Cyclophilin-promoted folding of mouse dihydrofolate reductase does not include the slow conversion of the late-folding intermediate to the active enzyme

Cyclophilins accelerate slow protein folding reactions in vitro by catalyzi ng the cis/trans isomerization of peptidyl-prolyl bonds. Cyclophilins were reported to be involved in a variety of cellular functions, including the p romotion of protein folding by use of the substrate mouse dihdrofolate redu ctase (DHFR). The interaction of cyclophilin with DHFR has only been studie d under limited conditions so far, not taking into account that native DHFR exists in equilibrium with a non-native late-folding intermediate. Here we report a systematic analysis of catalysis of DHFR folding by cyclophilins. The specific ligand methotrexate traps DHFR in its native state, permittin g a specific analysis of the action of cyclophilin on both denatured DHFR w ith non-native prolyl bonds and denatured DHFR with all-native prolyl bonds . Cyclophilins from yeast and Neurospora crassa as well as the related prol yl isomerase b from Escherichia coli promote the folding of different forms of DHFR to the enzymatically active form, demonstrating the generality of cyclophilin-catalyzed folding of DHFR. The slow equilibrium between the lat e-folding intermediate and native DHFR suggests that prolyl isomerization m ay be required for this final phase of conversion to native DHFR. However, by reversible trapping of the intermediate, we analyze the slow interconver sion between native and late-folding conformations in the backward and forw ard reactions and show a complete independence of cyclophilin. We conclude that cyclophilin catalyzes folding of DHFR, but surprisingly not in the las t slow folding step. (C) 2000 Academic Press.