The structural basis for catalysis and substrate specificity of a rhomboid protease

Abstract

Rhomboids are intramembrane proteases that use a catalytic dyad of serine and histidine for proteolysis. They are conserved in both prokaryotes and eukaryotes and regulate cellular processes as diverse as intercellular signalling, parasitic invasion of host cells, and mitochondrial morphology. Their widespread biological significance and consequent medical potential provides a strong incentive to understand the mechanism of these unusual enzymes for identification of specific inhibitors. In this study, we describe the structure of Escherichia coli rhomboid GlpG covalently bound to a mechanism-based isocoumarin inhibitor. We identify the position of the oxyanion hole, and the S 1 -and S 2 0 -binding subsites of GlpG, which are the key determinants of substrate specificity. The inhibitor-bound structure suggests that subtle structural change is sufficient for catalysis, as opposed to large changes proposed from previous structures of unliganded GlpG. Using bound inhibitor as a template, we present a model for substrate binding at the active site and biochemically test its validity. This study provides a foundation for a structural explanation of rhomboid specificity and mechanism, and for inhibitor design.

Catalytic Mechanism of Rhomboid Protease GlpG Probed by 3,4-Dichloroisocoumarin and Diisopropyl Fluorophosphonate

“…The crystal structure of a complex between GlpG and 7-amino-4-chloro-3-methoxyisocoumarin was reported recently (37). This represents a major breakthrough because it confirms structurally the acyl-enzyme mechanism for rhomboid protease.…”

“…3C). The flipping of the indole ring created a cavity between Trp-236 and Asn-154, which was proposed to constitute the binding subsite for the P2Ј side chain in the peptide substrate (37). In the DFP structure, this cavity is not present, and insertion of a hydrophobic side chain would have to disrupt the hydrogen bond between Trp-236 and Asn-154.…”

“…In the isocoumarin structure, His-254 is covalently bonded to the methyl acetate group of the inhibitor (37) (Fig. 3C): the phenol ring of Tyr-205 rotated toward the tryptophan and is hydrogenbonded to its backbone oxygen, and the indole ring of the tryptophan flipped outwards to make contact with Phe-153.…”

“…The side (20). F, structure of the isocoumarin complex (Protein Data Bank code 2XOW) (37). Note that the inhibitor is covalently bonded to both Ser-201 and His-254.…”

Topological constraints and modular structure in the folding and functional motions of GlpG, an intramembrane protease

“…We performed molecular dynamics simulations of a coarse-grained structure-based model (26) of GlpG based on the crystal structure with Protein Data Bank (PDB) ID code 2XOV (27). We carried out two parallel sets of simulations: one with an implicit membrane present and one without a membrane.…”