Substrate Recognition by a Colistin Resistance Enzyme from Moraxella catarrhalis
Lipid A phosphoethanolamine (PEtN) transferases render bacteria resistant to the last resort antibiotic colistin. The recent discoveries of pathogenic bacteria harboring plasmid-borne PEtN transferase (mcr) genes have illustrated the serious potential for wide dissemination of these resistance elements. The origin of mcr-1 is traced to Moraxella species co-occupying environmental niches with Enterobacteriaceae. Here, we describe the crystal structure of the catalytic domain of the chromosomally encoded colistin resistance PEtN transferase, ICRMc (for intrinsic colistin resistance) of Moraxella catarrhalis. The ICRMc structure in complex with PEtN reveals key molecular details including specific residues involved in catalysis and PEtN binding. It also demonstrates that ICRMc catalytic domain dimerization is required for substrate binding. Our structure-guided phylogenetic analysis provides sequence signatures defining potentially colistin-active representatives in this enzyme family. Combined, these results advance the molecular and mechanistic understanding of PEtN transferases and illuminate their origins.
<!>icrMc Confers Polymyxin Resistance.<!>MCR-1, MCR-2, and ICRMc Define a Distinct Group in the Broader PEtN Transferase Family.<!>Crystal structure of the catalytic domain of ICRMc.<!>Substrate Recognition Requires the Dimerization of the ICRMccat Domain.<!>Comparative Structural Analysis Highlights Signature Active Site Features of the PEtN Transferase Family.<!>MIC Determination.<!>Protein Expression and Purification.<!>Crystallization and Structure Determination.<!>Sequence and Structural Analysis.
<p>The polymyxin antibiotic colistin is classified as an antibiotic of critical importance by the World Health Organization due to its activity against multidrug resistant Gram-negative pathogens.1 Colistin is utilized as a treatment of last resort for recalcitrant infections such as those caused by carbapenem-resistant Enterobacteriaceae. Even though colistin is nephrotoxic, the seriousness of such infections and the dearth of alternative treatments has increasingly secured a place for this antibiotic in the infectious disease treatment armory in the absence of new antibiotics.2</p><p>Many species are intrinsically resistant to polymyxins, including Neisseria meningitidis, Helicobacter pylori, Burkholderia cepacia, and Moraxella catarrhalis.3,4 The mechanisms of resistance harbored by these bacteria include expression of efflux pumps, shedding of capsule polysaccharide, and modification of lipopolysaccharide (LPS), the latter of which appears the most prevalent (reviewed in ref 5). Diverse covalent modifications of LPS, which include among others the addition of phosphoethanolamine (PEtN) groups (reviewed in ref 6) are thought to alter the physical properties of the outer membrane, resulting in polymyxin resistance. The increased clinical and agricultural use of colistin has been linked to the mobilization and transfer of colistin resistance elements to human pathogenic bacteria such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii, representing serious threats to the effectiveness of polymyxins against these species.3</p><p>Well-characterized representatives of PEtN transferases include EptA from E. coli, Vibrio cholerae, and H. pylori, EptA (originally named LptA) from N. meningitidis, PmrC from A. baumannii, and CptA from Campylobacter jejuni (reviewed in ref 6). These chromosomally encoded enzymes catalyze the transfer of PEtN from phosphatidylethanolamine (PE) onto the lipid A moiety of LPS. E. coli EptB is another type of PEtN transferase, which specifically catalyzes the modification of the outer 3-deoxy-D-manno-octulosonic acid (Kdo) residue of LPS.7</p><p>The recent discovery of the PEtN transferase encoded by the mcr-1 gene8 mobilized on a plasmid raised the possibility of accelerated dissemination and acquisition of colistin resistance among pathogens or its collection into multidrug resistance cassettes.9 Indeed, this gene has since been detected in other Enterobacteriaceae species around the world, in additional patient isolates, and in retrospective searches of culture collections.10–17
mcr-1 has also been found on mobile genetic elements containing up to nine other antibiotic resistance genes, including extended-spectrum β-lactamases (ESBLs).10,14,18 As further evidence of the mobilization of colistin resistance, additional genes encoding PEtN transferases including mcr-2, which shares 81% sequence identity with mcr-1, plus the more sequence-distinct mcr-3, mcr-4, and mcr-5 have been recently discovered on Enterobacteriaceae plasmids.19–21 Thus, PEtN transferases have mobilized multiple times and are becoming worrisome resistance determinants that may have serious and long-lasting clinical implications.</p><p>Resistance genes manifesting in the clinic can often be traced to environmental and other bacteria, which represent a global reservoir of resistance genes that can be transferred between diverse genera via mobile genetic elements (reviewed in refs 22 and 23). For example, genes conferring clinical levels of antibiotic resistance such as qnrA and blaCTX-M were subsequently traced to aquatic bacteria (reviewed in ref 24). Along the same lines, environmental paralogues were suggested for mcr-1 and similar PEtN transferase genes. Sequence similarity searches identify close MCR-1 and MCR-2 orthologs (61–65% identity of the full-length protein sequences) in the Moraxellaceae family, specifically in the human pathogen Moraxella catarrhalis, the nematode symbiont Moraxella osloensis, and the environmental bacteria Enhydrobacter aerosaccus and Psychrobacter spp.8 Based on these observations, it was suggested that Moraxella species occupying the same ecological niche as E. coli encode the ancestral gene of mcr-1 and mcr-2;19 a subsequent study indeed detected mcr-1 orthologous genes in numerous Moraxella species.25 Thus, Moraxella or related species may represent an ecological reservoir for mobilization of PEtN transferases to other pathogens; however, the molecular and functional characterization of these ancestral MCR-1 proteins has been lacking.</p><p>According to sequence analysis, PEtN transferases are comprised of an N-terminal transmembrane region and a periplasm-facing C-terminal hydrolase domain belonging to the alkaline phosphatase superfamily. The periplasmic catalytic domains of MCR-1, MCR-2, EptA, and EptC have been recently structurally characterized. Some of the revealed common molecular features observed in the active site of these enzymes included a conserved catalytic threonine (often covalently linked to phosphate) and multiple Zn2+ binding sites.26–33 The crystal structures of PEtN catalytic domains also featured disulfide bridges between spatially colocalized cysteine residues. These structural data provided the molecular framework for understanding of PEtN transferases; however, the molecular information concerning substrate recognition is still lacking. Furthermore, significant sequence divergence between EptA and EptC and MCR-1 (EptA and EptC both share only 42% sequence identity to MCR-1 in the catalytic domain) necessitates further structural and functional studies into closer representatives of the latter enzyme to define the possible role of these enzymes in development of colistin resistance.</p><p>In this study, we characterized the crystal structure of the catalytic domain of the PEtN transferase from M. catarrhalis (which we designate as ICRMc), the protein that represents the closest known ortholog to MCR-1 and MCR-2 and is responsible for intrinsic colistin resistance of this bacterium. Our crystal structures also provide insights into the molecular basis of recognition of the PEtN substrate and identify a putative role of dimerization for resistance activity. Our findings show that MCR-1 and ICRMc share key structural and functional characteristics in their catalytic domains, supporting the hypothesis that Moraxellaceae are the environmental reservoir of PEtN transferases.</p><!><p>To test the ability of ICRMc to confer resistance, we expressed icrMc in E. coli BW25113 and compared its ability to grow in the presence of colistin and polymyxin B with that of the same E. coli strain expressing mcr-1. Both the icrMc and mcr-1 expressing strains showed 4–16-fold (depending on promoter strength/expression level34) higher resistance to these antibiotics compared to the E. coli strain with no expression vector (Table 1). Next we tested whether the expression of the icrMc or mcr-1 fragments corresponding to the PEtN transferase periplasmic catalytic domain (designed icrMccat and mcr-1cat) in the same E. coli strain is sufficient to confer polymyxin resistance. The results showed that E. coli harboring icrMccat or mcr-1cat did not show a polymyxin resistance phenotype. These results demonstrated that expression of icrMc confers polymyxin resistance comparable to that of mcr-1 and that both enzymes require their N-terminal transmembrane regions to confer drug resistance in the cell.</p><!><p>To clarify the phylogenetic relationship between ICRMc and other PEtN transferases, we performed a phylogenetic reconstruction of the PEtN transferase family, including 89 known MCR and related proteins (Figure 1). This analysis revealed five clades, which segregate along their substrate specificity. One of the clades is represented by MCR-3, MCR-4, and EptA, known to catalyze transfer of PEtN from PE to lipid A. The second one features orthologs of MCR-5 found in Legionella species. The third clade is represented by EptB, which acts on Kdo, while the fourth clade includes EptC/CptA, which target lipid A. The fifth clade contains orthologs of MCR-1 and MCR-2, which we termed as MCR-1-like. The five clades show significant divergence reflected in low amino acid sequence similarity between representatives of different clades. Specifically, EptB and MCR-3 shared only 25% and 32% sequence identity with MCR-1. Within the MCR-3/MCR-4/EptA clade, the MCR-3 and MCR-4 enzymes appear to be distinct from the rest of their orthologs, suggesting separate evolutionary paths or ancestral origins. These observations are consistent with multiple incidents of mobilization of intrinsic PEtN ancestral genes leading to diversification among members of this family and reflecting different functional properties.</p><p>Consistent with previous reports,8,25 our phylogenetic reconstruction recapitulated that the MCR-1-like clade contains a closely associated group of sequences from Gammaproteo-bacteria that share between 57% and 98% sequence identity with MCR-1, including MCR-1, MCR-2, and members in Moraxellaceae species: ICRMc, ICRM. osloensis, and ICRE.aerosaccus. Beyond this group, the MCR-1-like clade contained a group of uncharacterized proteins, including representatives from Sulfurospirillum spp., Arcobacter spp., Campylobacter sputurum and Marinobacter subterrani and these share between 41 and 79% identity with MCR-1. Notably, this group also shares significant similarity with the EptA clade. These observations indicate that the ability to confer colistin resistance established for MCR-1 and ICRMc may be shared by the wider sequence-related group of PEtN transferases, which forms a distinct clade in the PEtN transferase superfamily.</p><p>Alignment of PEtN transferase sequences (Figure S1) revealed higher degree of sequence conservation in the C-terminal region (i.e., 68% sequence identity between ICRMc and MCR-1 or MCR-2). Combined with the comparable resistance phenotype conferred by expression of these enzymes, this points to strong conservation of functional and structural features between the ICRMc and MCR-1/MCR-2 enzymes.</p><!><p>Next, we purified and crystallized the catalytic domain of ICRMc (ICRMccat) spanning residues 235 to 578 (Figure 2A). The crystal structure of this domain was determined to 2.61 Å by Molecular Replacement using the structure of N. meningitis EptA/LptA26 as a model. The X-ray crystallographic statistics are summarized in Table 2.</p><p>The crystal structure contains one copy of the ICRMccat domain in the asymmetric unit. Residues 244 through 578 could be traced in the electron density, while residues 235–243 appear disordered. The resolved ICRMccat residues adopted the common sulfatase/hydrolase fold, characterized by a central seven-stranded β-sheet mainly parallel in topology, with the edge strand β12 oriented in an antiparallel orientation (Figure 2A).</p><p>The structure of ICRMccat reveals two unambiguous disulfide bonds (Cys390 to Cys398, Cys448 to Cys456). Another pair of cysteine residues, Cys311 and Cys321 appear reduced; however the proximity of their side chains suggests the possibility of formation of an additional disulfide bond (Figure 2A). We interpreted additional difference electron density in the structure as six sulfate ions and one phosphate ion. Unexpectedly, the ICRMccat crystal structure did not contain additional electron density corresponding to the Zn2+ ion required for PEtN transferases.26,27,29–33 Interpreting this as possible depletion of Zn2+ ions in the course of ICRMccat purification, we soaked ICRMccat protein crystals in zinc chloride-supplemented buffer (see Methods for details). We detected significant anomalous diffraction at the Zn2+ dispersion absorption peak for such crystals and identified additional electron density corresponding to a single Zn2+ atom bound to ICRMccat active site (Figure 2A, Figure 3).</p><p>The active site region of ICRMccat localized to a small (~200 Å3) pocket at the "top" of the central β-sheet (as shown in Figure 2B) lined by residues from converging loops. This pocket was on the large, flat face of the ICRMccat domain close to its N-terminus. Apart from the Zn2+, the active site contains a phosphate ion at 3.7 Å distance from the Zn2+, which is anchored by a network of interactions with the active site residues (Figure 3). The Zn2+ ion is coordinated primarily by the side chains of Glu273, Thr315, Asp498, and His499. The phosphate forms interactions with the side chain hydroxyl and backbone amide of Thr315, the side chain of His511, and two ordered water molecules, one of which also interacted with the Zn2+ ion. The Zn2+ and phosphate-binding residues are in turn oriented by a network of interactions with other active site residues including Asn359, Ser360, and His424. Notably, Thr315 in the ICRMccat active site occupies the position corresponding to the phosphate-accepting nucleophilic residue in the other PEtN transferases.26,27,29–32 Analysis of ICRMccat residues interacting with both Zn2+ and the phosphate ions also highlights His429 as a functionally important residue, although its interaction with the Zn2+ was mediated through a hydrogen bond with the coordinated water molecule. In addition to the Zn2+ and phosphate ions, the ICRMccat active site contained a sulfate ion positioned at 11 Å distance from the phosphate ion described above. The sulfate forms close interactions with Arg436, which in turn forms a salt bridge with Glu397. In total, our analysis suggested that at least 18 ICRMc active site residues participate in interactions with the Zn2+, phosphate, and sulfate ligands.</p><!><p>To characterize the molecular basis of substrate recognition by ICRMccat, we crystallized the Thr315Ala ICRMccat variant in the presence of the disaccharide headgroup of lipid A or PEtN, a component of the lipid substrate phosphatidylethanolamine. Thr315 is predicted to be the nucleophilic residue that attacks the substrate phosphate group. Consistent with this prediction, mutation to Ala abolishes antibiotic resistance activity (Table 1). This crystal structure was determined to 1.64 Å (Table 2) and revealed a different unit cell and packing compared to the wild-type ICRMccat. In contrast to the ICRMccat apo structure, the ICRMccatThr315Ala.mono-Zn2+.PEtN complex structure features two ICRMccatThr315Ala molecules associated as a homodimer with ~1190 ~2 buried at the dimerization interface. The ICRMccat protein chains in the PEtN complex structure associate in a symmetrical "front-to-front" orientation, with the two active sites facing each other (Figure 4A,B). Dimerization involves interactions between 38 residues in each monomer and impacts both the shape and accessibility of the active sites. In the ICRMccat dimer, the entrance to the catalytic centers is buried along the dimerization interface with access through deep clefts formed by this interface (Figure 4C). Consistent with this observed association of ICRMccat domains, we also detected the formation of a dimer in solution by size exclusion chromatography, which is more prominent in the presence of Zn2+ ions (Figure S2).</p><p>Well-defined additional electron density allowed unambiguous positioning of one PEtN molecule in each active site of the ICRMccatThr315Ala dimer in the complex structure (Figure 5). The phosphate of the PEtN molecule forms interactions with the Zn2+ ion and the side chain of His511, as well with the backbone of Ala315 and three water molecules. Intriguingly, the Tyr338 from the partner monomer chain also contributes to coordination of the substrate molecule (Figure 5). Notably, the position of the phosphate group of the PEtN molecule in the complex structure corresponds to the position of the phosphate ion in the apo structure. Orientation of the PEtN molecule in the ICRMccat active site is consistent with the predicted catalytic mechanism of this family whereby a threonine residue (Thr315 in ICRMc) performs nucleophilic attack on the phosphate atom of the phosphatidylethanolamine substrate to generate a phosphoethanolamine–Thr intermediate species. We did not observe any interactions between the ethanolamine moiety of PEtN and the enzyme. Taken together, the structural analysis of the ICRMccatThr315Ala. PEtN complex suggests that dimerization of this domain may facilitate the coordination of the substrate providing an additional interaction partner (Tyr338) from the adjacent subunit that may be required for optimal positioning of the phosphatidylethanolamine substrate for catalysis.</p><p>One of the tested ICRMccatThr315Ala crystals obtained in the presence of lipid A headgroup lacked additional density corresponding to the substrate molecule but featured a second Zn2+ ion (Zn2 in Figure S3) in the active sites of the ICRMccat dimer. The position of Zn2 corresponds to that of the phosphate of PEtN in the complex structure. In this structure, the Zn2 is coordinated by side chains of residues Glu273, His429, and His511 and the backbone amide of Ala315. In this interaction, the His429 underwent a rotamer change relative to its position in the ICRMc apo structure. Thus, there could be an interplay between the binding of a second Zn2+ atom and the phosphate groups of the enzyme's substrates. Furthermore, the active sites of the ICRMccatThr315Ala.di-Zn2+ structure contain elongated difference electron density corresponding to a poly(ethylene glycol) molecule present in the crystallization solution (Figure S3). The position of this molecule in the active site approaches the catalytic center of the enzyme and forms a packing interaction against His429 and hydrophobic interactions with Met426 and the aliphatic region of the Lys378 side chain. Accordingly, we propose that the position of the poly(ethylene glycol) molecule may be indicative of the position of the lipid A headgroup in the ICRMccat active site.</p><p>Guided by our structural analysis, we tested the roles of ICRMccat residues implicated in interactions with Zn2+ and PEtN by site-directed mutagenesis. Residues Thr315, His429, Arg436, and Tyr338 were individually mutated to Ala in the context of the full-length ICRMc protein. The E. coli strains expressing the corresponding ICRMc variants completely lost resistance to colistin and polymyxin B compared to the strain carrying the wild-type enzyme, confirming the important role of these residues for ICRMc activity (Table 1). Based on these results, we postulate that coordination of Zn2+ binding by Thr315, His429, and Tyr338 residues and unexpectedly, interactions of residues Tyr338 and Arg436 with substrate molecules as facilitated by ICRMccat domain dimerization, may be essential for the catalytic activity of ICRMc.</p><!><p>We conducted a comparative analysis of this structure versus those of recently characterized MCR-1 and MCR-2 resistance enzymes as well as the structures of previously characterized EptA and EptC to clarify the shared and distinct features of these PEtN transferases.</p><p>The ICRMc structure superimposes with that of MCR-1 and MCR-2 with RMSD of 0.5 and 0.5 Å over 251 and 270 matching Cα atoms, respectively (Figure 6). This superimposition reveals only minor structural differences primarily in conformations of the β5–η4 and β6–η5 loops. More importantly, all 18 residues of the active site (Figure 6) are absolutely conserved across these three enzymes. Furthermore, these 18 amino acids, which include the histidine (His429) residue undergoing rotation between binding to PEtN or Zn2+ ion in ICRMc structures, are universally conserved across the MCR-1-like PEtN transferases (Figure S1). Interestingly, the Tyr338 residue, whose role in PEtN binding is facilitated by catalytic domain dimerization, is also conserved in the orthologs of ICRMc, MCR-1 and MCR-2. This observation prompted us to suggest that the active site architecture, binding mode of PEtN, and coordination of substrates across a dimer interface as visualized in the ICRMc cat–PEtN complex structure is conserved across the MCR-1-like clade of PEtN transferases defined above.</p><p>Expanding our structure-based sequence analysis to the recently identified MCR-3, MCR-4 and MCR-5 enzymes revealed conservation of 12 out of 18 active site residues invariable in MCR-1-like enzymes (Figure S1). These include residues involved in coordination of the Zn2+ ion and in catalysis. Notably, we were not able to identify the equivalent of ICRMccat residue Tyr338 in MCR-3 or MCR-4. These differences may be indicative of deviation in substrate-binding mode between MCR-1-like PEtN transferases and the MCR-3, MCR-4, and MCR-5 enzymes.</p><p>Detailed comparative analysis of ICRMc and EptA and EptC revealed differences in the conformations of the β5–η4 and β6–η5 loops and in active site composition (Figure S4). Of the 18 active site residues in MCR-1-like enzymes described above, only 11 are conserved in EptA and EptC (Figures S1 and S4). Once again, the conserved residues in EptA and EptC active sites cluster to the Zn2+ binding sites, consistent with a common catalytic mechanism shared across the entire PEtN transferase family. In contrast, the four nonconserved residues corresponding to ICRMc residues Asn359, Ser360, Lys378, and Met426 cluster together and away from the Zn2+ binding site. Identification of these subtle differences may be reflective of EptA and EptC deviating in substrate preference from MCR-1-like enzymes.</p><p>Our observation that the dimeric state of ICRMccatThr315Ala forms a solvent-occluded pocket containing a PEG molecule suggests that only in the dimer state of the catalytic domain or in the context of the full-length protein containing the transmembrane region is the protein capable of forming an active site structure that can recognize the larger lipid A substrate. Indeed, analysis of the crystal structure of EptA, the MCR-1 ortholog from N. meningitidis, suggested that the membrane-spanning portion and the linker to the catalytic domain are involved in mediating substrate interactions.35 The detergent (DDM) molecule trapped in the membrane-spanning portion of EptA is oriented toward the active site of the catalytic domain suggesting it mimics the position of enzyme's substrate (Figure 7). With our ICRMccatThr315Ala.PEtN crystal structure in hand, we are able to posit a model of the ternary complex with a lipid positioned to attack the phosphoethanolamine–threonine intermediate (Figure 7). This model was developed through a superposition of the structures of EptA. DDM and ICRMccatThr315Ala.PEtN, which showed that the position of these two ligands do not overlap: more specifically, the O3B of DDM and O3 of PEtN approached each other in this model, and thus this overlay can be used to interpret the position of a phosphorylated lipid molecule in the active site (Figure 7). This model also validates the prediction that EptA Glu114 interacts with the amine group of PEtN35 as this residue and its equivalent in ICRMc (E113) is positioned within bonding distance to this functional group. Consistent with its functional importance, this glutamate residue is conserved throughout the PEtN transferase family (except for the EptB subgroup), suggesting it plays a role in discrimination for PE. Further structural and enzymatic studies into PEtN transferase and their interactions with native substrates are required to further validate this model and reveal additional key details.</p><p>The binding of substrates may require ICRMccat domain dimerization, which according to our structural analysis provides additional interaction partners for the substrate molecules in both actives sites of the dimer. A molecular dynamics simulation of EptA35 showed that the soluble domain can adopt multiple conformations and "roll" over the simulated membrane surface, indicating that the dimerization interface we observed in ICRMc complex structure may be accommodated in such a fluid situation.</p><p>Our demonstration of the conservation of the molecular details of the ICRMc, MCR-1, and MCR-2 active sites supports the notion that Moraxella species form the reservoir of at least this group of PEtN transferases. This stresses that structural and functional studies of intrinsic resistance enzymes in species cohabitating environmental niches as human pathogens are key components of developing a better understanding of the origins and mechanisms of resistance impacting clinical management of pathogens. As has been evidenced in numerous examples, and even in the ongoing discovery of new mcr genes, the source of clinical antibiotic resistance is the environment. Progress toward developing a complete understanding of the broader resistome, such as our characterization of ICRMc, is a key component of resistance surveillance and anticipation of future mobilization events.</p><p>Finally, our structure-based phylogenetic analysis provides a foundational organization of PEtN transferases that clearly shows that these enzymes are diverse in sequence and each discovery of novel mcr genes identifies founding members of subclasses of PEtN transferases. This phenomenon parallels the expansion of β-lactamase families over the course of decades of β-lactam clinical deployment36 and underscores the extent of the dissemination of polymyxin resistance. As with β-lactamase family-specific properties, functional insights into individual PEtN transferases may not be conserved across the subgroups. Thus, appreciation of the structural and molecular diversity of MCR enzymes is critical for drug discovery efforts against these enzymes.</p><!><p>Full-length icrMc and its catalytic domain (icrMccat; residues 235–578) plus full-length E. coli mcr-1 and its catalytic domain (mcr-1cat; residues 208–541) were ligated into the pGDP1 and pGDP2 vectors (in house derivatives of the pBR322 vector; New England BioLabs). These plasmids utilize constitutive promoters of differing strength; the stronger β-lactamase Pbla promoter (pGDP1) and the weaker Plac promoter (pGDP2). Amino acid substitutions in icrMc were performed using Phusion High-Fidelity DNA Polymerase (Thermo Scientific) and the pGDP1:icrMc construct. Constructs were confirmed by sequencing (Central MOBIX facility, McMaster University). Susceptibility testing (in triplicate, with two biological replicates) was performed with E. coli BW25113 cultured in Mueller-Hinton II broth (cation adjusted) (Becton, Dickinson and Co, Franklin Lakes NJ) using the broth microdilution method according to CLSI guidelines. Inoculum was prepared using the colony resuspension method, and plates were incubated with aeration at 37 °C for 18 h.</p><!><p>cDNA of icrMc from Moraxella catarrhalis strain BC1 (Genbank accession EGE18576) coding for residues 235 to 578 of ICRMc was synthesized by Integrated DNA Technologies and subcloned into the pMCSG53 expression vector, which codes for an N-terminal His6-tag and a TEV protease cleavage site. icrMc was expressed in E. coli BL21(DE3) Gold cells, grown to an OD600 of 0.6 at 37 °C, chilled to 16 °C, and induced overnight with 500 μM isopropyl β-D-thiogalactopyranoside. Cells were harvested via centrifugation at 5000 × g, pellets were resuspended in binding buffer [50 mM Hepes (pH 7.5), 100–300 mM sodium chloride, 10 mM imidazole, and 2% glycerol (v/v)] and lysed by sonication, and cell debris was removed via centrifugation at 30000 × g. Cleared lysate was loaded onto a 5 mL Ni-NTA column (QIAGEN) pre-equilibrated with binding buffer and extensively washed with binding buffer containing 30 mM imidazole, and protein was eluted using the above buffer with 250 mM imidazole. Fractions containing ICRMc were identified by SDS–polyacrylamide gel electrophoresis and further purified via gel filtration on a HiLoad 16/60 Superdex75 prep-grade column [10 mM Hepes (pH 7.5) and 50 mM potassium chloride].</p><!><p>All crystallization was performed at RT using the sitting drop method and 2 μL of protein or protein/ligand mixture plus 2 μL of reservoir solution. Zn2+-free ICRMc was crystallized with reservoir solution 0.1 M sodium acetate, pH 4.6, and 1.2 M ammonium sulfate, and the crystal was cryoprotected in 8% glycerol, 8% ethylene glycol, and 8% sucrose. Crystals of Zn2+-bound mono-Zn2+.ICRMc were obtained by soaking apo crystals with 2.5 mM zinc chloride for 30 min followed by cryoprotection in the above cryosolution. Crystals of ICRMccatThr315Ala. mono-Zn2+.PEtN were obtained from reservoir solution 0.1 M MES, pH 6, 22% (w/v) PEG 400, 2.5 mM zinc chloride, and 5 mM PEtN, and the crystal was cryoprotected in paratone oil. Crystals of ICRMccatThr315Ala.di-Zn2+.PEG were obtained from reservoir solution 0.1 M Tris, pH 8, 28% (w/v) PEG 4000, 3 mM zinc chloride, and 2 mM lipid A headgroup (Glycobiotech GmbH), and the crystal was cryoprotected in paratone oil. All X-ray diffraction data at 100 K were collected at the Advanced Photon Source, Argonne National Laboratory, Life Sciences Collaborative Access Team beamline 21-ID-D at the following wavelengths corresponding to the measured zinc absorption peak: ICRMc.PO4, 1.27424 Å/9730 keV; ICRMc.mono-Zn2+, 1.27424 Å/9730 keV; ICRMccatThr315Ala.mono-Zn2+.PEtN, 1.27687 Å/9710 keV; ICRMccatThr315Ala.di-Zn2+.PEG, 1.27815 A/9700 keV. X-ray data were reduced with XDS37 and CCP4 Aimless38 or HKL-300039 for the di-Zn2+.PEG structure. The WT structures were solved by molecular replacement using the ICRMc sequence-substituted structure of Neisseria meningitis EptA/LptA (PDB id 4kay26) using Phenix.-phaser,40 while the ICRMccatThr315Ala structures were phased using the anomalous signal from Zn2. Refinement was completed with Phenix.refine and Coot.41 All B-factors were refined as isotropic with TLS parametrization. Zn2+, sulfate ions, and the positions of the six cysteine residues implicated in disulfide bond formation were verified by the presence of peaks in omit or anomalous difference maps. In the WT ICRMc structure, the peak in the anomalous difference electron density peak was 32σ, 0.148 e−/A3. With respect to Zn2+ atoms, in the ICRMccatThr315Ala.di-Zn2+ structure, the anomalous difference density peak heights were 91σ, 0.896 e−/A3, and 82σ, 0.827 e−/A3, in one active site and 85σ, 0.839 e−/A3, and 84σ, 0.828 e−/A3, in the second active site. In the ICRMccatThr315Ala.mono-Zn2+.PEtN complex, the anomalous different density peak heights were 95.0σ, 0.96 e−/A3, and 81.6σ, 0.86 e−/A3, in one active site and 85.3σ, 0.90 e−/A3, and 84.3σ, 0.89 e−/A3, in the second active site. All geometry was verified using the Phenix and the wwPDB server.</p><!><p>Sequence orthologs of ICRMc, MCR-1, MCR-2, MCR-3, MCR-4, EptA, EptB, and EptC, were identified in Genbank using BLAST. Sequence alignment was performed using Jalview42 and verified by eye and inspection of structural features of ICRMc. Sequence alignment was produced by ESPript.43 Phylogenetic reconstruction was conducted using the aligned full-length PEtN transferase sequences and the program Mr. Bayes.44 Structure similarity searches were performed using the Dali server.45 Active site volume was calculated by the CastP server.46 Model of full-length ICRMc onto EptA was constructing using Phyre2.47 Dimerization interface burial in the ICRMccatThr315Ala crystal structure was calculated using the PDBePISA server48 and experimentally tested in solution using a Superdex S200 size exclusion column.</p>