Crystal structure of the IMP-1 metallo beta-lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor: Binding determinants of a potent, broad-spectrum inhibitor

Metallo beta-lactamase enzymes confer antibiotic resistance to bacteria by catalyzing the hydrolysis of beta-lactam antibiotics. This relatively new f orm of resistance is spreading unchallenged as there is a current lack of p otent and selective inhibitors of metallo beta-lactamases. Reported here ar e the crystal structures of the native IMP-1 metailo beta-lactamase from Ps eudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor, 2-[5-(1-tetrazolylmethyl)thien-3-yl]-N-[2-(mer butyrylglycine)]. The struct ures were determined by molecular replacement, and refined to 3.1 Angstrom (native) and 2.0 Angstrom (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 g roup 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 thi olate bridges the two Zn(II) ions in the active site displacing the bridgin g water, and the phenlbutyryl side chain binds in a hydrophobic pocket (S1) at the base of the flap. The flap is displaced 2.9 Angstrom compared to th e unbound structure, allowing Trp28 to interact edge-to-face with the inhib itor'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-lac tamases bind their substrates by establishing a subset of binding interacti ons near the catalytic center with conserved characteristic chemical groups of the beta-lactam substrates, These interactions are complemented by addi tional nonspecific binding between the more variable groups in the substrat es and the flexible flap. This unique mode of binding of the mercaptocarbox ylate inhibitor in the enzyme active site provides a binding model for meta llo beta-lactamase inhibition with utility for future drug design.