Grafting of discontinuous sites: a protein modeling strategy.

A strategy for modeling continuous as well as discontinuous sites in protein structures has been developed. Central to this modeling strategy is the search algorithm of FITSITE, a program to search a given target structure for suitable combinations of backbone positions mirroring as closely as possible the geometric relationships of a source structural motif of interest. All target sites detected by FITSITE are further refined to mimic the source geometry. The sidechain rotamer library concept fails to precisely describe side chains involved in coordinative bonding (e.g, metal binding sites). Therefore an algorithm using detailed data-base bonding parameter information was applied for the side-chain construction. The FITSITE program and the subsequent processing of the program output are presented in a test case. The Rop protein, a four-helix bundle structure, served as the target protein. It was searched for candidate sites to model a variety of metal binding sites, with structures extracted from Brookhaven Protein Database entries. The preliminary protein models were investigated for structural overlaps with neighboring residues by interactive computer graphics; if required, additional changes were performed. A set of parameters for energy minimization with AMBER (including metal ions) was developed, and the completed Rop variants were energy minimized. Finally, 12 potentially metal binding Rop variants were selected for production via genetic engineering.