A Relay Mechanism in Rhomboid Intramembrane Protease

Abstract

Intramembrane proteases are a family of highly conserved membrane proteins that cleave other transmembrane (TM) helical segments within the plane of the lipid membrane. GlpG rhomboid protease, the best characterized of these intramembrane proteases, has six helical segments and a unique loop lying in the plane of the membrane. TM5 acts as the lateral gate that opens to allow docking of the incoming substrate; the role of the L1 loop, which extends away from the active site, is uncertain. Site-directed mutagenesis experiments have identified a triple serine L1 mutant (Y138S/F139S/L143S) with a significantly reduced catalytic activity, and a triple valine mutant of the TM5 gate (L229V/F232V/W236V) with enhanced activity relative to the wild-type protease (Baker et al, Proc. Natl. Acad. Sci. USA 104, 8257-8262, 2007). To dissect the roles of TM5 and L1, we performed all-atom molecular dynamics simulations of the L1 and TM5 mutants in hydrated bilayers of 1-palmytoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE). The results reveal a relay mechanism that transmits structural and dynamical perturbations between the remote TM5/L1 structural elements of the protease. Perturbation of L1 is transmitted to the active site and TM5 via intra-protein hydrogen bonds to which conserved amino acid residues contribute. Likewise, perturbation of TM5 leads to changes in protein dynamics and local structural rearrangements of the remote L1 loop. In the (inactive) L1 triple serine mutant, but not in the (highly active) TM5 triple valine mutant, several intra-protein interactions become locked in a new geometry. The communication between L1 and the TM5 helical gate TM5 suggests a regulatory role for loop L1.This work was supported by research grants from the National Institute for General Medical Sciences and the NIH National Center for Research Resources.