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Systematic substitutions at BLIP position 50 result in changes in binding specificity for class A ?-lactamases.

ABSTRACT: The production of ?-lactamases by bacteria is the most common mechanism of resistance to the widely prescribed ?-lactam antibiotics. ?-lactamase inhibitory protein (BLIP) competitively inhibits class A ?-lactamases via two binding loops that occlude the active site. It has been shown that BLIP Tyr50 is a specificity determinant in that substitutions at this position result in large differential changes in the relative affinity of BLIP for class A ?-lactamases.In this study, the effect of systematic substitutions at BLIP position 50 on binding to class A ?-lactamases was examined to further explore the role of BLIP Tyr50 in modulating specificity. The results indicate the sequence requirements at position 50 are widely different depending on the target ?-lactamase. Stringent sequence requirements were observed at Tyr50 for binding Bacillus anthracis Bla1 while moderate requirements for binding TEM-1 and relaxed requirements for binding KPC-2 ?-lactamase were seen. These findings cannot be easily rationalized based on the ?-lactamase residues in direct contact with BLIP Tyr50 since they are identical for Bla1 and KPC-2 suggesting that differences in the BLIP-?-lactamase interface outside the local environment of Tyr50 influence the effect of substitutions.Results from this study and previous studies suggest that substitutions at BLIP Tyr50 may induce changes at the interface outside its local environment and point to the complexity of predicting the impact of substitutions at a protein-protein interaction interface.

SUBMITTER: Adamski CJ

PROVIDER: S-EPMC5340008 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Systematic substitutions at BLIP position 50 result in changes in binding specificity for class A β-lactamases.

Adamski Carolyn J CJ Palzkill Timothy T

BMC biochemistry 20170306 1

<h4>Background</h4>The production of β-lactamases by bacteria is the most common mechanism of resistance to the widely prescribed β-lactam antibiotics. β-lactamase inhibitory protein (BLIP) competitively inhibits class A β-lactamases via two binding loops that occlude the active site. It has been shown that BLIP Tyr50 is a specificity determinant in that substitutions at this position result in large differential changes in the relative affinity of BLIP for class A β-lactamases.<h4>Results</h4>I ...[more]

PMID: 28264645

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Project description:Since the discovery and use of penicillin, the increase of antibiotic resistance among bacterial pathogens has become a major health concern. The most prevalent resistance mechanism in Gram-negative bacteria is due to ?-lactamase expression. Class D ?-lactamases are of particular importance due to their presence in multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa. The class D enzymes were initially characterized by their ability to efficiently hydrolyze isoxazolyl-type ?-lactams like oxacillin. Due to this substrate preference, these enzymes are traditionally referred to as oxacillinases or OXAs. However, this class is comprised of subfamilies characterized by diverse activities that include oxacillinase, carbapenemase, or cephalosporinase substrate specificity. OXA-1 represents one subtype of class D enzyme that efficiently hydrolyzes oxacillin, and OXA-24/40 represents another with weak oxacillinase, but increased carbapenemase, activity. To examine the structural basis for the substrate selectivity differences between OXA-1 and OXA-24/40, the X-ray crystal structures of deacylation-deficient mutants of these enzymes (Lys70Asp for OXA-1; Lys84Asp for OXA-24) in complexes with oxacillin were determined to 1.4 Å and 2.4 Å, respectively. In the OXA-24/40/oxacillin structure, the hydrophobic R1 side chain of oxacillin disrupts the bridge between Tyr112 and Met223 present in the apo OXA-24/40 structure, causing the main chain of the Met223-containing loop to adopt a completely different conformation. In contrast, in the OXA-1/oxacillin structure, a hydrophobic pocket consisting of Trp102, Met99, Phe217, Leu161, and Leu255 nicely complements oxacillin's nonpolar R1 side chain. Comparison of the OXA-1/oxacillin and OXA-24/40/oxacillin complexes provides novel insight on how substrate selectivity is achieved among subtypes of class D ?-lactamases. By elucidating important active site interactions, these findings can also inform the design of novel antibiotics and inhibitors.

Dataset Information

Systematic substitutions at BLIP position 50 result in changes in binding specificity for class A ?-lactamases.

Publications

Systematic substitutions at BLIP position 50 result in changes in binding specificity for class A β-lactamases.

Similar Datasets

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