antagonist/protein NMR
Materials and methods Samples of synthetic echistatin for NMR studies were prepared and NMR spectra acquired as previously described (Cooke et al., 1991). The 3-D structure of echistatin was determined using the YASAP protocol (Nilges etal., 1988) within the program XPLOR (Nilges, 1990). The NMR information used as restraints consisted of 314 NOEs from two-dimensional spectra, 20 dihedral angle restraints derived from N H - a C H coupling constants and the six hydrogen bonds that could be inferred from the secondary structure of the protein (Cooke et al., 1991). NOE cross peaks were classified as strong, medium or weak and assigned distance restraints of 1.8-3.0, 1.8-3.5 or 1.8-5.0 A respectively. The numbers of strong, medium and weak NOEs were 21, 145 and 148 respectively; of these, 229 were between resonances of groups separated by less than five residues in the sequence, the remaining 85 defining long-range interactions. The upper bounds of NOE restraints involving inon-stereospecifically assigned methyl and methylene proton resonances were increased to allow for centre averaging (Wagner et al., 1987), and 0.5 A was added to restraints involving methyl groups to allow for their higher relative NOE intensities (Clore etal.. 1987). Initially 50 structures were generated with random / and \f/ angles. No NMR evidence was observed for c/'s-peptide bonds; consequently, all peptide bonds were set to trans-(\80 ± 5°). Each structure then underwent the following, (i) 50 steps of conjugate gradient minimization, (ii) 7500 steps (15 ps) of molecular dynamics at 1000 K. The potential energy target function used during these stages was a sum of covalent terms [equations 4 to 6 in Nilges (1990)], a quartic van der Waals repulsive term [equation 10 in Nilges (1990)] and a soft-square NOE potential [equations 70 to 72 in Nilges (1990)]. An NOE force constant of 50 kcal/mol-A", with a low asymptotic constant of 0.1 kcal/mol-A2 and a near-zero repulsive potential force constant were used. (iii) 5000 steps (10 ps) of dynamics at 1000 K with the NOE asymptotic constant and repulsive force constant gradually increased to final values of 1.0 and 0.1 kcal/mol-A2 respectively. (iv) 1450 steps (2.9 ps) of dynamics during which the temperature was incrementally reduced to 300 K. At this stage the NOE potential was switched from soft-square to square-well [equations 70 and 72 in Nilges (1990)] with the same force constant. A torsion angle restraint potential [equations 74 and 75 in Nilges (1990)] with force constant of 200 kcal/mol-rad2 was incorporated into the potential energy target function. The repulsive force was increased to 4 kcal/mol-A4 while the hard sphere van der Waals radii were reduced by 20%. (v) 500 steps of conjugate gradient minimisation. In these calculations no disulphide pattern was imposed upon the structures and the molecular topology was modified to allow cysteine sulphur atoms to approach within 2.0 A. Following a
R.H.Cooke et al.
Echisiatin
ECESGPCCRNCKFLKEGTICKRARGDDMDDYCNGKTCDCPRNPHKGPAT
Kislrin
GKECDCSSPENPCCDAATCKLRPGAQCGEGLCCEQCKFSRAGKICRIPRGDMPDDRCTGQSADCPRYH
r
Albolabrin
EAGEDCDCGSPANPCCDAATCKLLPGAQCGEGLCCDQCSFMKKGTICRRARGDDLDDYCNGISAGCPRNPLHA
Flavoridin
GEECDCGSPSNPCCDAATCKLRPGAQCADGLCCDQCRFKKKTGICRIARGDFPDDRCTGLSNDCPRWNDL
Applagm
EAGEECDCGSPENPCCDAATCKLRPGAQCAEGLCCDQCKFMKEGTVCRARGDDVNDYCNGISAGCPRNPFH
Trigramin Batroxostatin Eleganiin
EAGEDCDCGSPANPCCDAATCKLIPGAQCGEGLCCDQCSFIEEGTVCRIARGDDLDDYCNGRSAGCPRNPFH EAGEECDCGTPENPCCDAATCKLRPGAQCAEGLCCDQCRFKGAGKICRRARGDNPDDRCTGQSADCPRNRF EAGEECDCGSPENPCCDAATCKLRPGAQCADGLCCDQCRFKKKRTICRRARGDNPDDRCTGQSADCPRNGLYS
Conserved
EAG-
-CDC- P N P C C D A A T C K L - P G A - C -
-G-CC--C-F
C- - RGD- - - D C - G • - • - C P R •
Table I. Analysis of possible disulphide bonding patterns in echistatina RMSD (A)b
Disulphide pattern 2-11 2-11 2-11 2-11 2-11 2-11 2-37 2-8
7-32 7-39 7-20 7-8 7-37 7-8 7-32 7-32
8-37 8-37 8-37 20-39 8-32 20-32 8-11 11-37
20-39 20-32 32-39 32-37 20-39 37-39 20-39 20-39
4.19 5.03 5.29 5.52 5.65 5.67 6.17 7.07
Mean S —S separations (A)c Bond 1 Bond 2 5.9 ± 5.9 ± 5.9 59 5.9 5.9 12.1 8.5 ± 2.3
4.2 7.5 7.9 6.9 7.9 6.9 4.2 42
± ± ± ± ± ± ± ±
.0 .3 .4 .2 .1 2 1.0 1.0
Bond 3
Bond 4
80 ± 2.2 8.0 2.2 80 2.2 3.7 14 10.2 1.2 4.7 16 7.9 1.6 2.3 137
3.7 4.7 5.5 10 5 3.7 10 6 37 3.7
± ± ± ± ± ± ± ±
1 4 1.6 1.8 0.9 1.4 1.1 1.4 14
21 1 0 0 0 0 0 0
"The 22 acceptable echistatin structures calculated in the absence of disulphide bridges were analysed in terms of S - S bond lengths for possible permutations. The structures were examined to determine which bridges would give the minimum deviation from ideal bond lengths, both individually and as a set. and the extent to which a single pattern would be favoured. The seven most favourable patterns and one equivalent to the homologous protein kistrin (ranked 14th) are presented here. ^*he average RMSD from an ideal S —S separation (2.02 A) for all hypothetical bridges in all structures The mean S—S separation for each hypothetical bridge in all structures. d The number of structures for which the particular disulphide pattern gave the minimum RMSD from ideal S —S bond lengths.
series of statistical analyses (MJ.Hartshorn el al., manuscript in preparation), the most likely pairings of cysteines in disulphide bridges were predicted. Fifty structures were then generated for each of these disulphide patterns as before, but with three additional restraints for each bridge: Sj-Sj, S, — C/Sj and Sj-C/3,, with target values of 2.02 ± 0.02, 2.99 ± 0.5 and 2.99 ± 0.5 A respectively (Nilges et al., 1988b). The disulphide bond restraints were then removed and the molecular topology modified to incorporate covalent disulphide bridges. An additional 500 steps of conjugate gradient minimization were then performed. Results and discussion A feature of computational methods for generating an ensemble of protein structures is a need to assess the merit of individual structures, and thus select a family of those representative of the physical system. In this study the validity of structures was checked by plotting their potential energies versus the number of NOE restraint violations. This yielded a cluster of 'good' structures with low values of each, typically numbering between 40 and 60% of the total. In the case of the 50 echistatin structures 474
generated without pre-defined disulphide bridges 22 satisfactory examples were thus selected. The 22 satisfactory echistatin structures were examined to determine the most likely pairings of cysteine residues in disulphide bridges. The only bridge which can be unequivocally assigned from NMR data alone is Cys2 —Cysl 1 (Cooke et al., 1991): NOEs are observed between the aCH resonance of Cys2 and the /3CH resonance of Cys 11, the aCH resonance of Cys 11 and a /3CH resonance of Cys2 and the 0CH resonances of the two cysteines. For the remaining cysteine residues either no NOEs to resonances of other cysteines are observed or NOEs are observed to resonances of more than one other cysteine. Elucidation of the pairings in the remaining bridges thus required the use of a series of statistical analyses (M.J.Hartshorn et al., manuscript in preparation) based on the separation of S7 atoms of cysteines in the calculated structures (Table I). All 105 possible arrangements of the four bridges were considered, even though pairings such as Cys7—Cys8 and Cys37—Cys39 could be discounted on geometric grounds. Searching for the lowest mean S 7 - S 7 separation identified the Cys20-Cys39 pairing. Selecting the smallest separation from the remaining cysteines yielded Cys7-Cys32, then Cys2-Cysl 1 and finally Cys8-Cys37. The
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Fig. 1. The aligned amino acid sequences of several anticoagulant snake toxins (Gan el al.. 1988; Chao et al., 1989: Dennis et al., 1989; Huang et al.. 1989; Musial el al., 1990; Rucinski et al., 1990; Williams el al., 1990). Residues which are totally conserved are indicated in the bottom line. The cysteine residues of echistatin are numbered, with the disulphide bridges predicted in this study marked. Disulphide bridges are also marked for kistrin (Adler et al.. 1991) and albolabrin (Calvette et al., 1991). For the two occurrences of adjacent cysteines in albolabrin the bridges could not be distinguished and could be with either cysteine.
Solution structure of echistatin
pattern pairing Cys2-Cysl 1, Cys7-Cys32, Cys8-Cys37 and Cys20-Cys39 produced the lowest RMS deviation from ideal Sy — S7 distance averaged over all bridges and all structures, and in 21 of 22 structures this pattern gave the lowest RMSD from ideal S 7 - S 7 distances (Table I). This pattern is thus the most likely to exist in solution. Geometric restraints appropriate for bridges pairing Cys2 - Cys 11, Cys7 - Cys32, Cys8 - Cys37 and Cys20 - Cys39 were then incorporated and a further set of structures generated. From this set 24 low energy/low violation structures were selected and are overlaid in Figure 2(A). The energies, convergence and violations of experimental distance restraints for these structures
are presented as pattern A in Table II. In almost every respect these structures are better than those calculated without disulphide bond restraints. It is apparent from Figure 2(A) that while some regions of the echistatin structures consistently converge to a common fold, other regions vary considerably in conformation. The most divergent parts consist of the N-terminus, a longer stretch at the C-terminus and the loop bearing the RGD recognition sequence. These correspond to regions where there are relatively few interresidue NOEs other than between sequentially adjacent residues, and it is likely that the divergence may be a realistic representation of the relative disorder of these regions in solution. The backbone 475
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Fig. 2. (A) A stereo view of the backbone atoms (Ca. N, C) of 24 cchistatin structures derived from simulated annealing of the NMR data with the disulphide pattern 2—11. 7 — 32, 8 — 37 and 20 — 39 The structures were superimposed using the backbone atoms of residues 4 — 23 and 28—41. yielding an RMSD of 0 875 A The approximate positions of the N- and C-termini and the RGD loop are marked. (B) The echistatin structure with Ihe lowest RMS restraint violation and final energy. Only the Ca atoms and the four disulphide bridges are shown The eight cysteine residues are numbered, as are Glu3 and Pro6 (discussed in the text) and Gly25 (the central residue of the RGD sequence) The orientation of (B) differs from that of (A) by an ~ 90° rotation about the vertical axis.
R.H.Cooke el al.
Table II. Comparison of echistatin structures determined with different disulphide patterns
No. analysed" No. violations'1 RMSV (A)c Fm (kcal/mol)d F N 0 E (kcal/mol)c F lor (kcal/mol)c ^repcl (kcal/mol)c £L.j (kcal/mol)f Bond dev (A)8 Angle dev. (deg.)g Imp. dev. (deg.)8 RMSD (A)h
No disulphides
Pattern A
Pattern B
Pattern C
22 6 0.0089 7.2 1.34 0.43 3.35 -118.3 0.007 2.07 0.141 1.37
24 2 0.0075 0.0 0.93 0.27 2.93 -124.6 0.007 1.92 0.127 0.88
24 28 0 0137 42.4 3.10 2.66 5.29 -123.6 0009 2.52 0.187 1.53
29 8 0.0088 11 0 1.32 1.10 3 38 -124.7 0.007 2.10 0.146 0.93
± ±
0.0018 46
± 0.56 ± 0.19 ± 0.42 ± 16.8 ± 0 003 ± 0.39
± 0.053 ±
0.38
± 0.0012 ± 34
± 0.28 ± ± ± ± ± ± ±
0.10 0.43 8.6 0.003 0.39 0.046 0.13
± 0.0013 ± 15.6 ± 0.58 ± 1.14 ± 0.94 ± 9.4 ± 0.004 ± 0 99 ± 0.120 ± 0 35
± 0.0019 ± 5.3 ± 0.56 ± 0.33 ± 0.48 ±99 ± 0.003 ± 0.46 ± 0.070 ± 0.20
atoms of residues 4 - 2 3 and 28-41 are, however, well defined, and the disulphide bridge between Cys2 and Cysll partly ties down the N-terminus. A turn is located at residues 6 - 7 followed by a type-II' turn leading from residue 8 to residue 13. Residues 16-20 are hydrogen-bonded with 30-33 to form an anti-parallel /3-sheet with a j3-bulge at residues 17 and 18. Residues 21-29 form a highly charged loop presenting the RGD sequence (residues 24-26) at one end of the molecule. The chain fold reverses between residues 33 and 37, placing residue 38 adjacent to residue 6. From residue 42 on, the protein is essentially disordered. The four disulphide bridges in echistatin form a 'ladder' (Figure 2B) and, whilst a protein this small and extended cannot be considered to possess a distinct core, the ordered part of the protein is arranged around three of the bridges: 7 — 32, 8-37 and 20-39. This may explain why some of the most pronounced secondary shifts in the NMR spectrum of echistatin are observed for Cys /3CH resonances (Cooke etal., 1991). A similar conformation for echistatin in solution, arising from a distance geometry treatment of NMR data, has previously been reported by Saudek etal. (1991). As in our studies, prior knowledge of the disulphide bridges was not available, but they were unable to distinguish between two possible arrangments: 2 - 1 1 , 7 - 3 2 , 8-37 and 20-39, or 2 - 1 1 , 7 - 3 7 , 8-32 and 20-39. More recently Chen etal. (1991) reported similar findings. The first arrangement is that predicted in our studies while the second is far less favoured (Table I). A structure determination including appropriate geometric restraints for the second set of bridges with our NMR data produced less acceptable structures on all criteria (Table II, pattern B). The second is thus unlikely to exist in solution. An NMR/distance geometry structure has also been reported for the larger snake toxin, kistrin (Adler et al., 1991). Again prior knowledge of the pattern of disulphide bridges was not available, but NOEs were observed between resonances of paired cysteines in five out of six cases, and an unequivocal pattern of bridges was derived from the conformation of the folded protein. 476
As in echistatin, the RGD region of kistrin lies at the end of a flexible loop and, within the well-defined regions, the fold of our echistatin structure is very similar to that of the homologous regions of kistrin. Although Cys37 in echistatin is replaced by Ala in kistrin, the other seven cysteines are conserved. Conventional wisdom would predict that the bridges corresponding to residues 2—11, 7 — 32 and 20 — 39 in echistatin occur in kistrin, with the cysteine corresponding to residue 8 forming a bridge to the N-terminal domain. The pattern of disulphides determined for kistrin, however, corresponds to pairing cysteines 2—8, 7—32 and 20-39 in echistatin, with the cysteine corresponding to residue 11 in echistatin bridged to the N-terminal domain in kistrin. We find no NOEs to support a 2 — 8 bridge in echistatin, while the 2— 11 pairing is the most unequivocal from NMR data alone (Chen et al., 1991; Cooke et al., 1991; Dal vit et al., 1991). Table I confirms that a disulphide pattern equivalent to that of kistrin is unlikely in echistatin (i.e. 2 - 8 , 7 - 3 2 , 11-37 and 20-39), and a structure determination combining geometric restraints for this disulphide pattern with our NMR data produced less satisfactory structures (Table II, pattern C). Thus two homologous proteins, with similar chain folds and similar activities, possess different patterns of disulphide bridges. The reasons for this are not clear. It could result from a marginal stability of the 2 - 1 1 bridge, which is located at one end of echistatin (Figure 2B). Once Cys37 is removed, bridging residues 2 and 8 and allowing Cys 11 to link with the N-terminal domain may be a more stable arrangement. Alternatively, it may result from other sequence variations: an unfavourable distortion in the backbone of Glu3 was observed when applying the kistrin pattern of disulphides to echistatin. The substitution of this residue with glycine in kistrin may allow the backbone to adopt a different conformation and promote a different set of disulphides. The change from Pro to Leu at position 6 may also contribute to this. A qualitative comparison of the echistatin and kistrin structures suggest that the most significant differences occur for residues 1-7 (echistatin numbering).
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Pattern A: 2 - 1 1 , 7-32, 8-37, 20-39, pattern B: 2 - 1 1 . 7-37, 8-32, 20-39; pattern C. 2 - 8 , 7-32, 11-37. 20-39. "The number of structures found to have low restraints violations and potential energies, these were selected for the analyses presented in this table. ^The number of NOE violations >0.1 A summed over all analysed structures. c The average r.m.s. violations of NOE restraint upper limits. d The average value of the potential energy target function [cf. equation 1 to 5 in Dnscoll et al. (1989)] at the end of the structure determination, relative to that of disulphide pattern A. e The average values of the square-well NOE restraint potentials (F N O E) a n d torsion angle restraint potentials (Flor) were calculated with force constants of 50 and 200 kcal/mol-A2 respectively. The quartic van der Waals repulsive term (F2repe|) was calculated with a force constant of 4 kcal/molA4 with the hardsphere van der Waals radii set to 0.8 times the standard values used in the CHARMM empirical energy function (Brooks el al., 1983) f The average Lennard-Jones van der Waals energy calculated with the CHARMM empirical energy function (Brooks et al.. 1983). It was not included in the potential energy function during the simulated annealing. ^Deviations from the ideal bond lengths, bond angles and improper dihedral angels of the XPLOR parameter set. h Average root mean square deviation of the analysed structures from the mean for each disulphide pattern, calculated using the backbone atoms (N. Ca, C) or residues 4 - 2 3 and 2 8 - 4 1 .
Solution structure of echistatin
Disulphide bridges may be added to or removed from homologous proteins without disrupting the pattern of other bridges (Thornton, 1981). More significantly, the disulphide topology changes between mouse and human IL-4 because of the deletion of one cysteine and the insertion of another (Carr etal., 1991). The structure of IL-4 (Redfield etal., 1991) suggests this may be accommodated by a small change in the position of the one end of one bridge without disrupting the others. The disulphide rearrangements described in this paper are even more dramatic, in that the changes in topology are more extensive than the minimum required to allow for cysteine insertions and deletions. It is not clear how widespread this phenomenon is, but it reinforces arguments that disulphide bridges are not powerful determinants of protein structure (Schulz and Schirmer, 1979; Creighton, 1984). It also sounds a cautionary note in modelling homologous protein structures: rather than assuming homologous disulphide bridges, close attention must be paid to the spatial proximity of bridges and the minor sequence changes that may alter disulphide patterns.
Chen.Y., Pitzenberger.S.M., Garsky.V.M.. Lumma.P.K., Sanyal.G. and BaumJ. (1991) Biochemistry, 30, 11625-11636. Clore.G M., Gronenbom.A.M.. Nilges.M. and Ryan.C.A. (1987) Biochemistry, 26, 8012-8023. Cooke.R.M., Carter.B.G., Martin.D.M., Weir.M.P. and Murray-Rust.P. (1991) Eur. J. Biochem., 202, 32-328. Creighton,T.E. (\9M) Proteins. Structures and Molecular Principles. Freeman. New York. Dalvit.C, Widmer.H., Bovermann.G. Breckenridge.R. and Mettemich.R. (1991) Eur. J. Biochem., 202, 315-321. Dennis.M.S.. Henzel.W.J., Pitti,R.M., Lipari.M.T.. Napier.M.A., Deisher.T.A., Bunting.S. and Lazarus,R.A. (1989) Proc. Natl Acad. Sci. USA, 87, 2471-2475. Dnscoll.P.C, Gronenborn,A.M., Beress.L. and Clore.G.M. (1989) Biochemistry. 28, 2188-2198. Gan,Z -R., Gould,R.J., Jacobs.J.W., Friedman.P.A and Polokoff.M.A. (1988) J. Biol. Chem., 263, 19827-19832. Huang,T.-F.. Holt.J.C, Kirby.E.P. and Niewiarowski.S. (1989) Biochemistry. 28, 661-666. Kornblihtt.A.R., Umezawa.K., Vibe-Pedersen,K. and Baralle.F.E (1985) EMBO / , 4, 1755-1759. Musail.J., Niewiarowski.S., Rucinski.B.. Stewart.G.J., Cook.J.J.. Williams.J.A. and Edmunds.L.H. (1990) Circulation, 82, 261-273. Nilges.M. (1990) In Brunger.A.T. (ed.), Xptor Version 2.1 User Manual. Yale University Press, New Haven, CT, pp. 256-263. Nilges.M., Clore,G.M. and Gronenborn.A.M. (1988a) FEBS Lett.. 239. 129-136. Nilges.M., Gronenbom.A.M., Brunger.A.T. and Clore.G.M. (1988b) Protein Engng, 2, 27-38. Redfield.C, Smith,L.J., Boyd.J., Lawrence.G.M.P., Edwards,R.G. Smith.R.A.G. and Dobson.C.M. (1991) Biochemistry, 30, 11029-11035. Rucinski.B., Niewiarowski.S., Holt.J.C., Soska.T. and Knudsen.K.A. (1990) Biochim. Biophys. Ada, 1054, 257-262. Ruostahti,E and Pierschbacher.M.D. (1987) Science, 338, 491-497. Saudek.V., Atkinson.R.A. and Pelton.J.T. (1991) Biochemistry, 30, 7369-7372 Schultz.G.E. and Schirmer.R.H. (1979) Principles of Protein Structure Springer Verlag, New York. Thornton.J.M. (1981)/ Mol. Biol, 151, 261-287. Wagner.G., Braun.W., Havel.T.F., Schaumann.T , Go.N. and Wuthrich.K (1987)7. Mol. Biol. 196, 611-619. Williams.J., Rucinski.B., Holt.J. and Niewiarowski.S. (1990) Biochim. Biophvs. Acta, 1039, 81-89 Wuthnch.K. (1986) NMR of Proteins and Nucleic Acids. John Wiley, New York. Received on May 8, 1992, revised on June 20, 1992: accepted on June 30, 1992
Acknowledgements We thank Guy Dodson, Alan Tonge and Malcom Weir for helpful discussions. Axel Brunger for the program XPLOR and advice and Molecular Simulations for the use of the graphics program QUANTA. Financial support to MJH and REH was provided by the SERC.
References Adler.M., Lazarus,R A . Dennis.M.S. and Wagner.G (1991) Science, 253, 445-448. Brooks,B.R., Bruccolen.R.E.. Olafson.B.D., States.D.J.. Swaminathan.S and Karplus.M. (1983)/ Comp Chem., 4, 187-217. Calvete.J.J., Schafer.W., Soszka.T.. Lu.W., Cook.J.J.. Jameson.B.A. and Niewiariowski.S. (1991) Biochemists, 30, 5225-5229. Carr.C, Aykent.S , Kimack.N.M. and Levine.A.D. (1991) Biochemistry, 30. 1515-1523 Chao.B.H.. JaukowskiJ.A.. Savage.B , Chow.E.P., Marzec.U.M., Harker.L.A. and Maraganore.J.M. (1989) Proc Natl Acad. Set. USA, 86. 8050-8054.
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A partial disulphide pattern has also been reported for albolabrin (Calvete etal, 1991), which is homologous to echistatin and even more so to kistrin (Figure 1). However, the albolabrin pattern comprises (in echistatin numbering) Cys2-Cys32, Cys20-Cys39, Cysll-Cys7/8, with the remaining one of Cys7/8 bridging to the N-terminus. This is distinct from either the kistrin or the echistatin patterns; a Cys2-Cys32 bridge in echistatin would be particularly difficult to form. It will be interesting to see the results of disulphide determinations for other members of this family, to ascertain the degree of heterogeneity. It is possible that the diversity of bridges within these snake toxins is related to the spatial separation of the disulphide 'ladder' from the recognition site, the RGD sequence. Rearranging the bridges could have little effect on the structural or dynamic properties of the RGD sequence. The major function of the bridges could be the generally accepted one of stabilizing the folded state by reducing the entropy of the unfolded protein (Schulz and Schirmer, 1979). An additional role in constraining the remainder of the molecule to restrict degradation should also be considered; echistatin displays abnormally high resistance to enzymatic degradation (D.M.A.Martin, personal communication). The independence of the recognition site from the disulphide bridges is supported by the observation that the RGD sequence in fibronectin is contained within a cysteine-free domain (Kornblihtt et at, 1985), while their role in stabilizing folding could account for the loss of activity in echistatin upon reduction (Gan etal, 1988).
The solution structure of echistatin: evidence for disulphide bond rearrangement in homologous snake toxins
Robert M.Cooke, Brian G.Carter, Peter Murray-Rust, Michael J.Hartshorn1, Pawel Herzyk1 and Roderick E.Hubbard1 Glaxo Group Research, Greenford Road, Greenford, Middlesex UB6 OHE and 'Department of Chemistry, University of York, Heslington, York YO1 5DD, UK
Introduction Considerable pharmaceutical interest was aroused by the discovery that the 49-residue snake toxin echistatin is a potent fibrinogen antagonist (Gan etal., 1988). This was attributed to the presence of an arginine—glycine —aspartic acid (RGD) sequence, a characteristic receptor-binding motif in many extracellular proteins including fibronectin, collagen and fibrinogen (Ruoslahti and Pierschbacher, 1987). The RGD sequence enables echistatin to compete with fibrinogen in binding to the Gp IIB/IIIA receptor on the surface of platelets and thus inhibit the blood clotting process (Gan et al., 1988). Amino acid sequencing of echistatin (Gan etal., 1988) revealed a high proportion of charged residues but showed that cysteine was the most common component (Figure 1). There is no overall sequence homology between echistatin and fibrinogen, but there is extensive homology between echistatin and other anti-coagulant snake toxins (Figure 1). Considerable efforts have been directed towards producing fibrinogen antagonists from smaller peptides containing an RGD sequence. We have studied the solution conformation of echistatin by nuclear magnetic resonance spectroscopy (NMR). The 'H NMR spectrum of echistatin has been assigned and the secondary structure of the protein deduced (Cooke et al., 1991). We have now determined the tertiary structure of echistatin by a simulated annealing analysis of the NMR data (Wuthrich, 1986; Nilges et al., 1988). In addition, the pattern of disulphide bridges, which could not be determined by classical methods of digestion and mass spectrometic analysis, has been predicted. This pattern is distinct from that in the closely related protein kistrin (Adler et al., 1991), contradicting conventional beliefs that homologous proteins possess, where possible, the same topology of disulphide bridging (Creighton, 1984). © Oxford University Press
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The solution structure of the fibrinogen antagonist, echistatin, has been determined by a combination of NMR and simulated annealing methods. While the structure of the disulphidelinked core is well-defined by the NMR data, the N- and Ctermini and the loop bearing the RGD sequence (which is responsible for the fibrinogen antagonist properties) are poorly defined. The pattern of disulphide bridges, which could not be determined by classical methods, was predicted by a statistical analysis of the simulated annealing structures. This pattern is distinct from that for the homologous protein kistrin, leading to the novel suggestion that homologous proteins possess non-conserved patterns of disulphide bridges. Key words: disulphide bonds/fibrinogen antagonist/protein NMR
Materials and methods Samples of synthetic echistatin for NMR studies were prepared and NMR spectra acquired as previously described (Cooke et al., 1991). The 3-D structure of echistatin was determined using the YASAP protocol (Nilges etal., 1988) within the program XPLOR (Nilges, 1990). The NMR information used as restraints consisted of 314 NOEs from two-dimensional spectra, 20 dihedral angle restraints derived from N H - a C H coupling constants and the six hydrogen bonds that could be inferred from the secondary structure of the protein (Cooke et al., 1991). NOE cross peaks were classified as strong, medium or weak and assigned distance restraints of 1.8-3.0, 1.8-3.5 or 1.8-5.0 A respectively. The numbers of strong, medium and weak NOEs were 21, 145 and 148 respectively; of these, 229 were between resonances of groups separated by less than five residues in the sequence, the remaining 85 defining long-range interactions. The upper bounds of NOE restraints involving inon-stereospecifically assigned methyl and methylene proton resonances were increased to allow for centre averaging (Wagner et al., 1987), and 0.5 A was added to restraints involving methyl groups to allow for their higher relative NOE intensities (Clore etal.. 1987). Initially 50 structures were generated with random / and \f/ angles. No NMR evidence was observed for c/'s-peptide bonds; consequently, all peptide bonds were set to trans-(\80 ± 5°). Each structure then underwent the following, (i) 50 steps of conjugate gradient minimization, (ii) 7500 steps (15 ps) of molecular dynamics at 1000 K. The potential energy target function used during these stages was a sum of covalent terms [equations 4 to 6 in Nilges (1990)], a quartic van der Waals repulsive term [equation 10 in Nilges (1990)] and a soft-square NOE potential [equations 70 to 72 in Nilges (1990)]. An NOE force constant of 50 kcal/mol-A", with a low asymptotic constant of 0.1 kcal/mol-A2 and a near-zero repulsive potential force constant were used. (iii) 5000 steps (10 ps) of dynamics at 1000 K with the NOE asymptotic constant and repulsive force constant gradually increased to final values of 1.0 and 0.1 kcal/mol-A2 respectively. (iv) 1450 steps (2.9 ps) of dynamics during which the temperature was incrementally reduced to 300 K. At this stage the NOE potential was switched from soft-square to square-well [equations 70 and 72 in Nilges (1990)] with the same force constant. A torsion angle restraint potential [equations 74 and 75 in Nilges (1990)] with force constant of 200 kcal/mol-rad2 was incorporated into the potential energy target function. The repulsive force was increased to 4 kcal/mol-A4 while the hard sphere van der Waals radii were reduced by 20%. (v) 500 steps of conjugate gradient minimisation. In these calculations no disulphide pattern was imposed upon the structures and the molecular topology was modified to allow cysteine sulphur atoms to approach within 2.0 A. Following a
R.H.Cooke et al.
Echisiatin
ECESGPCCRNCKFLKEGTICKRARGDDMDDYCNGKTCDCPRNPHKGPAT
Kislrin
GKECDCSSPENPCCDAATCKLRPGAQCGEGLCCEQCKFSRAGKICRIPRGDMPDDRCTGQSADCPRYH
r
Albolabrin
EAGEDCDCGSPANPCCDAATCKLLPGAQCGEGLCCDQCSFMKKGTICRRARGDDLDDYCNGISAGCPRNPLHA
Flavoridin
GEECDCGSPSNPCCDAATCKLRPGAQCADGLCCDQCRFKKKTGICRIARGDFPDDRCTGLSNDCPRWNDL
Applagm
EAGEECDCGSPENPCCDAATCKLRPGAQCAEGLCCDQCKFMKEGTVCRARGDDVNDYCNGISAGCPRNPFH
Trigramin Batroxostatin Eleganiin
EAGEDCDCGSPANPCCDAATCKLIPGAQCGEGLCCDQCSFIEEGTVCRIARGDDLDDYCNGRSAGCPRNPFH EAGEECDCGTPENPCCDAATCKLRPGAQCAEGLCCDQCRFKGAGKICRRARGDNPDDRCTGQSADCPRNRF EAGEECDCGSPENPCCDAATCKLRPGAQCADGLCCDQCRFKKKRTICRRARGDNPDDRCTGQSADCPRNGLYS
Conserved
EAG-
-CDC- P N P C C D A A T C K L - P G A - C -
-G-CC--C-F
C- - RGD- - - D C - G • - • - C P R •
Table I. Analysis of possible disulphide bonding patterns in echistatina RMSD (A)b
Disulphide pattern 2-11 2-11 2-11 2-11 2-11 2-11 2-37 2-8
7-32 7-39 7-20 7-8 7-37 7-8 7-32 7-32
8-37 8-37 8-37 20-39 8-32 20-32 8-11 11-37
20-39 20-32 32-39 32-37 20-39 37-39 20-39 20-39
4.19 5.03 5.29 5.52 5.65 5.67 6.17 7.07
Mean S —S separations (A)c Bond 1 Bond 2 5.9 ± 5.9 ± 5.9 59 5.9 5.9 12.1 8.5 ± 2.3
4.2 7.5 7.9 6.9 7.9 6.9 4.2 42
± ± ± ± ± ± ± ±
.0 .3 .4 .2 .1 2 1.0 1.0
Bond 3
Bond 4
80 ± 2.2 8.0 2.2 80 2.2 3.7 14 10.2 1.2 4.7 16 7.9 1.6 2.3 137
3.7 4.7 5.5 10 5 3.7 10 6 37 3.7
± ± ± ± ± ± ± ±
1 4 1.6 1.8 0.9 1.4 1.1 1.4 14
21 1 0 0 0 0 0 0
"The 22 acceptable echistatin structures calculated in the absence of disulphide bridges were analysed in terms of S - S bond lengths for possible permutations. The structures were examined to determine which bridges would give the minimum deviation from ideal bond lengths, both individually and as a set. and the extent to which a single pattern would be favoured. The seven most favourable patterns and one equivalent to the homologous protein kistrin (ranked 14th) are presented here. ^*he average RMSD from an ideal S —S separation (2.02 A) for all hypothetical bridges in all structures The mean S—S separation for each hypothetical bridge in all structures. d The number of structures for which the particular disulphide pattern gave the minimum RMSD from ideal S —S bond lengths.
series of statistical analyses (MJ.Hartshorn el al., manuscript in preparation), the most likely pairings of cysteines in disulphide bridges were predicted. Fifty structures were then generated for each of these disulphide patterns as before, but with three additional restraints for each bridge: Sj-Sj, S, — C/Sj and Sj-C/3,, with target values of 2.02 ± 0.02, 2.99 ± 0.5 and 2.99 ± 0.5 A respectively (Nilges et al., 1988b). The disulphide bond restraints were then removed and the molecular topology modified to incorporate covalent disulphide bridges. An additional 500 steps of conjugate gradient minimization were then performed. Results and discussion A feature of computational methods for generating an ensemble of protein structures is a need to assess the merit of individual structures, and thus select a family of those representative of the physical system. In this study the validity of structures was checked by plotting their potential energies versus the number of NOE restraint violations. This yielded a cluster of 'good' structures with low values of each, typically numbering between 40 and 60% of the total. In the case of the 50 echistatin structures 474
generated without pre-defined disulphide bridges 22 satisfactory examples were thus selected. The 22 satisfactory echistatin structures were examined to determine the most likely pairings of cysteine residues in disulphide bridges. The only bridge which can be unequivocally assigned from NMR data alone is Cys2 —Cysl 1 (Cooke et al., 1991): NOEs are observed between the aCH resonance of Cys2 and the /3CH resonance of Cys 11, the aCH resonance of Cys 11 and a /3CH resonance of Cys2 and the 0CH resonances of the two cysteines. For the remaining cysteine residues either no NOEs to resonances of other cysteines are observed or NOEs are observed to resonances of more than one other cysteine. Elucidation of the pairings in the remaining bridges thus required the use of a series of statistical analyses (M.J.Hartshorn et al., manuscript in preparation) based on the separation of S7 atoms of cysteines in the calculated structures (Table I). All 105 possible arrangements of the four bridges were considered, even though pairings such as Cys7—Cys8 and Cys37—Cys39 could be discounted on geometric grounds. Searching for the lowest mean S 7 - S 7 separation identified the Cys20-Cys39 pairing. Selecting the smallest separation from the remaining cysteines yielded Cys7-Cys32, then Cys2-Cysl 1 and finally Cys8-Cys37. The
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Fig. 1. The aligned amino acid sequences of several anticoagulant snake toxins (Gan el al.. 1988; Chao et al., 1989: Dennis et al., 1989; Huang et al.. 1989; Musial el al., 1990; Rucinski et al., 1990; Williams el al., 1990). Residues which are totally conserved are indicated in the bottom line. The cysteine residues of echistatin are numbered, with the disulphide bridges predicted in this study marked. Disulphide bridges are also marked for kistrin (Adler et al.. 1991) and albolabrin (Calvette et al., 1991). For the two occurrences of adjacent cysteines in albolabrin the bridges could not be distinguished and could be with either cysteine.
Solution structure of echistatin
pattern pairing Cys2-Cysl 1, Cys7-Cys32, Cys8-Cys37 and Cys20-Cys39 produced the lowest RMS deviation from ideal Sy — S7 distance averaged over all bridges and all structures, and in 21 of 22 structures this pattern gave the lowest RMSD from ideal S 7 - S 7 distances (Table I). This pattern is thus the most likely to exist in solution. Geometric restraints appropriate for bridges pairing Cys2 - Cys 11, Cys7 - Cys32, Cys8 - Cys37 and Cys20 - Cys39 were then incorporated and a further set of structures generated. From this set 24 low energy/low violation structures were selected and are overlaid in Figure 2(A). The energies, convergence and violations of experimental distance restraints for these structures
are presented as pattern A in Table II. In almost every respect these structures are better than those calculated without disulphide bond restraints. It is apparent from Figure 2(A) that while some regions of the echistatin structures consistently converge to a common fold, other regions vary considerably in conformation. The most divergent parts consist of the N-terminus, a longer stretch at the C-terminus and the loop bearing the RGD recognition sequence. These correspond to regions where there are relatively few interresidue NOEs other than between sequentially adjacent residues, and it is likely that the divergence may be a realistic representation of the relative disorder of these regions in solution. The backbone 475
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Fig. 2. (A) A stereo view of the backbone atoms (Ca. N, C) of 24 cchistatin structures derived from simulated annealing of the NMR data with the disulphide pattern 2—11. 7 — 32, 8 — 37 and 20 — 39 The structures were superimposed using the backbone atoms of residues 4 — 23 and 28—41. yielding an RMSD of 0 875 A The approximate positions of the N- and C-termini and the RGD loop are marked. (B) The echistatin structure with Ihe lowest RMS restraint violation and final energy. Only the Ca atoms and the four disulphide bridges are shown The eight cysteine residues are numbered, as are Glu3 and Pro6 (discussed in the text) and Gly25 (the central residue of the RGD sequence) The orientation of (B) differs from that of (A) by an ~ 90° rotation about the vertical axis.
R.H.Cooke el al.
Table II. Comparison of echistatin structures determined with different disulphide patterns
No. analysed" No. violations'1 RMSV (A)c Fm (kcal/mol)d F N 0 E (kcal/mol)c F lor (kcal/mol)c ^repcl (kcal/mol)c £L.j (kcal/mol)f Bond dev (A)8 Angle dev. (deg.)g Imp. dev. (deg.)8 RMSD (A)h
No disulphides
Pattern A
Pattern B
Pattern C
22 6 0.0089 7.2 1.34 0.43 3.35 -118.3 0.007 2.07 0.141 1.37
24 2 0.0075 0.0 0.93 0.27 2.93 -124.6 0.007 1.92 0.127 0.88
24 28 0 0137 42.4 3.10 2.66 5.29 -123.6 0009 2.52 0.187 1.53
29 8 0.0088 11 0 1.32 1.10 3 38 -124.7 0.007 2.10 0.146 0.93
± ±
0.0018 46
± 0.56 ± 0.19 ± 0.42 ± 16.8 ± 0 003 ± 0.39
± 0.053 ±
0.38
± 0.0012 ± 34
± 0.28 ± ± ± ± ± ± ±
0.10 0.43 8.6 0.003 0.39 0.046 0.13
± 0.0013 ± 15.6 ± 0.58 ± 1.14 ± 0.94 ± 9.4 ± 0.004 ± 0 99 ± 0.120 ± 0 35
± 0.0019 ± 5.3 ± 0.56 ± 0.33 ± 0.48 ±99 ± 0.003 ± 0.46 ± 0.070 ± 0.20
atoms of residues 4 - 2 3 and 28-41 are, however, well defined, and the disulphide bridge between Cys2 and Cysll partly ties down the N-terminus. A turn is located at residues 6 - 7 followed by a type-II' turn leading from residue 8 to residue 13. Residues 16-20 are hydrogen-bonded with 30-33 to form an anti-parallel /3-sheet with a j3-bulge at residues 17 and 18. Residues 21-29 form a highly charged loop presenting the RGD sequence (residues 24-26) at one end of the molecule. The chain fold reverses between residues 33 and 37, placing residue 38 adjacent to residue 6. From residue 42 on, the protein is essentially disordered. The four disulphide bridges in echistatin form a 'ladder' (Figure 2B) and, whilst a protein this small and extended cannot be considered to possess a distinct core, the ordered part of the protein is arranged around three of the bridges: 7 — 32, 8-37 and 20-39. This may explain why some of the most pronounced secondary shifts in the NMR spectrum of echistatin are observed for Cys /3CH resonances (Cooke etal., 1991). A similar conformation for echistatin in solution, arising from a distance geometry treatment of NMR data, has previously been reported by Saudek etal. (1991). As in our studies, prior knowledge of the disulphide bridges was not available, but they were unable to distinguish between two possible arrangments: 2 - 1 1 , 7 - 3 2 , 8-37 and 20-39, or 2 - 1 1 , 7 - 3 7 , 8-32 and 20-39. More recently Chen etal. (1991) reported similar findings. The first arrangement is that predicted in our studies while the second is far less favoured (Table I). A structure determination including appropriate geometric restraints for the second set of bridges with our NMR data produced less acceptable structures on all criteria (Table II, pattern B). The second is thus unlikely to exist in solution. An NMR/distance geometry structure has also been reported for the larger snake toxin, kistrin (Adler et al., 1991). Again prior knowledge of the pattern of disulphide bridges was not available, but NOEs were observed between resonances of paired cysteines in five out of six cases, and an unequivocal pattern of bridges was derived from the conformation of the folded protein. 476
As in echistatin, the RGD region of kistrin lies at the end of a flexible loop and, within the well-defined regions, the fold of our echistatin structure is very similar to that of the homologous regions of kistrin. Although Cys37 in echistatin is replaced by Ala in kistrin, the other seven cysteines are conserved. Conventional wisdom would predict that the bridges corresponding to residues 2—11, 7 — 32 and 20 — 39 in echistatin occur in kistrin, with the cysteine corresponding to residue 8 forming a bridge to the N-terminal domain. The pattern of disulphides determined for kistrin, however, corresponds to pairing cysteines 2—8, 7—32 and 20-39 in echistatin, with the cysteine corresponding to residue 11 in echistatin bridged to the N-terminal domain in kistrin. We find no NOEs to support a 2 — 8 bridge in echistatin, while the 2— 11 pairing is the most unequivocal from NMR data alone (Chen et al., 1991; Cooke et al., 1991; Dal vit et al., 1991). Table I confirms that a disulphide pattern equivalent to that of kistrin is unlikely in echistatin (i.e. 2 - 8 , 7 - 3 2 , 11-37 and 20-39), and a structure determination combining geometric restraints for this disulphide pattern with our NMR data produced less satisfactory structures (Table II, pattern C). Thus two homologous proteins, with similar chain folds and similar activities, possess different patterns of disulphide bridges. The reasons for this are not clear. It could result from a marginal stability of the 2 - 1 1 bridge, which is located at one end of echistatin (Figure 2B). Once Cys37 is removed, bridging residues 2 and 8 and allowing Cys 11 to link with the N-terminal domain may be a more stable arrangement. Alternatively, it may result from other sequence variations: an unfavourable distortion in the backbone of Glu3 was observed when applying the kistrin pattern of disulphides to echistatin. The substitution of this residue with glycine in kistrin may allow the backbone to adopt a different conformation and promote a different set of disulphides. The change from Pro to Leu at position 6 may also contribute to this. A qualitative comparison of the echistatin and kistrin structures suggest that the most significant differences occur for residues 1-7 (echistatin numbering).
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Pattern A: 2 - 1 1 , 7-32, 8-37, 20-39, pattern B: 2 - 1 1 . 7-37, 8-32, 20-39; pattern C. 2 - 8 , 7-32, 11-37. 20-39. "The number of structures found to have low restraints violations and potential energies, these were selected for the analyses presented in this table. ^The number of NOE violations >0.1 A summed over all analysed structures. c The average r.m.s. violations of NOE restraint upper limits. d The average value of the potential energy target function [cf. equation 1 to 5 in Dnscoll et al. (1989)] at the end of the structure determination, relative to that of disulphide pattern A. e The average values of the square-well NOE restraint potentials (F N O E) a n d torsion angle restraint potentials (Flor) were calculated with force constants of 50 and 200 kcal/mol-A2 respectively. The quartic van der Waals repulsive term (F2repe|) was calculated with a force constant of 4 kcal/molA4 with the hardsphere van der Waals radii set to 0.8 times the standard values used in the CHARMM empirical energy function (Brooks el al., 1983) f The average Lennard-Jones van der Waals energy calculated with the CHARMM empirical energy function (Brooks et al.. 1983). It was not included in the potential energy function during the simulated annealing. ^Deviations from the ideal bond lengths, bond angles and improper dihedral angels of the XPLOR parameter set. h Average root mean square deviation of the analysed structures from the mean for each disulphide pattern, calculated using the backbone atoms (N. Ca, C) or residues 4 - 2 3 and 2 8 - 4 1 .
Solution structure of echistatin
Disulphide bridges may be added to or removed from homologous proteins without disrupting the pattern of other bridges (Thornton, 1981). More significantly, the disulphide topology changes between mouse and human IL-4 because of the deletion of one cysteine and the insertion of another (Carr etal., 1991). The structure of IL-4 (Redfield etal., 1991) suggests this may be accommodated by a small change in the position of the one end of one bridge without disrupting the others. The disulphide rearrangements described in this paper are even more dramatic, in that the changes in topology are more extensive than the minimum required to allow for cysteine insertions and deletions. It is not clear how widespread this phenomenon is, but it reinforces arguments that disulphide bridges are not powerful determinants of protein structure (Schulz and Schirmer, 1979; Creighton, 1984). It also sounds a cautionary note in modelling homologous protein structures: rather than assuming homologous disulphide bridges, close attention must be paid to the spatial proximity of bridges and the minor sequence changes that may alter disulphide patterns.
Chen.Y., Pitzenberger.S.M., Garsky.V.M.. Lumma.P.K., Sanyal.G. and BaumJ. (1991) Biochemistry, 30, 11625-11636. Clore.G M., Gronenbom.A.M.. Nilges.M. and Ryan.C.A. (1987) Biochemistry, 26, 8012-8023. Cooke.R.M., Carter.B.G., Martin.D.M., Weir.M.P. and Murray-Rust.P. (1991) Eur. J. Biochem., 202, 32-328. Creighton,T.E. (\9M) Proteins. Structures and Molecular Principles. Freeman. New York. Dalvit.C, Widmer.H., Bovermann.G. Breckenridge.R. and Mettemich.R. (1991) Eur. J. Biochem., 202, 315-321. Dennis.M.S.. Henzel.W.J., Pitti,R.M., Lipari.M.T.. Napier.M.A., Deisher.T.A., Bunting.S. and Lazarus,R.A. (1989) Proc. Natl Acad. Sci. USA, 87, 2471-2475. Dnscoll.P.C, Gronenborn,A.M., Beress.L. and Clore.G.M. (1989) Biochemistry. 28, 2188-2198. Gan,Z -R., Gould,R.J., Jacobs.J.W., Friedman.P.A and Polokoff.M.A. (1988) J. Biol. Chem., 263, 19827-19832. Huang,T.-F.. Holt.J.C, Kirby.E.P. and Niewiarowski.S. (1989) Biochemistry. 28, 661-666. Kornblihtt.A.R., Umezawa.K., Vibe-Pedersen,K. and Baralle.F.E (1985) EMBO / , 4, 1755-1759. Musail.J., Niewiarowski.S., Rucinski.B.. Stewart.G.J., Cook.J.J.. Williams.J.A. and Edmunds.L.H. (1990) Circulation, 82, 261-273. Nilges.M. (1990) In Brunger.A.T. (ed.), Xptor Version 2.1 User Manual. Yale University Press, New Haven, CT, pp. 256-263. Nilges.M., Clore,G.M. and Gronenborn.A.M. (1988a) FEBS Lett.. 239. 129-136. Nilges.M., Gronenbom.A.M., Brunger.A.T. and Clore.G.M. (1988b) Protein Engng, 2, 27-38. Redfield.C, Smith,L.J., Boyd.J., Lawrence.G.M.P., Edwards,R.G. Smith.R.A.G. and Dobson.C.M. (1991) Biochemistry, 30, 11029-11035. Rucinski.B., Niewiarowski.S., Holt.J.C., Soska.T. and Knudsen.K.A. (1990) Biochim. Biophys. Ada, 1054, 257-262. Ruostahti,E and Pierschbacher.M.D. (1987) Science, 338, 491-497. Saudek.V., Atkinson.R.A. and Pelton.J.T. (1991) Biochemistry, 30, 7369-7372 Schultz.G.E. and Schirmer.R.H. (1979) Principles of Protein Structure Springer Verlag, New York. Thornton.J.M. (1981)/ Mol. Biol, 151, 261-287. Wagner.G., Braun.W., Havel.T.F., Schaumann.T , Go.N. and Wuthrich.K (1987)7. Mol. Biol. 196, 611-619. Williams.J., Rucinski.B., Holt.J. and Niewiarowski.S. (1990) Biochim. Biophvs. Acta, 1039, 81-89 Wuthnch.K. (1986) NMR of Proteins and Nucleic Acids. John Wiley, New York. Received on May 8, 1992, revised on June 20, 1992: accepted on June 30, 1992
Acknowledgements We thank Guy Dodson, Alan Tonge and Malcom Weir for helpful discussions. Axel Brunger for the program XPLOR and advice and Molecular Simulations for the use of the graphics program QUANTA. Financial support to MJH and REH was provided by the SERC.
References Adler.M., Lazarus,R A . Dennis.M.S. and Wagner.G (1991) Science, 253, 445-448. Brooks,B.R., Bruccolen.R.E.. Olafson.B.D., States.D.J.. Swaminathan.S and Karplus.M. (1983)/ Comp Chem., 4, 187-217. Calvete.J.J., Schafer.W., Soszka.T.. Lu.W., Cook.J.J.. Jameson.B.A. and Niewiariowski.S. (1991) Biochemists, 30, 5225-5229. Carr.C, Aykent.S , Kimack.N.M. and Levine.A.D. (1991) Biochemistry, 30. 1515-1523 Chao.B.H.. JaukowskiJ.A.. Savage.B , Chow.E.P., Marzec.U.M., Harker.L.A. and Maraganore.J.M. (1989) Proc Natl Acad. Set. USA, 86. 8050-8054.
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A partial disulphide pattern has also been reported for albolabrin (Calvete etal, 1991), which is homologous to echistatin and even more so to kistrin (Figure 1). However, the albolabrin pattern comprises (in echistatin numbering) Cys2-Cys32, Cys20-Cys39, Cysll-Cys7/8, with the remaining one of Cys7/8 bridging to the N-terminus. This is distinct from either the kistrin or the echistatin patterns; a Cys2-Cys32 bridge in echistatin would be particularly difficult to form. It will be interesting to see the results of disulphide determinations for other members of this family, to ascertain the degree of heterogeneity. It is possible that the diversity of bridges within these snake toxins is related to the spatial separation of the disulphide 'ladder' from the recognition site, the RGD sequence. Rearranging the bridges could have little effect on the structural or dynamic properties of the RGD sequence. The major function of the bridges could be the generally accepted one of stabilizing the folded state by reducing the entropy of the unfolded protein (Schulz and Schirmer, 1979). An additional role in constraining the remainder of the molecule to restrict degradation should also be considered; echistatin displays abnormally high resistance to enzymatic degradation (D.M.A.Martin, personal communication). The independence of the recognition site from the disulphide bridges is supported by the observation that the RGD sequence in fibronectin is contained within a cysteine-free domain (Kornblihtt et at, 1985), while their role in stabilizing folding could account for the loss of activity in echistatin upon reduction (Gan etal, 1988).
