The human rotamase or peptidyl-prolyl cis-trans isomerase Pin1 is a conserved mitotic regulator essential for the G2/M transition of the eukaryotic cell cycle. We report the 1.35 A crystal structure of Pin1 complexed with an AlaPro dipeptide and the initial characterization of Pin1's functional properties. The crystallographic structure as well as pH titration studies and mutagenesis of an active site cysteine suggest a catalytic mechanism that includes general acid-base and covalent catalysis during peptide bond isomerization. Pin1 displays a preference for an acidic residue N-terminal to the isomerized proline bond due to interaction of this acidic side chain with a basic cluster. This raises the possibility of phosphorylation-mediated control of Pin1-substrate interactions in cell cycle regulation.

Pin1 is an essential and conserved mitotic peptidyl-prolyl isomerase (PPIase) that is distinct from members of two other families of conventional PPIases, cyclophilins and FKBPs (FK-506 binding proteins). In response to their phosphorylation during mitosis, Pin1 binds and regulates members of a highly conserved set of proteins that overlaps with antigens recognized by the mitosis-specific monoclonal antibody MPM-2. Pin1 is here shown to be a phosphorylation-dependent PPIase that specifically recognizes the phosphoserine-proline or phosphothreonine-proline bonds present in mitotic phosphoproteins. Both Pin1 and MPM-2 selected similar phosphorylated serine-proline-containing peptides, providing the basis for the specific interaction between Pin1 and MPM-2 antigens. Pin1 preferentially isomerized proline residues preceded by phosphorylated serine or threonine with up to 1300-fold selectivity compared with unphosphorylated peptides. Pin1 may thus regulate mitotic progression by catalyzing sequence-specific and phosphorylation-dependent proline isomerization.