Mechanistic insights into intramembrane proteolysis by E. coli site-2 protease homolog RseP

Abstract

Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including Escherichia coli RseP. Structure-based chemical modification and cross-linking experiments indicated that the RseP domains surrounding the active center undergo conformational changes to expose the substrate-binding site, suggesting that RseP has a gating mechanism to regulate substrate entry. Furthermore, mutational analysis suggests that a conserved electrostatic linkage between the transmembrane and peripheral membrane-associated domains mediates the conformational changes. In vivo cleavage assays also support that the substrate transmembrane helix is unwound by strand addition to the intramembrane β sheet of RseP and is clamped by a conserved asparagine residue at the active center for efficient cleavage. This mechanism underlying the substrate binding, i.e., unwinding and clamping, appears common across distinct families of intramembrane proteases that cleave transmembrane segments.

In vivo characterization of the bacterial intramembrane-cleaving protease RseP using the heme binding tag-based assay iCliPSpy

“…Molecular dynamics (MD) simulations of the AlphaFold 29 , 30 structure of RseP (with bound Zn 2+ ion) in a lipid bilayer with a lipid composition mimicking E. coli membranes show that residue Tyr69 is located in the membrane/cytosol environment near the active site and may play a role in positioning the substrate peptide through hydrophobic interactions. These results are in agreement with the recently published crystal structure of RseP and the model proposed for substrate binding, in which the so-called edge strand of RseP (residues Gly67 to Val70 including Tyr69 and part of the β-MRE) forms an antiparallel β-sheet with the substrate TMD 22 . Our data show that iCliPSpy is a simple and sensitive assay to identify and study I-CLiP activities in vivo.…”

“…Next, we focused on a more detailed characterization of residues Tyr69 and Tyr428 by MD simulations and site-directed mutagenesis. Residue Tyr428 of TMD 4 was included in our studies because it is localized on the opposite side of Tyr69 at the putative substrate binding groove, and Tyr69 and Tyr428 together may work in gating substrates 22 into the binding groove (Fig. 6d ).…”

“…6f and Supplementary Fig. 5 ), because the pdb file for the crystal structure of RseP 22 was only available as we were working on the revised version of the manuscript but not in the beginning of our studies. Later, it also turned out that the AlphaFold structure prediction for the membrane core of RseP is very similar to the published crystal structure of RseP (Supplementary Fig.…”

“…When the photoreactive hydrophobic amino acid, p-benzoyl-L-phenylalanine (pBPA) was incorporated at position 69, 71 or 74 of RseP ( e.g . RseP Y69pBPA), RseP retained its proteolytic activity and the substrate RseA was photo-crosslinked to RseP, leading to a model in which the β-MRE binds to substrate TMDs and stabilizes their extended conformation 19 , 20 , 22 . In the recently proposed mechanism for substrate accommodation and cleavage in RseP, it is assumed that RseP is in equilibrium between a gate-open and gate-closed conformation, that the substrate is bound by the edge residues in the gate-open conformation, and that in a final step the transmembrane segment of the bound substrate is extended by the edge strand to promote proteolytic cleavage 22 .…”

“…In addition, the GFG motif of RseP and a membrane-bound amphiphilic helix (residues Pro323 to Thr350) contribute to substrate discrimination and binding 20 , 21 . The recently deposited crystal structure of RseP with the inhibitor batimastat provides mechanistic insights into intramembrane proteolysis and suggests a gating mechanism to regulate substrate entry 22 .…”