THE MUTANT ESCHERICHIA-COLI F112W CYCLOPHILIN BINDS CYCLOSPORINE-A INNEARLY IDENTICAL CONFORMATION AS HUMAN CYCLOPHILIN

The periplasmic Escherichia coil cyclophilin is distantly related to h uman cyclophilin (34% sequence identity). Peptidyl-prolyl isomerase ac tivity, cyclosporin A binding, and inhibition of the calcium-dependent phosphatase calcineurin are compared for human and E. coil wild-type and mutant proteins. Like human cyclophilin, the E. coil protein is a cis-trans peptidyl-prolyl isomerase. However, while the human protein binds cyclosporin A tightly (K-d 17 nM), the E. coil protein does not (K-d 3.4 mu M). The mutant F112W E. coil cyclophilin has enhanced cycl osporin binding (K-d 170 nM). As for the human protein, the complex of the E. coli mutant with cyclosporin A inhibits calcineurin. Here we d escribe the structure at pH 6.2 of cyclosporin A bound to the mutant E . coil cyclophilin as solved with solution NMR methods. Despite the lo w overall sequence identity, the structure of the bound cyclosporin A is virtually identical in both proteins. To assess differences of the cyclosporin binding site, the solution structure of wild-type E. coil cyclophilin was compared with structures of uncomplexed human cyclophi lin A and with cyclosporin bound. Despite the structural similarity of bound cyclosporin A, the architecture of the binding site in the E. c oil protein is substantially different at the site most distant to try ptophan 121 (human sequence). This site is constructed by a five-resid ue insertion in a loop of the E. coil protein, replacing another loop in the human protein.