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A DNA aptamer reveals an allosteric site for inhibition in metallo-?-lactamases.

ABSTRACT: The hydrolysis of ?-lactam antibiotics by ?-lactamase enzymes is the most prominent antibiotic resistance mechanism for many pathogenic bacteria. Out of this broad class of enzymes, metallo-?-lactamases are of special clinical interest because of their broad substrate specificities. Several in vitro inhibitors for various metallo-?-lactamases have been reported with no clinical efficacy. Previously, we described a 10-nucleotide single stranded DNA aptamer (10-mer) that inhibits Bacillus cereus 5/B/6 metallo-?-lactamase very effectively. Here, we find that the aptamer shows uncompetitive inhibition of Bacillus cereus 5/B/6 metallo-?-lactamase during cefuroxime hydrolysis. To understand the mechanism of inhibition, we report a 2.5 Å resolution X-ray crystal structure and solution-state NMR analysis of the free enzyme. Chemical shift perturbations were observed in the HSQC spectra for several residues upon titrating with increasing concentrations of the 10-mer. In the X-ray crystal structure, these residues are distal to the active site, suggesting an allosteric mechanism for the aptamer inhibition of the enzyme. HADDOCK molecular docking simulations suggest that the 10-mer docks 26 Å from the active site. We then mutated the three lysine residues in the basic binding patch to glutamine and measured the catalytic activity and inhibition by the 10-mer. No significant inhibition of these mutants was observed by the 10-mer as compared to wild type. Interestingly, mutation of Lys50 (Lys78; according to standard MBL numbering system) resulted in reduced enzymatic activity relative to wild type in the absence of inhibitor, further highlighting an allosteric mechanism for inhibition.

SUBMITTER: Khan NH

PROVIDER: S-EPMC6476477 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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A DNA aptamer reveals an allosteric site for inhibition in metallo-β-lactamases.

Khan Nazmul H NH Bui Anthony A AA Xiao Yang Y Sutton R Bryan RB Shaw Robert W RW Wylie Benjamin J BJ Latham Michael P MP

PloS one 20190422 4

The hydrolysis of β-lactam antibiotics by β-lactamase enzymes is the most prominent antibiotic resistance mechanism for many pathogenic bacteria. Out of this broad class of enzymes, metallo-β-lactamases are of special clinical interest because of their broad substrate specificities. Several in vitro inhibitors for various metallo-β-lactamases have been reported with no clinical efficacy. Previously, we described a 10-nucleotide single stranded DNA aptamer (10-mer) that inhibits Bacillus cereus 5 ...[more]

PMID: 31009467

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Project description:BackgroundGenes conferring antibiotic resistance to groups of bacterial pathogens are cause for considerable concern, as many once-reliable antibiotics continue to see a reduction in efficacy. The recent discovery of the metallo β-lactamase blaNDM-1 gene, which appears to grant antibiotic resistance to a variety of Enterobacteriaceae via a mobile plasmid, is one example of this distressing trend. The following work describes a computational analysis of pathogen-borne MBLs that focuses on the structural aspects of characterized proteins.ResultsUsing both sequence and structural analyses, we examine residues and structural features specific to various pathogen-borne MBL types. This analysis identifies a linker region within MBL-like folds that may act as a discriminating structural feature between these proteins, and specifically resistance-associated acquirable MBLs. Recently released crystal structures of the newly emerged NDM-1 protein were aligned against related MBL structures using a variety of global and local structural alignment methods, and the overall fold conformation is examined for structural conservation. Conservation appears to be present in most areas of the protein, yet is strikingly absent within a linker region, making NDM-1 unique with respect to a linker-based classification scheme. Variability analysis of the NDM-1 crystal structure highlights unique residues in key regions as well as identifying several characteristics shared with other transferable MBLs.ConclusionsA discriminating linker region identified in MBL proteins is highlighted and examined in the context of NDM-1 and primarily three other MBL types: IMP-1, VIM-2 and ccrA. The presence of an unusual linker region variant and uncommon amino acid composition at specific structurally important sites may help to explain the unusually broad kinetic profile of NDM-1 and may aid in directing research attention to areas of this protein, and possibly other MBLs, that may be targeted for inactivation or attenuation of enzymatic activity.

Dataset Information

A DNA aptamer reveals an allosteric site for inhibition in metallo-?-lactamases.

Publications

A DNA aptamer reveals an allosteric site for inhibition in metallo-β-lactamases.

Similar Datasets

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