title: 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.
authors.affiliation: School of Cellular and Molecular Medicine, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Philip
authors.initials: P
authors.lastname: Hinchliffe
authors.affiliation: Institute of Infection and Immunity, Cardiff University, Cardiff CF14 4XN, UK.
authors.forename: Qiu E
authors.initials: QE
authors.lastname: Yang
authors.affiliation: Institute of Infection and Immunity, Cardiff University, Cardiff CF14 4XN, UK.
authors.forename: Edward
authors.initials: E
authors.lastname: Portal
authors.affiliation: School of Chemistry, University of Bristol, Bristol BS8 1TS, UK.
authors.forename: Tom
authors.initials: T
authors.lastname: Young
authors.affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.affiliation: School of Cellular and Molecular Medicine, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Tooke
authors.affiliation: Institute of Infection and Immunity, Cardiff University, Cardiff CF14 4XN, UK.
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Carvalho
authors.affiliation: Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK.
authors.forename: Neil G
authors.initials: NG
authors.lastname: Paterson
authors.affiliation: Department of Chemistry, University of Oxford, Oxford OX1 3TA, UK.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Brem
authors.affiliation: Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok, 65000, Thailand.
authors.forename: Pannika R
authors.initials: PR
authors.lastname: Niumsup
authors.affiliation: Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok, 65000, Thailand.
authors.forename: Uttapoln
authors.initials: U
authors.lastname: Tansawai
authors.affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Lei
authors.affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors.forename: Mei
authors.initials: M
authors.lastname: Li
authors.affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors.forename: Zhangqi
authors.initials: Z
authors.lastname: Shen
authors.affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of Oxford, Oxford OX1 3TA, UK.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Schofield
authors.affiliation: School of Chemistry, University of Bristol, Bristol BS8 1TS, UK.
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Mulholland
authors.affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Shen
authors.affiliation: School of Chemistry, University of Bristol, Bristol BS8 1TS, UK.
authors.forename: Natalie
authors.initials: N
authors.lastname: Fey
authors.affiliation: Institute of Infection and Immunity, Cardiff University, Cardiff CF14 4XN, UK.
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Walsh
authors.affiliation: School of Cellular and Molecular Medicine, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: James
authors.initials: J
authors.lastname: Spencer
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D020134:Catalytic Domain; D003091:Colistin / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D004220:Disulfides / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008667:Metalloproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D004220:Disulfides; D029968:Escherichia coli Proteins; C000614037:MCR-1 protein, E coli; D008667:Metalloproteins; D019704:Protein Disulfide-Isomerases; D015032:Zinc; D003091:Colistin