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Dynamic Behavior and Substrate Interactions of the Polymyxin Resistance Determinant MCR-1 Investigated by Molecular
Emily Lythell1,2, Jack Badley1, Reynier Suardíaz1,3,4
1Centre for Computational Chemistry, School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, U.K.
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.
Insights
Mobile Colistin Resistance (MCR) enzymes confer antibiotic resistance. Molecular dynamics simulations reveal limited domain movement and a "ping-pong" mechanism, with the monozinc MCR-1 enzyme ready for the first reaction stage.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Mobile Colistin Resistance (MCR) enzymes are plasmid-borne phosphoethanolamine (PEtN) transferases conferring resistance to colistin, a critical antibiotic, particularly in *Escherichia coli*.
- Understanding the full-length MCR enzyme structure and mechanism is limited, with prior studies focusing on isolated catalytic domains or related enzymes.
- Bacterial PEtN transferases possess both catalytic and integral membrane domains, influencing their interaction with cellular membranes.
Purpose of the Study:
- To investigate the dynamic behavior of full-length MCR-1 using molecular dynamics simulations.
- To explore the impact of zinc stoichiometry and substrate binding on MCR-1 structure and function.
- To elucidate the mechanism of colistin resistance conferred by MCR enzymes.
Main Methods:
- Molecular dynamics simulations of a full-length MCR-1 model in a simulated membrane environment.
- Analysis of enzyme dynamics under varying zinc concentrations and with PEtN donor substrate (POPE).
- Site-directed mutagenesis of key residues (e.g., Glu116) to assess functional impact.
Main Results:
- Simulations showed limited domain movement in full-length MCR-1, suggesting a relatively stable structure.
- Palmitoyloleoyl phosphoethanolamine (POPE) binding was observed in an orientation suitable for PEtN transfer to the Thr285 residue in the monozinc state.
- A second zinc ion binding, stabilized by Glu116 in the transmembrane domain, was observed under specific conditions; mutation of Glu116 abolished MCR-1 activity.
Conclusions:
- MCR enzyme domain motions are condition-dependent, supporting a "ping-pong" reaction mechanism.
- The monozinc form of MCR-1 is competent for the initial step of the catalytic cycle.
- The transmembrane domain, particularly Glu116, plays a crucial role in MCR-1 function and colistin resistance.
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