Structure of a lipid A phosphoethanolamine transferase suggests how conformational changes govern substrate binding

Abstract

Multidrug-resistant (MDR) gram-negative bacteria have increased the prevalence of fatal sepsis in modern times. Colistin is a cationic antimicrobial peptide (CAMP) antibiotic that permeabilizes the bacterial outer membrane (OM) and has been used to treat these infections. The OM outer leaflet is comprised of endotoxin containing lipid A, which can be modified to increase resistance to CAMPs and prevent clearance by the innate immune response. One type of lipid A modification involves the addition of phosphoethanolamine to the 1 and 4′ headgroup positions by phosphoethanolamine transferases. Previous structural work on a truncated form of this enzyme suggested that the full-length protein was required for correct lipid substrate binding and catalysis. We now report the crystal structure of a full-length lipid A phosphoethanolamine transferase from Neisseria meningitidis, determined to 2.75-Å resolution. The structure reveals a previously uncharacterized helical membrane domain and a periplasmic facing soluble domain. The domains are linked by a helix that runs along the membrane surface interacting with the phospholipid head groups. Two helices located in a periplasmic loop between two transmembrane helices contain conserved charged residues and are implicated in substrate binding. Intrinsic fluorescence, limited proteolysis, and molecular dynamics studies suggest the protein may sample different conformational states to enable the binding of two very different-sized lipid substrates. These results provide insights into the mechanism of endotoxin modification and will aid a structure-guided rational drug design approach to treating multidrug-resistant bacterial infections.lipid modification | multidrug resistance | molecular dynamics | Neisseria | membrane protein structure

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

“…The current structure represents a first step towards this goal. Anandan et al, 2017) are superposed. The membrane domain of EptA is coloured grey; zinc ions are shown as grey spheres and bound DDM as purple sticks.…”

“…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. This full-length structure confirmed the prediction (Hinchliffe et al, 2017) that the active site is proximal to the membrane.…”

Antibiotic exposure elicits the emergence of colistin- and carbapenem-resistant Escherichia coli coharboring MCR-1 and NDM-5 in a patient

“…Unfortunately, it seems likely that the clinical use of carbapenems and colistin has been significantly challenged by the occurrence of mobilized colistin resistance determinant (MCR-1) in clinical carbapenem-resistant isolates producing New Delhi metallo- β -lactamase 1 (NDM-1) [6,7] or its variant NDM-5 [8,9]. In addition to the intrinsic determinants of colistin resistance (e.g., EptA [10,11] and ICR-Mo [12]), the transferable determinants (MCR-1 and MCR-2 [10,13,14]) consistently encode members of phosphoethanolamine (PEA)-lipid A transferases, which adopt a ‘ping-pong’ catalysis reaction in transferring of the PEA moiety to the 4ʹ-phosphate position of the lipopolysaccharide (LPS)-lipid A species anchored on bacterial surface [4,5]. …”

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

“…Meanwhile, there are still many differences between the two structures. We also conducted a comparison of cMCR-1-D-xylose complex with phosphoethanolamine transferase A (EptA) from Neisseria meningitides (PDB entry 5FGN [17]), the only structure of a full-length phosphoethanolamine transferase so far. The structure of cMCR-1-D-xylose complex can be well superimposed with the structure of EptA catalytic domain, with a Cα root-mean-square deviation of 2.0 Å as revealed by Dali server (http://ekhidna.biocenter.helsinki.fi/dali_server/start) [18] (Figure 4g).…”

“…The structure of cMCR-1-D-xylose complex can be well superimposed with the structure of EptA catalytic domain, with a Cα root-mean-square deviation of 2.0 Å as revealed by Dali server (http://ekhidna.biocenter.helsinki.fi/dali_server/start) [18] (Figure 4g). Anandan et al [17] have shown that detergent dodecyl-β-D-maltoside (DDM) could bind in a substrate pocket of EptA, and the pocket bound by DDM was probably the phosphoethanolamine (PEA) binding pocket near the putative lipid A-binding pocket.…”