Crystal Structure of the IMP-1 Metallo β-Lactamase from Pseudomonas aeruginosa and Its Complex with a Mercaptocarboxylate Inhibitor:  Binding Determinants of a Potent, Broad-Spectrum Inhibitor^,

Abstract

Metallo beta-lactamase enzymes confer antibiotic resistance to bacteria by catalyzing the hydrolysis of beta-lactam antibiotics. This relatively new form of resistance is spreading unchallenged as there is a current lack of potent and selective inhibitors of metallo beta-lactamases. Reported here are the crystal structures of the native IMP-1 metallo beta-lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor, 2-[5-(1-tetrazolylmethyl)thien-3-yl]-N-[2-(mercaptomethyl)-4 -(phenylb utyrylglycine)]. The structures were determined by molecular replacement, and refined to 3.1 A (native) and 2.0 A (complex) resolution. Binding of the inhibitor in the active site induces a conformational change that results in closing of the flap and transforms the active site groove into a tunnel-shaped cavity enclosing 83% of the solvent accessible surface area of the inhibitor. The inhibitor binds in the active site through interactions with residues that are conserved among metallo beta-lactamases; the inhibitor's carboxylate group interacts with Lys161, and the main chain amide nitrogen of Asn167. In the "oxyanion hole", the amide carbonyl oxygen of the inhibitor interacts through a water molecule with the side chain of Asn167, the inhibitor's thiolate bridges the two Zn(II) ions in the active site displacing the bridging water, and the phenylbutyryl side chain binds in a hydrophobic pocket (S1) at the base of the flap. The flap is displaced 2.9 A compared to the unbound structure, allowing Trp28 to interact edge-to-face with the inhibitor's thiophene ring. The similarities between this inhibitor and the beta-lactam substrates suggest a mode of substrate binding and the role of the conserved residues in the active site. It appears that the metallo beta-lactamases bind their substrates by establishing a subset of binding interactions near the catalytic center with conserved characteristic chemical groups of the beta-lactam substrates. These interactions are complemented by additional nonspecific binding between the more variable groups in the substrates and the flexible flap. This unique mode of binding of the mercaptocarboxylate inhibitor in the enzyme active site provides a binding model for metallo beta-lactamase inhibition with utility for future drug design.

The 1.5-Å Structure of Chryseobacterium meningosepticum Zinc β-Lactamase in Complex with the Inhibitor, D-Captopril

“…However, in the presence of an inhibitor, the loop is stabilized (30), transforming the active site groove into a tunnel-shaped cavity. In the BlaB structure, the loop is also well defined, probably due to the presence of D-captopril in the active site (Fig.…”

“…In this context, the analysis of the three-dimensional structures of MBLs in complex with potential inhibitors becomes a powerful tool for rational drug design. At present, the structures of Bacteroides fragilis CcrA complexed with Mes, biphenyl tetrazole, or a tricyclic natural product (32) and of (23,31) Pseudomonas aeruginosa IMP-1 complexed with mercaptocarboxylate (30) or with succinic acid derivatives (33) have been reported.…”

Crystal Structures of Pseudomonas aeruginosa GIM-1: Active-Site Plasticity in Metallo-β-Lactamases

“…The phase problem was solved by PHASER (16) using IMP-1 (Protein Data Bank [PDB] entry 1DDK) as a search model (17), and the structures were refined in Phenix (18) and manually built in WinCoot (19).…”

Structural and functional characterization of Salmonella enterica serovar Typhimurium YcbL: An unusual Type II glyoxalase

“…All family members also contain a binuclear metal binding site, with the zinc b-lactamases containing either a mono or dizinc center. [7][8][9][10] GLX2s contain a mixed zinc-iron binuclear center, 11 whereas the A-type flavoproteins contain a diiron site. 12 Structural alignment suggests that it is the subtle variation in the type of the ligand-coordinating amino acid that provides the metal specificity.…”