Conformational Studies of Cyclic Peptide Structures in Solution from 'H-Nmr Data by Distance Geometry Calculation and Restrained Energy Minimization HANS SENN,' HANS-RUDOLF LOOSLI, zyxwvuts ' MICHEL SANNER, ' and WERNER BRAUN* ' zyxwvutsrqpo Sandoz Ltd., Preclinical Research, CH-4002 Basle, 'lnstitut fur Molekularbiologie, Eidgenijssische Technische Hochschule, CH-8093 Zurich, Switzerland SYNOPSIS The three-dimensional structure of a cyclic bouvardin anaLgue, cyclo ( -Pro-MeTyr-Ala- MeTyr-MeTyr-D-Ala- ) has been determined by distance geometry calculation and restrained energy minimization from nmr data. The preparation ofthe input for the distance geometry calculations, the modification of the amino acid library, and the analysis of the structures were done with the aid of a recently developed software package, GEOM. A great variety of different initial structures were explored to check the uniqueness of the determined solution structure. Calculations with 500 different initial structures and two different strat- egies led to a uniquely determined backbone conformation with a root mean square deviations value of 0.4 A. The backbone structure consists of two @-turns, a 13-11 turn at Pro'-MeTyr', and a @-VI turn at MeTyr4-MeTyr5. The efficiency of the two calculation strategies were compared in order to propose an optimal means for performing distance geometry calcu- lations with cyclic structures. INTRODUCTION In typical drug design situations with polypeptides, the occurrence of cyclic structures and of chemically modified amino acid residues requires special care in performing distance geometry calculations from nmr data. zyxwvutsrq A flexible software package, GEOM, spe- cifically developed for support in the determination of three-dimensional structures of a great variety of covalent structures, has been tested and described.' We have used this tool to determine the three-di- mensional structure of the Bouvardin analogue 2 12, cyclo ( zyxwvutsrqp -Pro-MeTyr-Ala-MeTyr-MeTyr-D-Ala- ) , where MeTyr designates the substitution of an amide proton by a methyl group. In the structure calculation of this cyclic peptide, we did not assume any hypothetical initial model or available x-ray structure. Distance geometry calculations were per- (c, 1990 John Wiley & Sons, Inc. zyxwvutsrqpo ccc 0006-3525/90/10-111387-14 $04.00 Biopolymers, Vol. 29, 1387-1400 (1990) formed with the program DISMAN,2i3 where the residue library had been generated by GEOM. Pre- vious studies with cyclic peptides 4-7 did not include a systematic variation of the initial structure, to show that the final result is not biased by the initial structure. Exhaustive methods for grid search can only be applied in practice to small rings because of the ex- ponentially increasing number of conformations that have to be considered.8 By carefully checking the van der Waals radii and by using experimental data early in the tree search, the number of confor- mations to be looked at can be dramatically de- ~reased.~ But it has still not been shown if the re- duction of the number of conformations is sufficient for practical application with typical nmr data sets. The method we have applied is also not exhaustive, but it definitely explores a much greater variety of initial structures than has been previously possible.s The procedure is similar in spirit to the approach followed by others, lo where sequential and short- range information are carefully examined so as to discard forbidden conformations. but it has the ad- 1387