title: Structural and biochemical characterization of the interaction between KPC-2 beta-lactamase and beta-lactamase inhibitor protein.
abstract: KPC beta-lactamases hydrolyze the "last resort" beta-lactam antibiotics (carbapenems) used to treat multidrug resistant infections and are compromising efforts to combat life-threatening Gram-negative bacterial infections in hospitals worldwide. Consequently, the development of novel inhibitors is essential for restoring the effectiveness of existing antibiotics. The beta-lactamase inhibitor protein (BLIP) is a competitive inhibitor of a number of class A beta-lactamases. In this study, we characterize the previously unreported interaction between KPC-2 beta-lactamase and BLIP. Biochemical results show that BLIP is an extremely potent inhibitor of KPC enzymes, binding KPC-2 and KPC-3 with subnanomolar affinity. To understand the basis of affinity and specificity in the beta-lactamase-BLIP system, the crystallographic structure of the KPC-2-BLIP complex was determined to 1.9 A resolution. Computational alanine scanning was also conducted to identify putative hot spots in the KPC-2-BLIP interface. Interestingly, the two complexes making up the KPC-2-BLIP asymmetric unit are distinct, and in one structure, the BLIP F142 loop is absent, in contrast to homologous structures in which it occupies the active site. This finding and other sources of structural plasticity appear to contribute to BLIP's promiscuity, enabling it to respond to mutations at the beta-lactamase interface. Given the continuing emergence of antibiotic resistance, the high-resolution KPC-2-BLIP structure will facilitate its use as a template for the rational design of new inhibitors of this problematic enzyme.
authors.affiliation: Biophysics Graduate Group, University of California, Berkeley, California 94729, USA.
authors.forename: Melinda S
authors.initials: MS
authors.lastname: Hanes
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Jude
authors.forename: James M
authors.initials: JM
authors.lastname: Berger
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bonomo
authors.forename: Tracy M
authors.initials: TM
authors.lastname: Handel
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000502:physiology; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry; D007711:Klebsiella pneumoniae / Q000201:enzymology; D008969:Molecular Sequence Data; D011487:Protein Conformation; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry* / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors; C065596:beta-lactamase-inhibitor protein, Streptomyces; C491478:beta-lactamase KPC-2; C054882:beta-lactamase PIT-2; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1