title: Structural and computational characterization of the SHV-1 beta-lactamase-beta-lactamase inhibitor protein interface.
abstract: Beta-lactamase inhibitor protein (BLIP) binds a variety of class A beta-lactamases with affinities ranging from micromolar to picomolar. Whereas the TEM-1 and SHV-1 beta-lactamases are almost structurally identical, BLIP binds TEM-1 approximately 1000-fold tighter than SHV-1. Determining the underlying source of this affinity difference is important for understanding the molecular basis of beta-lactamase inhibition and mechanisms of protein-protein interface specificity and affinity. Here we present the 1.6A resolution crystal structure of SHV-1.BLIP. In addition, a point mutation was identified, SHV D104E, that increases SHV.BLIP binding affinity from micromolar to nanomolar. Comparison of the SHV-1.BLIP structure with the published TEM-1.BLIP structure suggests that the increased volume of Glu-104 stabilizes a key binding loop in the interface. Solution of the 1.8A SHV D104K.BLIP crystal structure identifies a novel conformation in which this binding loop is removed from the interface. Using these structural data, we evaluated the ability of EGAD, a program developed for computational protein design, to calculate changes in the stability of mutant beta-lactamase.BLIP complexes. Changes in binding affinity were calculated within an error of 1.6 kcal/mol of the experimental values for 112 mutations at the TEM-1.BLIP interface and within an error of 2.2 kcal/mol for 24 mutations at the SHV-1.BLIP interface. The reasonable success of EGAD in predicting changes in interface stability is a promising step toward understanding the stability of the beta-lactamase.BLIP complexes and computationally assisted design of tight binding BLIP variants.
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California 92093-0684, USA.
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Reynolds
authors.forename: Jodi M
authors.initials: JM
authors.lastname: Thomson
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Corbett
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Bethel
authors.forename: James M
authors.initials: JM
authors.lastname: Berger
authors.forename: Jack F
authors.initials: JF
authors.lastname: Kirsch
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 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003198:Computer Simulation; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D016415:Sequence Alignment; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors; C065596:beta-lactamase-inhibitor protein, Streptomyces; C054882:beta-lactamase PIT-2; D001618:beta-Lactamases