Structure of Rhomboid Protease in Complex with β-Lactam Inhibitors Defines the S2′ Cavity

Abstract

SummaryRhomboids are evolutionarily conserved serine proteases that cleave transmembrane proteins within the membrane. The increasing number of known rhomboid functions in prokaryotes and eukaryotes makes them attractive drug targets. Here, we describe structures of the Escherichia coli rhomboid GlpG in complex with β-lactam inhibitors. The inhibitors form a single bond to the catalytic serine and the carbonyl oxygen of the inhibitor faces away from the oxyanion hole. The hydrophobic N-substituent of β-lactam inhibitors points into a cavity within the enzyme, providing a structural explanation for the specificity of β-lactams on rhomboid proteases. This same cavity probably represents the S2′ substrate binding site of GlpG. We suggest that the structural changes in β-lactam inhibitor binding reflect the state of the enzyme at an initial stage of substrate binding to the active site. The structural insights from these enzyme-inhibitor complexes provide a starting point for structure-based design for rhomboid inhibitors.

Intramembrane proteases as drug targets

“…One problem may be the plasticity of the IMPs. Inhibitor-rhomboid complexes have revealed that 'recognition pockets' near the active site may only form upon interaction with a substrate or inhibitor [86], which complicates in silico screening of inhibitors.…”

Inhibitors of rhomboid proteases

“…Structures with several other inhibitors have also been elucidated: with DFP (7) [41], the phosphonate CAPF (8) [65], the submicromolar isocoumarin S016 (3) [37], several N-carbamoylated b-lactams (5) [53] and a tetrapeptide chloromethyl ketone (10) [34]. The similarities between the structures are striking and several interesting observations can be made.…”

“…Binding of b-lactams, for example, led to rotation of W236 together with lifting of the L5 cap, opening the S2 0 pocket (Fig. 5G) [53]. A crystal structure of a tetrapeptide chloromethyl ketone, the most substrate-like structure to date, revealed that the S4 subsite adjusts itself depending on the P4 residue, by rearrangements of residues in the L1 loop [34].…”

“…5C) [34,37,41,51,65]. Only in the blactam structures, the oxygen points away from these residues [53]. In all inhibitor-rhomboid complexes, loop L5 that covers the active site is partially lifted or disordered (Fig.…”

“…3C) [43]. The N-sulfonyl group can be replaced by a carbamate group (5) [43,53]; both are electron withdrawing groups that activate the lactam electrophile. Upon reaction with the active site serine, the lactam ring opens, which was corroborated by X-ray crystallography (see paragraph 5) (Fig.…”