Abstract
The hydrolysis of beta-lactam antibiotics by class A beta-lactamases is a common cause of bacterial resistance to these agents. The beta-lactamase inhibitory protein (BLIP) is able to bind and inhibit several class A beta-lactamases, including TEM-1 beta-lactamase and SME-1 beta-lactamase. Although the TEM-1 and SME-1 enzymes share 33% amino acid sequence identity and a similar fold, they differ substantially in surface electrostatic properties and the conformation of a loop-helix region that BLIP binds. Alanine-scanning mutagenesis was performed to identify the residues on BLIP that contribute to its binding affinity for each of these enzymes. The results indicate that the sequence requirements for binding are similar for both enzymes with most of the binding free energy provided by two patches of aromatic residues on the surface of BLIP. Polar residues such as several serines in the interface do not make significant contributions to affinity for either enzyme. In addition, the specificity of binding is significantly altered by mutation of two charged residues, Glu73 and Lys74, that are buried in the structure of the TEM-1.BLIP complex as well as by residues located on two loops that insert into the active site pocket. Based on the results, a E73A/Y50A double mutant was constructed that exhibited a 220,000-fold change in binding specificity for the TEM-1 versus SME-1 enzymes.
Highlights
Protein-protein interactions play a central role in many cellular processes, such as signal transduction, the immune response, and biochemical regulation of enzyme function
The specificity of binding is significantly altered by mutation of two charged residues, Glu73 and Lys74, that are buried in the structure of the TEM-11⁄7BLIP complex as well as by residues located on two loops that insert into the active site pocket
It was found that the specificity of binding is significantly altered by mutation of two charged residues, Glu73 and Lys74, that are buried in the structure of the TEM-11⁄7BLIP complex as well as by residues located on two loops that insert into the active site pocket
Summary
CTT GC a The coding sequences for these mutants overlap with MALbli-2 primer. these constructions were performed by one-step PCR with the PDbla-1 primer and modified Malbli-2 primers as described in Experimental Procedures. The apparent binding constants (Ki) of wild type BLIP and 23 alanine mutants for the interaction with TEM-1 and SME-1 were determined using a kinetic assay of -lactamase inhibition. It was found that the specificity of binding is significantly altered by mutation of two charged residues, Glu and Lys, that are buried in the structure of the TEM-11⁄7BLIP complex as well as by residues located on two loops that insert into the active site pocket. This information was used to engineer a BLIP molecule with a 220,000-fold change in binding specificity relative to wild type
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