Dissecting the protein-protein interface between beta-lactamase inhibitory protein and class A beta-lactamases.
beta-Lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A beta-lactamases at a wide range of affinities. Alanine-scanning mutagenesis was previously performed to identify the amino acid sequence requirements of BLIP for inhibiting TEM-1 beta-lactamase and SME-1 beta-lactamase. Two hotspots of binding energy, one from each domain of BLIP, were identified (Zhang, Z., and Palzkill, T. (2003) J. Biol. Chem. 278, 45706-45712). This study has been extended to examine the amino acid sequence requirements of BLIP for binding to the SHV-1 beta-lactamase, which is a poor binding substrate (Ki= 1.1 microm), and the Bacillus anthracis Bla1 enzyme (Ki= 2.5 nm). The two hotspots previously identified as important for binding TEM-1 and SME-1 beta-lactamase were also found to be important for binding Bla1. The hotspot from the second domain of BLIP, however, does not make substantial contributions to SHV-1 binding. This may explain why BLIP binds to SHV-1 beta-lactamase with much weaker affinity than to the other three enzymes. Three regions, including two loops that insert into the active pocket of TEM-1 beta-lactamase and the Glu-73-Lys-74 buried charge motif, exhibit strikingly different effects on the binding affinity of BLIP toward the various enzymes when mutated and, therefore, act as specificity determinants. Analysis of double mutants of BLIP that combine specificity-determining residues suggests that these residues contribute to the poor affinity between the second domain of BLIP and SHV-1 beta-lactamase.
Structural and Computational Biology and Molecular Biophysics Program, Baylor College of Medicine, Houston, Texas 77030, USA.
Zhen
Z
Zhang
Timothy
T
Palzkill
D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D001408:Bacillus anthracis / Q000378:metabolism; D003001:Cloning, Molecular; D005998:Glycine / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013816:Thermodynamics; D001618:beta-Lactamases / Q000737:chemistry* / Q000145:classification / Q000378:metabolism
C094219:beta-lactamase Sme-1; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1; D000409:Alanine; D005998:Glycine