proteins STRUCTURE O FUNCTION O BIOINFORMATICS Constructing ensembles of flexible fragments in native proteins by iterative stochastic elimination is relevant to protein–protein interfaces Efrat Noy, Tal Tabakman, and Amiram Goldblum * Department of Medicinal Chemistry and the David R. Bloom Center for Pharmacy, School of Pharmacy, The Hebrew University of Jerusalem, Israel 91120 INTRODUCTION The formation of specific complexes between proteins has major roles in many biological processes. Structural information of pro- tein–protein complexes is extremely valuable both in providing insights into the interactions between the partners and in supplying a basis for designing compounds that might interfere with these interactions. Nonetheless, information on structures of protein complexes is largely missing. It is due to the greater difficulty in crystallizing protein complexes, 1,2 a limitation which will probably remain in the near future. Therefore, there has been much interest in developing computational methods for protein–protein docking, which predict the structure of protein–protein complexes, starting from known structures of the unbound partners. 1,3,4 Structures of protein–protein complexes have been examined and shown to be often accompanied by conformational changes in receptor and ligand compared with their unbound structures (UbS). 5,6 Main-chain and side-chain atoms can have significant shifts upon complex formation. 5 The interplay between protein flexibility and protein–protein recognition is currently one of the largest obstacles to understanding and to reliable prediction of the structures of protein complexes. 4,7–9 Even small local changes of 1–2 A ˚ in the positions of backbone atoms might affect the dynam- ics of contact formation upon docking and reduce shape comple- mentarity to a level where it can no longer reliably predict the cor- rect form of the complex. 2,10 Results of protein–protein docking algorithms are in general satisfactory when reconstructing known complexes. In some cases docking algorithms work well also on Abbreviations: BS, bound structure; FF, flexible fragment; ISE, Iterative Stochastic Elimination; MD, molecular dynamics; PDB, protein data bank; RMSD, root mean square deviation; UbS, unbound structure. The Supplementary Material referred to in this article can be found at http://www.interscience. wiley.com/jpages/0887-3585/suppmat/ Grant sponsor: Israel Science Foundation (ISF); Grant number: 608/02. *Correspondence to: Amiram Goldblum, Department of Medicinal Chemistry and the David R. Bloom Center for Pharmacy, School of Pharmacy, The Hebrew University of Jerusalem, Israel 91120. E-mail: amiram@vms.huji.ac.il Received 14 August 2006; Revised 11 January 2007; Accepted 19 January 2007 Published online 17 May 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.21437 ABSTRACT We investigate the extent to which ensembles of flexible fragments (FF), generated by our loop con- formational search method, include conformations that are near experimental and reflect conforma- tional changes that these FFs undergo when binary protein–protein complexes are formed. Twenty-eight FFs, which are located in protein–protein interfaces and have different conformations in the bound structure (BS) and unbound structure (UbS) were extracted. The conformational space of these frag- ments in the BS and UbS was explored with our method which is based on the iterative stochastic elimination (ISE) algorithm. Conformational search of BSs generated bound ensembles and conforma- tional search of UbSs produced unbound ensembles. ISE samples conformations near experimental (less than 1.05 A ˚ root mean square deviation, RMSD) for 51 out of the 56 examined fragments in the bound and unbound ensembles. In 14 out of the 28 unbound fragments, it also samples conformations within 1.05 A ˚ from the BS in the unbound ensem- ble. Sampling the bound conformation in the unbound ensemble demonstrates the potential bio- logical relevance of the predicted ensemble. The 10 lowest energy conformations are the best choice for docking experiments, compared with any other 10 conformations of the ensembles. We conclude that generating conformational ensembles for FFs with ISE is relevant to FF conformations in the UbS and BS. Forming ensembles of the isolated proteins with our method prior to docking represents more com- prehensively their inherent flexibility and is expected to improve docking experiments compared with results obtained by docking only UbSs. Proteins 2007; 68:702–711. V V C 2007 Wiley-Liss, Inc. Key words: loop conformational search; flexible fragments; conformational ensembles; protein–pro- tein docking; conformational changes. 702 PROTEINS V V C 2007 WILEY-LISS, INC.