Conformational Distributions of Protease−Serpin Complexes: A Partially Translocated Complex†
journal contributionposted on 12.09.2006, 00:00 by Lu Liu, Nicole Mushero, Lizbeth Hedstrom, Anne Gershenson
Serpins regulate serine proteases by forming metastable covalent complexes with their targets. The protease docks with the serpin and cleaves the serpin's reactive center loop (RCL) forming an acylenzyme intermediate. Cleavage triggers insertion of the RCL into β sheet A, translocating the attached protease ∼70 Å and disrupting the protease active site, trapping the acylenzyme intermediate. Using single-pair Förster resonance energy transfer (spFRET), we have measured the conformational distributions of trypsin and α1-proteinase inhibitor (α1PI) covalent complexes. Bovine trypsin (BTryp) complexes display a single set of conformations consistent with the full translocation of BTryp (EfullI*). However, the range of spFRET efficiencies is large, suggesting that the region around the trypsin label is mobile. Most complexes between α1PI variants and the more stable rat trypsin (RTryp) also show a single set of conformations, but the conformational distribution is narrower, indicating less disruption of RTryp. Surprisingly, RTryp complexes containing α1PI labeled at Cys232 with a cationic fluorophore display two equally populated conformations, EfullI* and a conformation in which RTryp is only partially translocated (EpartI*). Destabilizing the RTryp active site, by substituting Ala for Ile16, increases the EfullI* population. Thus, interactions between anionic RTryp and cationic dyes likely exert a restraining force on α1PI, increasing the energy needed to translocate trypsin, and this force can be counteracted by active site destabilization. These results highlight the role of protease stability in determining the conformational distributions of protease−serpin covalent complexes and show that full translocation is not required for the formation of metastable complexes.