Insights into the Mechanistic Basis of Plasmid-Mediated Colistin Resistance from Crystal Structures of the Catalytic Domain of MCR-1

Abstract

The polymixin colistin is a “last line” antibiotic against extensively-resistant Gram-negative bacteria. Recently, the mcr-1 gene was identified as a plasmid-mediated resistance mechanism in human and animal Enterobacteriaceae, with a wide geographical distribution and many producer strains resistant to multiple other antibiotics. mcr-1 encodes a membrane-bound enzyme catalysing phosphoethanolamine transfer onto bacterial lipid A. Here we present crystal structures revealing the MCR-1 periplasmic, catalytic domain to be a zinc metalloprotein with an alkaline phosphatase/sulphatase fold containing three disulphide bonds. One structure captures a phosphorylated form representing the first intermediate in the transfer reaction. Mutation of residues implicated in zinc or phosphoethanolamine binding, or catalytic activity, restores colistin susceptibility of recombinant E. coli. Zinc deprivation reduces colistin MICs in MCR-1-producing laboratory, environmental, animal and human E. coli. Conversely, over-expression of the disulphide isomerase DsbA increases the colistin MIC of laboratory E. coli. Preliminary density functional theory calculations on cluster models suggest a single zinc ion may be sufficient to support phosphoethanolamine transfer. These data demonstrate the importance of zinc and disulphide bonds to MCR-1 activity, suggest that assays under zinc-limiting conditions represent a route to phenotypic identification of MCR-1 producing E. coli, and identify key features of the likely catalytic mechanism.

Substrate analog interaction with MCR‐1 offers insight into the rising threat of the plasmid‐mediated transferable colistin resistance

“…Although several structures of PEA transferases have been determined (30‐33, 53, 54), little is known about their substrate‐binding sites; the understanding of which is crucial for understanding the catalytic process and for designing inhibitory drugs. We studied lipid A and phosphatidylethanolamine under various conditions for cocrystallization with cMCR‐1, but the electron‐density map failed to show the presences of any substrates.…”

“…S3 B ). Based on the crystal structure and information from a previous study (33), the Zn 2+ ion was coordinated by conserved residues E246, D465, H466, and T285, stabilizes the nucleophilic T285, and is crucial for MCR‐1 activity. In addition, E246, T285, H478, and the adjacent N329, K333, and H395 are thought to be the PEA‐binding region, which integrates with the Zn 2+ binding region.…”

“…The structure of the catalytic domains of MCR‐1 and 2 homologs, EptA and EptC, have been previously determined (30‐33, 53, 54). Recently, the first full‐length crystal structure of a member of the PEA transferase family, EptA, and its dynamic states were reported (25).…”

1.12 Å resolution crystal structure of the catalytic domain of the plasmid-mediated colistin resistance determinant MCR-2

“…MCR-1 catalyses the transfer of positively charged phosphoethanolamine onto lipid A, which is subsequently incorporated into the outer membrane, reducing the net negative charge and preventing colistin binding (Hinchliffe et al, 2017;Liu et al, 2016). It is an integral, metal-dependent innermembrane protein, with a large periplasmic domain containing the catalytic centre and the conserved Thr285 that is likely to act as the acceptor for the phosphoethanolamine group during the transfer reaction (Hinchliffe et al, 2017).…”

Crystal Structure of the Catalytic Domain of MCR-1 (cMCR-1) in Complex with d-Xylose

“…Although several structures of MCR-1 catalytic domain (namely cMCR-1) have been determined [5][6][7][8], few effective inhibitors for MCR-1 are known. A recent co-crystallization study [9] showed that two substrate analogues of MCR-1, ethanolamine and D-glucose, could specifically bind to cMCR-1.…”