Crystal Structure of the IMP-1 Metallo -Lactamase from Pseudomonas aeruginosa and Its Complex with a Mercaptocarboxylate Inhibitor: Binding Determinants of a Potent, Broad-Spectrum Inhibitor ²,‡ Ne ´stor O. Concha,* ,§ Cheryl A. Janson, | Pam Rowling, | Stewart Pearson, ⊥ Christy A. Cheever, ⊥ Brian P. Clarke, # Ceri Lewis, | Moreno Galleni, @ Jean-Marie Fre `re, @ David J. Payne, ⊥ John H. Bateson, # and Sherin S. Abdel-Meguid § Departments of Structural Biology, Protein Biochemistry, Anti-InfectiVes, and Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, PennsylVania 19406, and Laboratoire d’Enzymologie & Centre d’Inge ´ nierie des Prote ´ ines, Institut de Chimie, B6 Sart Tilman, UniVersite ´ de Lie ` ge, B-4000 Lie ` ge, Belgium ReceiVed NoVember 8, 1999; ReVised Manuscript ReceiVed January 31, 2000 ABSTRACT: Metallo -lactamase enzymes confer antibiotic resistance to bacteria by catalyzing the hydrolysis of -lactam antibiotics. This relatively new form of resistance is spreading unchallenged as there is a current lack of potent and selective inhibitors of metallo -lactamases. Reported here are the crystal structures of the native IMP-1 metallo -lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor, 2-[5-(1-tetrazolylmethyl)thien-3-yl]-N-[2-(mercaptomethyl)-4-(phenyl- butyrylglycine)]. The structures were determined by molecular replacement, and refined to 3.1 Å (native) and 2.0 Å (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 -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 Å 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 -lactam substrates suggest a mode of substrate binding and the role of the conserved residues in the active site. It appears that the metallo -lactamases bind their substrates by establishing a subset of binding interactions near the catalytic center with conserved characteristic chemical groups of the -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 -lactamase inhibition with utility for future drug design. Bacterial resistance to the most common and potent anti- biotics has been rising in recent years (1). Selective pressure in hospitals and infant- and child-care facilities is selecting pathogenic microorganisms carrying resistance genes. Re- sistance against antibiotics occurs through a variety of mech- anisms, one of which is the production of -lactamases (2, 3). -Lactamases catalyze the hydrolysis of the amide bond of the -lactam ring to produce the corresponding -amino acid devoid of antibacterial activity (4). Of the four structural classes of -lactamases (5-7), classes A, C, and D catalyze the hydrolysis of -lactams using a serine-dependent mech- anism and proceeding through an acyl-enzyme intermediate (8-10). Class B enzymes, the metallo -lactamases, require zinc, cobalt, cadmium, or manganese for catalysis (11, 12) and hydrolyze most classes of -lactams, including the broad- spectrum carbapenems (13, 14). First discovered in Bacillus cereus (12), metallo -lactamase activity has now been found in several pathogenic species of Bacteroides (15-20), Pseudomonas (21, 22), Stenotrophomonas (23), Aeromonas (24), Chryseobacterium (25), Serratia (26), Klebsiella (27), and Shigella (28). Unlike the serine -lactamases, metallo -lactamases are not inhibited by the classic -lactamase inhibitors clavulanic acid, sulbactam, and tazobactam, but ² Work by M.G. and J.-M.F. was funded in part by EU Grant ERB- FMRX-CT98-0232. ‡ The atomic coordinates of the native and complex crystal structures have been deposited in the Protein Data Bank (accession codes 1DD6 and 1DDK, respectively). * To whom correspondence should be addressed: Mail Code UE0447, 709 Swedeland Rd., King of Prussia, PA 19406. Telephone: (610) 270-7462. Fax: (610) 270-4091. E-mail: nestor_o_concha@ sbphrd.com. § Department of Structural Biology, SmithKline Beecham Pharma- ceuticals. | Department of Protein Biochemistry, SmithKline Beecham Phar- maceuticals. ⊥ Department of Anti-Infectives, SmithKline Beecham Pharmaceu- ticals. # Department of Medicinal Chemistry, SmithKline Beecham Phar- maceuticals. @ Universite ´ de Lie `ge. 4288 Biochemistry 2000, 39, 4288-4298 10.1021/bi992569m CCC: $19.00 © 2000 American Chemical Society Published on Web 03/25/2000