Grafting of discontinuous sites: a protein modeling strategy

A strategy for modeling continuous as well as discontinuoussites in protein structures has been developed. Central to thismodeling strategy is the search algorithm of FITSITE, a programto search a given target structure for suitable combinationsof backbone positions mirroring as closely as possible the geometricrelationships of a source structural motif of interest. Alltarget sites detected by FITSITE are further refined to mimicthe source geometry. The side-chain rotamer library conceptfails to precisely describe side chains involved in coordinativebonding (e.g. metal binding sites). Therefore an algorithm usingdetailed database bonding parameter information was appliedfor the side-chain construction. The FITSITE program and thesubsequent processing of the program output are presented ina test case. The Rop protein, a four-helix bundle structure,served as the target protein. It was searched for candidatesites to model a variety of metal binding sites, with structuresextracted from Brookhaven Protein Database entries. The preliminaryprotein models were investigated for structural overlaps withneighboring residues by interactive computer graphics; if required,additional changes were performed. A set of parameters for energyminimization with AMBER (including metal ions) was developed,and the completed Rop variants were energy minimized. Finally,12 potentially metal binding Rop variants were selected forproduction via genetic engineering.