Dynamic Behavior and Substrate Interactions of the Polymyxin Resistance Determinant MCR-1 Investigated by Molecular Dynamics Simulations in the Membrane Environment.

Abstract

The Mobile Colistin Resistance (MCR) phosphoethanolamine (PEtN) transferase is a plasmid-borne enzyme responsible for colistin antibiotic resistance in Escherichia coli, the most important antimicrobial-resistant bacterial pathogen worldwide. Bacterial PEtN transferases like MCR comprise periplasmic catalytic and integral membrane domains, with mechanistic understanding largely based on studies of the former and limited information on the full-length enzyme. Previous investigations of a Neisseria meningitidis PEtN transferase identified that the catalytic domain can effectively dissociate from the transmembrane component and instead make extensive contacts with the membrane surface. Here, we report molecular dynamics simulations of a model of full-length MCR-1 in a representative membrane comprising 80% of a PEtN donor substrate, palmitoyloleoyl phosphoethanolamine (POPE), that explore the dynamic behavior of the enzyme and the impact upon it of zinc stoichiometry and PEtN addition to the Thr285 acceptor residue. The results identify only limited movement of the two domains relative to one another, and that POPE can bind the likely "resting" state of the enzyme (monozinc with unmodified Thr285) in an orientation compatible with PEtN transfer to Thr285. Stable binding of a second zinc equivalent occurred only with application of restraints and involved Glu116 from the transmembrane domain. Mutation of this residue abolished MCR-1-mediated protection of recombinant E. coli from colistin. Our data suggest domain motions in bacterial PEtN transferases to be condition-dependent and support a proposed "ping-pong" reaction mechanism, with the monozinc enzyme competent to undertake the first stage.