Dissecting the Microscopic Steps of the Cyclophilin A Enzymatic Cycle on the Biological HIV-1 Capsid Substrate by NMR

Peptidyl–prolyl isomerases (PPIases) are emerging as key regulators of many diverse biological processes. Elucidating the role of PPIase activity in vivo has been challenging because mutagenesis of active-site residues not only reduces the catalytic activity of these enzymes but also dramatically affects substrate binding. Employing the cyclophilin A PPIase together with its biologically relevant and natively folded substrate, the N-terminal domain of the human immunodeficiency virus type 1 capsid (CAN) protein, we demonstrate here how to dissect residue-specific contributions to PPIase catalysis versus substrate binding utilizing NMR spectroscopy. Surprisingly, a number of cyclophilin A active-site mutants previously assumed to be strongly diminished in activity toward biological substrates based only on a peptide assay catalyze the human immunodeficiency virus capsid with wild-type activity but with a change in the rate-limiting step of the enzymatic cycle. The results illustrate that a quantitative analysis of catalysis using the biological substrates is critical when interpreting the effects of PPIase mutations in biological assays. [Display omitted] ► Cyclophilin A binding and catalysis can be quantified by NMR exchange experiments. ► Cyclophilin A binding and catalysis is examined with a biological protein substrate. ► Several cyclophilin A mutants unexpectedly retain wild-type catalytic efficiency.