Crystal structures of VIM-1 complexes explain active site heterogeneity in VIM-class metallo-β-lactamases.

Ramya Salimraj,Magda Kosmopoulou,Anil Verma,Christopher J Schofield,Raymond J Owens,James Spencer,Jonathan M Tyrrell,Sander S Van Berkel,Jürgen Brem,Timothy R Walsh,Michael A Mcdonough,Philip Hinchliffe

doi:10.1111/febs.14695

Abstract

Metallo‐β‐Lactamases (MBLs) protect bacteria from almost all β‐lactam antibiotics. Verona integron‐encoded MBL (VIM) enzymes are among the most clinically important MBLs, with VIM‐1 increasing in carbapenem‐resistant Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae) that are among the hardest bacterial pathogens to treat. VIM enzymes display sequence variation at residues (224 and 228) that in related MBLs are conserved and participate in substrate binding. How they accommodate this variability, while retaining catalytic efficiency against a broad substrate range, has remained unclear. Here, we present crystal structures of VIM‐1 and its complexes with a substrate‐mimicking thioenolate inhibitor, ML302F, that restores meropenem activity against a range of VIM‐1 producing clinical strains, and the hydrolysed product of the carbapenem meropenem. Comparison of these two structures identifies a water‐mediated hydrogen bond, between the carboxylate group of substrate/inhibitor and the backbone carbonyl of the active site zinc ligand Cys221, that is common to both complexes. Structural comparisons show that the responsible Cys221‐bound water is observed in all known VIM structures, participates in carboxylate binding with other inhibitor classes, and thus effectively replicates the role of the conserved Lys224 in analogous complexes with other MBLs. These results provide a mechanism for substrate binding that permits the variation at positions 224 and 228 that is a hallmark of VIM MBLs.Enzymes EC 3.5.2.6 DatabasesCo‐ordinates and structure factors for protein structures described in this manuscript have been deposited in the Protein Data Bank (www.rcsb.org/pdb) with accession codes 5N5G (VIM‐1), 5N5H (VIM‐1:ML302F complex) and 5N5I (VIM‐1‐hydrolysed meropenem complex).

Highlights

Antibiotic resistance is of immediate and growing concern to global public health [1]
More) were observed in four cases, with one strain apparently unaffected. These results show that ML302F can potentiate the activity of meropenem against clinical isolates of Verona Integron-encoded MBL (VIM)-1 expressing Enterobacteriaceae, and that this compound can penetrate clinically relevant bacteria to act as an effective inhibitor of VIM-1 in the bacterial host
While VIM-2 Arg228 is proposed to contact the substrate carboxylate as a functional equivalent of Lys224 in other B1 MBLs [24,39], other VIM variants can be expected to interact with this key b-lactam functional group in different ways

Summary

Introduction

Antibiotic resistance is of immediate and growing concern to global public health [1]. B-Lactams, which continue to form over half of the global antibacterial market [3], remain key agents for treatment of such infections, with the carbapenems in particular rapidly supplanting third-generation cephalosporins as first-choice drugs. In pathogens such as the Enterobacteriaceae (e.g. Escherichia coli; Klebsiella pneumoniae) or nonfermenting organisms (e.g. Pseudomonas aeruginosa; Acinetobacter baumannii) production of b-lactamases that inactivate b-lactams via hydrolysis of the b-lactam ring [4], is the major form of b-lactam resistance [5]. Our results reveal how substrate/inhibitor binding by VIM enzymes is tolerant of substitutions at sequence positions that in other MBLs make interactions, notably those involving the b-lactam carboxylate, that are crucial for activity

Results

Discussion

Experimental procedures

Conflicts of interest

Graphical Abstract