title: Identification of the β-lactamase inhibitor protein-II (BLIP-II) interface residues essential for binding affinity and specificity for class A β-lactamases.
abstract: The interactions between β-lactamase inhibitory proteins (BLIPs) and β-lactamases have been used as model systems to understand the principles of affinity and specificity in protein-protein interactions. The most extensively studied tight binding inhibitor, BLIP, has been characterized with respect to amino acid determinants of affinity and specificity for binding β-lactamases. BLIP-II, however, shares no sequence or structural homology to BLIP and is a femtomolar to picomolar potency inhibitor, and the amino acid determinants of binding affinity and specificity are unknown. In this study, alanine scanning mutagenesis was used in combination with determinations of on and off rates for each mutant to define the contribution of residues on the BLIP-II binding surface to both affinity and specificity toward four β-lactamases of diverse sequence. The residues making the largest contribution to binding energy are heavily biased toward aromatic amino acids near the center of the binding surface. In addition, substitutions that reduce binding energy do so by increasing off rates without impacting on rates. Also, residues with large contributions to binding energy generally exhibit low temperature factors in the structures of complexes. Finally, with the exception of D206A, BLIP-II alanine substitutions exhibit a similar trend of effect for all β-lactamases, i.e., a substitution that reduces affinity for one β-lactamase usually reduces affinity for all β-lactamases tested.
authors.affiliation: Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: Nicholas G
authors.initials: NG
authors.lastname: Brown
authors.forename: Dar-Chone
authors.initials: DC
authors.lastname: Chow
authors.forename: Kevin E
authors.initials: KE
authors.lastname: Ruprecht
authors.forename: Timothy
authors.initials: T
authors.lastname: Palzkill
mesh_terms: D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D004926:Escherichia coli; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D013302:Streptomyces; D013816:Thermodynamics; D018440:beta-Lactam Resistance; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases
keywords: Alanine Scanning Mutagenesis; Antibiotic Resistance; Beta-Lactam Antibiotic; Beta-Lactamase; Binding Hot Spot; Drug Discovery; Enzyme Inhibitors; Enzyme Kinetics; Molecular Recognition; Protein-Protein Interactions