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

Abstract

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/B/6 metallo-β-lactamase very effectively. Here, we find that the aptamer shows uncompetitive inhibition of Bacillus cereus 5/B/6 metallo-β-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.

Discovery of a Novel Natural Allosteric Inhibitor That Targets NDM-1 Against Escherichia coli

“…In the complex, the binding sites of residues were Thr76, Lys78, Phe103, Lys104, Lys107, and Tyr208. In our work, the NDM-1-CA complex allosteric sites Phe46, Tyr64, Leu65, Asp66, and Thr94 were mainly located in L3 and α1 helix, which are obviously different with the results of the previous literatures ( Sohier et al, 2013 ; Khan et al, 2019 ; Supplementary Figure S1 ). Therefore, we believed that the binding site of the NDM-1-CA complex is the novel allosteric site of NDM-1.…”

“…The residues of T107YVF110 contributed the major binding affinity for the ligand binding with the protein. In addition, DNA aptamer can be used as an allosteric inhibitor to bind Loop 4 and Loop 6 of 5/B/6 metallo-β-lactamase ( Khan et al, 2019 ). In the complex, the binding sites of residues were Thr76, Lys78, Phe103, Lys104, Lys107, and Tyr208.…”

“…In addition, Chen Cheng et al found that disulfiram is a promising candidate for the development of NDM-1 inhibitors, which can covalently bind to NDM-1 by forming an S–S bond with Cys208 residue at the active site ( Chen et al, 2020b ). At the same time, some metal complexes (cisplatin and Palladium(II) complexes) ( Chen et al, 2020a ) and DNA aptamers ( Khan et al, 2019 ) have also been found to have inhibitory effects on NDM-1. Among them, the metal complexes inhibited MβLs through a new inhibition mode, which binds to the Cys208 residue in the active site, causing Zn 2+ to be replaced by other ions.…”

Can We Exploit β-Lactamases Intrinsic Dynamics for Designing More Effective Inhibitors?

“…Three azolylthioacetamide derivatives reported by Xiang et al were characterized as inhibitors with mixed inhibition mechanism (competitive and uncompetitive), however, their uncompetitive inhibition mechanism and binding site has not been reported [ 81 ]. At the time of writing, allosteric inhibition has proven a viable strategy only by using macromolecules, such as DNA nanoribbons, DNA aptamers, camelid nanobodies, graphene oxides and nanotubes [ 82 , 83 , 84 , 85 ].…”

A Bottom-Up Approach for Developing Aptasensors for Abused Drugs: Biosensors in Forensics

“…The secondary structure of the aptamer can be computationally predicted by using Mfold [68], RNAstructure [69], and ValFold [70]. Circular dichroism (CiD) [71], nuclear magnetic resonance (NMR) [72], X-ray crystallography [73] as well as molecular modeling [74] are useful to probe aptamer-target interactions. Using the structural information, the full-length aptamer can be subjected to post-SELEX to obtain the optimal sequence [75][76][77][78][79][80].…”