High resolution crystal structure of the catalytic domain of MCR-1

Abstract

The newly identified mobile colistin resistant gene (mcr-1) rapidly spread among different bacterial strains and confers colistin resistance to its host, which has become a global concern. Based on sequence alignment, MCR-1 should be a phosphoethanolamine transferase, members of the YhjW/YjdB/YijP superfamily and catalyze the addition of phosphoethanolamine to lipid A, which needs to be validated experimentally. Here we report the first high-resolution crystal structure of the C-terminal catalytic domain of MCR-1 (MCR-1C) in its native state. The active pocket of native MCR-1C depicts unphosphorylated nucleophilic residue Thr285 in coordination with two Zinc ions and water molecules. A flexible adjacent active site loop (aa: Lys348-365) pose an open conformation compared to its structural homologues, suggesting of an open substrate entry channel. Taken together, this structure sets ground for further study of substrate binding and MCR-1 catalytic mechanism in development of potential therapeutic agents.

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

“…Protein was loaded onto a Superdex 75 size-exclusion column equilibrated in 50 mM HEPES pH 7.5, 150 mM NaCl, 100 mM ZnCl 2 . As for MCR-1 CD (Hinchliffe et al, 2017;Ma et al, 2016), MCR-2 CD eluted from the Superdex 75 column as a monomer. Peak fractions were concentrated to 15 mg ml À1 by centrifugation.…”

“…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). We recently described two crystal structures of the MCR-1 catalytic domain (MCR-1 CD ), revealing the presence of one (PDB entry 5lrn; MCR-1 5LRN ) or two (PDB entry 5lrm; MCR-1 5LRM ) zinc ions in the active site (Hinchliffe et al, 2017) containing additional zinc ions, and one with two active-site zinc ions and both phosphorylated and nonphosphorylated Thr285 (PDB entry 5grr; MCR-1 5GRR ; Ma et al, 2016). More recently, the full-length, detergent-solubilized crystal structure of an MCR homologue (EptA; 36% sequence identity to MCR-2) was solved (Anandan et al, 2017) with a single zinc ion, a nonphosphorylated Thr285 and a bound molecule of dodecyl maltoside (DDM) in the active site.…”

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

“…The data set of X-ray diffraction had a resolution range from 43.71 Å to 1.82 Å with 3.5% R merge and 99.0% completeness. To elucidate the structure of cMCR-1-D-xylose complex, we employed molecular replacement method using cMCR-1 monomer (PDB entry 5GRR [7]) as a search model and obtained a clear solution. We confirmed the occurrence of a single protein molecule in the asymmetric unit by cross-rotation and translation-function calculations; the corresponding solvent content was 29.13%.…”

“…The data were indexed, integrated, and scaled using HKL-2000 (HKL Research, Inc., Charlottesville, VA, USA) [10] and iMosflm programs [11]. The structure was solved by molecular replacement with Phaser [12] using a single monomer of cMCR-1 (PDB entry 5GRR [7]) as the search model. The structure model was constructed using alternating manual building in Coot [13] and restrained refinement in PHENIX [14].…”

“…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.…”