Interaction between the P14 residue and strand 2 of beta-sheet B is critical for reactive center loop insertion in plasminogen activator inhibitor-2

The molecular interactions driving reactive center loop (RCL) insertion are of considerable interest in gaining a better understanding of the serpin i nhibitory mechanism. Previous studies have suggested that interactions in t he proximal hinge/breach region may be critical determinants of RCL inserti on in serpins. In this study, conformational and functional changes in plas minogen activator inhibitor-2 (PAI-2) following incubation with a panel of synthetic RCL peptides indicated that the P14 residue is critical for RCL i nsertion, and hence inhibitory activity, in PAI-2. Only RCL peptides with a P14 threonine were able to induce the stressed to relaxed transition and a bolish inhibitory activity in PAI-2, indicating that RCL insertion into bet a -sheet A of PAI-2 is dependent upon this residue. The recently solved cry stal structure of relaxed PAI-2 (PAI-2-RCL peptide complex) allowed detaile d analysis of molecular interactions involving P14 related to RCL insertion . Of most interest is the rearrangement of hydrogen bonding around the brea ch region that accompanies the stressed to relaxed transition, in particula r the formation of a side chain hydrogen bond between the threonine at P14 and an adjacent tyrosine on strand 2 of beta -sheet B in relaxed PAI-2. Str uctural alignment of known serpin sequences showed that this pairing (or th e equivalent serine/threonine pairing) is highly conserved (similar to 87%) in inhibitory serpins and may represent a general structural basis for ser pin inhibitory activity.