Noncovalent binding of a cyclic peptide inhibitor to the peptidyl-prolyl isomerase Pin1, explored by hydrogen exchange mass spectrometry

Abstract

Pin1 is a peptidyl-prolyl isomerase (PPIase) that plays a central role in eukaryotic cell cycle regulation, making this protein an interesting target for cancer therapy. Pin1 exhibits high specificity for substrates where proline is preceded by phosphoserine or phosphothreonine. The protein comprises an N-terminal WW (tryptophan–tryptophan) domain and a C-terminal PPIase domain. The cyclic peptide [CRYPEVEIC] (square brackets are used to denote the cyclic structure) represents a lead compound for a new class of nonphosphorylated Pin1 inhibitors. Unfortunately, it has not been possible thus far to characterize the Pin1–[CRYPEVEIC] complex by X-ray crystallography. Thus, the exact binding mode remains unknown. The current work employs hydrogen/deuterium exchange mass spectrometry for gaining insights into the Pin1–[CRYPEVEIC] interactions. The WW domain shows extensive conformational dynamics, both in the presence and in the absence of ligand. In contrast, profound changes in deuteration kinetics are observed in the PPIase domain after the addition of [CRYPEVEIC]. The secondary structure elements β2, α3, and α4 exhibit markedly reduced deuteration, consistent with their postulated involvement in ligand binding. Unexpectedly, [CRYPEVEIC] destabilizes the range of residues 61–86, a segment that comprises basic side chains that normally interact with the substrate phosphate. This destabilization is likely caused by steric clashes with Y3 or E5 of the inhibitor. Ligand-induced destabilization has previously been reported for a few other proteins, but effects of this type are not very common. Our findings suggest that future crystallization trials on Pin1 variants deleted for residues in the 61–86 range might provide a path towards high-resolution X-ray structures of Pin1 bound to cyclic peptide inhibitors.