1
244 TIBS - September 1980 Prediction of natural binding site structures An example of the power of the art 26 is the prediction of serine protease structure. The aim was to deduce the form of the binding sites of serine proteases, so per- mitting the design of antagonists to these sites. The methodology used for PTI was also employed here. It seems to be a practi- cal proposition to predict the shape and constitution of binding site as part of the prediction of the conformation as a whole, providing that a homologous protein of known conformation is used as the starting point for energy calculation. The calcu- lation is, however, not trivial, because amino acid substitutions may significantly affect conformation, because such confor- mational changes may imply significant dif- ferences in the binding site of the protein of unknown conformation, and because the conformations of the insertion regions are unknown. Nevertheless, using the known structure of elastase as a starting point, and then using the energy calculation method of Ref. 9, the structure of trypsin was pre- dicted to within 2.4/~ rms (as defined in Ref. 6 but employing Ca carbons rather than the sidechains), of the trypsin struc- ture as determined experimentally ~6. As the result was known in advance, this study with David Tunms of I.C.I. was actually a control experiment to assess the validity of our similar type of prediction for thrombin., The fact that the distances between crucial catalytic residues of the serine proteases, (apparently highly conserved by evolution) were reproduced without prior constraint in both the trypsin and thrombin predic- tions suggests that the approach was productive. Unfortunately, this prediction does not yet include the small, associated A chain of thrombin, for which there is no extensive homologous conformation and which may help to determine thrombin specificity. Energy calculations on this must be carried out without the support of detailed experimental data, but obviously the problem closely resembles that of pre- dicting the bound conformation of any polypeptide mediator and should help to test further and develop design tools. No specific antagonists have yet been pro- posed to be tested on the basis of this study, but there is every reason to hope that a combination of the tools and ideas discus - sed in this review will lead to useful results. It must be admitted that for predicting. binding sites in general, this is hardly likely to be the method of choice. In most cases, the primary structure of the receptor pro- tein will not be known, and even if it were, there would probably be no homologous protein of known structure. Fortunately, equally objective methods seem to be poss- ible for inferring the pharmacophore struc- ture indirectly, from the binding data. Crippen" has proposed a method based on the use of distance constraints on the poss- ible conformations of the ligand. The dis- tances between ligand atoms are treated as the conformational variables, and from knowledge of the molecular structures the maximum and minimum possible distances in the various ligands are examined in the light of the binding studies. Since the initial test study was in terms of inhibitors of serine protease activity, it would be of interest to apply the method to the particu- lar case of thrombin and compare the pre- diction of the basic pharmacophore features with that of the above 'direct' approach. Extending the activities of poly~ptides with cofactors Polypeptides possess more than suf- ficient molecular potential for carrying out reactions such as hydrolyses, but they are not particularly well suited to decarboxy- lations, transaminations, oxidation- reduction reactions, oxygen binding and so on. In Nature, this is overcome by the use of non-peptide cofactors. A saving principle here is that some reactions car- ded out by enzymes with metal cofactors can be duplicated, with less specificity and no control, merely by adding the metal ion to the substrate. Proteins with organic and metal ion cofactors in intimate association can also often have their actions duplicated by adding the cofactor-ion complex to the substrate. Thus, the design process need not proceed ab hzitio. An excellent example of using cofactors in the design of an active structure is the successful synthesis of a synthetic 'haemo- globin m. Inspection of the natural system suggested the use ofa haem group with his- tidine residues poised above and below the iron atom in the plane of the porphyrin ring. A ~mple polypeptide scaffold was designed t~ place these in the appropriate spatial relationship, and in this case the polypeptide was also linked to a polyethylene glycol support. Not only was reversible oxygen binding achieved, but several other properties, including curious spectroscopic features, modelled those of the natural molecule. This also illustrates the potential value of synthesizing simple molecular systems in order to identify the crucial aspects of the biological counter- part. Further, it suggests that even more progress can be expected when natural cofactors are exploited in the design pro- cess. Certainly, this is one of the more con- vincing attempts to reproduce a biological function artificially. References 1 Robson,B. (1976) Trends Biochem. Sci. 3, 49-51 2 Chakravarty, P. K., Mathur, K. B. and Dhar, M.M. (1973) Erperientia 29, 786-788 3 Gune, B., D,~umigen, M. and Wittschieber, E. (1979) Nature (London) 281,650-655 4 Warshel, A. and Levitt, M. (1976) J. Mol. Biol. 103,227-249 5 Chakravarty, P. K., Mathur, K. B. and Dhar, M. M. (1973) Indian J. Biochem. Biophy.s. 10, 233-238 6 Levitt, M. and Warshel, A. (1975) Nature (London) 253,694-698 7 Tanaka, S. and Scheraga, t L A. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3802-3806 8 Kuntz, I. D., Crippen, G. M., Kollman,P. A. and Kimelman, D.L Mot. Biol. 106,983-984 9 Robson, B. and Osguthorpe, D. J. (1979)./. Mol. BioL 132, 19-51 10 Ptitsyn,O. B. and Rashin. A. A. (1975) Biophys. Chem. 3, 1-20 l 1 Cohen, F. E., Richmond, T. J. and Richards,F. M. (1979)Z MoL Biol. 132, 275-288 12 Robson, B. (1975) Nan,re (London) 254, 386-388 13 N6methy,G. and Scheraga, H. A. (1977) Quart. Rev. Biophys. 10, 239-352 14 Hagler, A. T. and Honig, B. (1978) Proc. Natl. Acad. ScL U.S.A. 75,554-558 15 Kotelchuck, D. and Scheraga, H. A. (1979) Proc. Natl. Acad. Sci. U.S.A. 62, 14-21 16 Robson, B. and Pain, R. tl. (1971) J. Mot. BioL 58, 237-259 17 Chou, P. Y. and Fasman, G. D. Biochemistry 13, 222-245 18 Gamier, J., Osguthorpe, D. J. and Robson, B. (1978)./. Mol. Biol. 190,97-120 19 Chakravarty, P. K., Mathur, K. B. and Dhar, M. M. (1974)Indian L Chem. 12,464-470 20 Rohlfing, D. L. and Fox,S. W. (1969)Adv. Catal. 20, 373-418 21 Naithani,V. K. and Dhar, M. M. (1967) Biochem. Biopl,ys. Res. Commun. 29, 368-369 22 Montgomery, J. A., Mayo, J. G. and Hansch, C. (1974)J. Med. Chem. 17, 477-480 23 Beddell, C. R., Clark, R. B., Lowe, L. A., Wilkinson, S., Chang, K. J., Cuatrecasas, P. and Miller, R. (1977) Br. J.Pharmacol. 61,351-356 24 Smith,G. D. and Griffin, J. F. (1978)Science 199, 1214-1216 25 Isogai, Y., N6methy, G. and Seheraga, H. A. (1977) Proc. Natl. Acad. Sci. U.S.A. 74,414-418 26 Robson, B., Stem, P. S., HiUier, I. H., Osguthorpe, D. J. and Hagler, A. T. (1979) J. Chim. Phys. 76, 831--834 27 Hagler, A. T., Huler, E. and Lifson, S. (1974) J. Am. Chem. Soc. 96, 5319-5326 28 Hagler,A. T. and Lifson,S. (1974) J. Am. Chem. Soc. 96, 5327-5335 29 HiUier, I. H. and Robson, B. (1979) J. 77zeor. Biol. 76, 83-98 30 Hagler, A. T., Stem, P. S., Sharon, R., Becker, J. M. and Naider, F. (1979) J. Am. Chem. Soc. 10l, 6842-6852 31 Hagler, A. T., Osguthorpe, D. and Robson, B. (1980) Science 208, 599--601 32 Crippen, G. M. (1979) J. Med. Chem. 22, 988-997 33 Bayer, E. and Holzbach, G. (1977)Angew~ Chem. 89, 120-122 Erratum Paul A. Srere, T1BS, May 1980, p. 121, col. 2, line 22, should read: 10 -6 to 10 -s M.

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244 T I B S - S e p t e m b e r 1 9 8 0

Prediction of natural binding site structures

An example of the power of the art 26 is the prediction of serine protease structure. The aim was to deduce the form of the binding sites of serine proteases, so per- mitting the design of antagonists to these sites. The methodology used for PTI was also employed here. It seems to be a practi- cal proposition to predict the shape and constitution of binding site as part of the prediction of the conformation as a whole, providing that a homologous protein of known conformation is used as the starting point for energy calculation. The calcu- lation is, however, not trivial, because amino acid substitutions may significantly affect conformation, because such confor- mational changes may imply significant dif- ferences in the binding site of the protein of unknown conformation, and because the conformations of the insertion regions are unknown. Nevertheless, using the known structure of elastase as a starting point, and then using the energy calculation method of Ref. 9, the structure of trypsin was pre- dicted to within 2 .4 /~ rms (as defined in Ref. 6 but employing Ca carbons rather than the sidechains), of the trypsin struc- ture as determined experimentally ~6. As the result was known in advance, this study with David Tunms of I.C.I. was actually a control experiment to assess the validity of our similar type of prediction for thrombin., The fact that the distances between crucial catalytic residues of the serine proteases, (apparently highly conserved by evolution) were reproduced without prior constraint in both the trypsin and thrombin predic- tions suggests that the approach was productive. Unfortunately, this prediction does not yet include the small, associated A chain of thrombin, for which there is no extensive homologous conformation and which may help to determine thrombin specificity. Energy calculations on this must be carried out without the support of detailed experimental data, but obviously the problem closely resembles that of pre- dicting the bound�9 conformation of any polypeptide mediator and should help to test further and develop design tools. No specific antagonists have yet been pro- posed to be tested on the basis of this study, but there is every reason to hope that a combination of the tools and ideas discus -�9 sed in this review will lead to useful results.

It must be admitted that for predicting. binding sites in general, this is hardly likely to be the method of choice. In most cases, the primary structure of the receptor pro- tein will not be known, and even if it were, there would probably be no homologous protein of known structure. Fortunately,

equally objective methods seem to be poss- ible for inferring the pharmacophore struc- ture indirectly, from the binding data. Cr ippen" has proposed a method based on the use of distance constraints on the poss- ible conformations of the ligand. The dis- tances between ligand atoms are treated as the conformational variables, and from knowledge of the molecular structures the maximum and minimum possible distances in the various ligands are examined in the light of the binding studies. Since the initial test study was in terms of inhibitors of serine protease activity, it would be of interest to apply the method to the particu- lar case of thrombin and compare the pre- diction o f the basic pharmacophore features with that of t h e above 'direct' approach.

Extending the activities of poly~ptides with cofactors

Polypeptides possess more than suf- ficient molecular potential for carrying out reactions such as hydrolyses, but they are not particularly well suited to decarboxy- lations, transaminations, oxidation- reduction reactions, oxygen binding and so on. In Nature, this is overcome by the use of non-peptide cofactors. A saving principle here is that some reactions car- ded out by enzymes with metal cofactors can be duplicated, with less specificity and no control, merely by adding the metal ion to the substrate. Proteins with organic and metal ion cofactors in intimate association can also often have their actions duplicated by adding the cofactor-ion complex to the substrate. Thus, the design process need not proceed ab hzitio.

An excellent example of using cofactors in the design of an active structure is the successful synthesis of a synthetic 'haemo- globin m. Inspection of the natural system suggested the use o f a haem group with his- tidine residues poised above and below the iron atom in the plane of the porphyrin ring. A ~mple polypeptide scaffold was designed t~ place these in the appropriate spatial relationship, and in this case the polypeptide was also linked to a polyethylene glycol support. Not only was reversible oxygen binding achieved, but several other properties, including curious spectroscopic features, modelled those of the natural molecule. This also illustrates the potential value of synthesizing simple molecular systems in order to identify the crucial aspects of the biological counter- part. Further, it suggests that even more progress can be expected when natural cofactors are exploited in the design pro-

cess. Certainly, this is one of the more con- vincing attempts to reproduce a biological function artificially.

References 1 Robson, B. (1976) Trends Biochem. Sci. 3, 49-51 2 Chakravarty, P. K., Mathur, K. B. and Dhar,

M.M. (1973) Erperientia 29, 786-788 3 Gune, B., D,~umigen, M. and Wittschieber, E.

(1979) Nature (London) 281,650-655 4 Warshel, A. and Levitt, M. (1976) J. Mol. Biol.

103,227-249 5 Chakravarty, P. K., Mathur, K. B. and Dhar,

M. M. (1973) Indian J. Biochem. Biophy.s. 10, 233-238

6 Levitt, M. and Warshel, A. (1975) Nature (London) 253,694-698

7 Tanaka, S. and Scheraga, t L A. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3802-3806

8 Kuntz, I. D., Crippen, G. M., Kollman, P. A. and Kimelman, D.L Mot. Biol. 106,983-984

9 Robson, B. and Osguthorpe, D. J. (1979)./. Mol. BioL 132, 19-51

10 Ptitsyn, O. B. and Rashin. A. A. (1975) Biophys. Chem. 3, 1-20

l 1 Cohen, F. E., Richmond, T. J. and Richards, F. M. (1979)Z MoL Biol. 132, 275-288

12 Robson, B. (1975) Nan,re (London) 254, 386-388

13 N6methy, G. and Scheraga, H. A. (1977) Quart. Rev. Biophys. 10, 239-352

14 Hagler, A. T. and Honig, B. (1978) Proc. Natl. Acad. ScL U.S.A. 75,554-558

15 Kotelchuck, D. and Scheraga, H. A. (1979) Proc. Natl. Acad. Sci. U.S.A. 62, 14-21

16 Robson, B. and Pain, R. tl. (1971) J. Mot. BioL 58, 237-259

17 Chou, P. Y. and Fasman, G. D. Biochemistry 13, 222-245

18 Gamier, J., Osguthorpe, D. J. and Robson, B. (1978)./. Mol. Biol. 190, 97-120

19 Chakravarty, P. K., Mathur, K. B. and Dhar, M. M. (1974)Indian L Chem. 12,464-470

20 Rohlfing, D. L. and Fox, S. W. (1969)Adv. Catal. 20, 373-418

21 Naithani, V. K. and Dhar, M. M. (1967) Biochem. Biopl,ys. Res. Commun. 29, 368-369

22 Montgomery, J. A., Mayo, J. G. and Hansch, C. (1974)J. Med. Chem. 17, 477-480

23 Beddell, C. R., Clark, R. B., Lowe, L. A., Wilkinson, S., Chang, K. J., Cuatrecasas, P. and Miller, R. (1977) Br. J.Pharmacol. 61,351-356

24 Smith, G. D. and Griffin, J. F. (1978)Science 199, 1214-1216

25 Isogai, Y., N6methy, G. and Seheraga, H. A. (1977) Proc. Natl. Acad. Sci. U.S.A. 74,414-418

26 Robson, B., Stem, P. S., HiUier, I. H., Osguthorpe, D. J. and Hagler, A. T. (1979) J. Chim. Phys. 76, 831--834

27 Hagler, A. T., Huler, E. and Lifson, S. (1974) J. Am. Chem. Soc. 96, 5319-5326

28 Hagler, A. T. and Lifson, S. (1974) J. Am. Chem. Soc. 96, 5327-5335

29 HiUier, I. H. and Robson, B. (1979) J. 77zeor. Biol. 76, 83-98

30 Hagler, A. T., Stem, P. S., Sharon, R., Becker, J. M. and Naider, F. (1979) J. Am. Chem. Soc.

�9 10l, 6842-6852 31 Hagler, A. T., Osguthorpe, D. and Robson, B.

(1980) Science 208, 599--601 32 Crippen, G. M. (1979) J. Med. Chem. 22,

988-997 33 Bayer, E. and Holzbach, G. (1977)Angew~ Chem.

89, 120-122

Erra tum

Paul A. Srere, T 1 B S , May 1980, p. 121, col. 2, line 22, should read: 10 -6 to 10 -s M.