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Substrate recognition by a colistin resistance enzyme from Moraxella catarrhalis
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Peter J Stogios, Georgina Cox, Haley L. Zubyk, Elena Evdokimova, Zdzislaw Wawrzak, Gerard D Wright, and Alexei Savchenko
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ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/ acschembio.8b00116 • Publication Date (Web): 09 Apr 2018 Downloaded from http://pubs.acs.org on April 10, 2018
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the
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ACS Chemical Biology Page 4 of 35 577
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ACS Chemical Biology
ACS Paragon Plus Environment
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Page 6 of 35
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Page 9 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
1
Substrate recognition by a colistin resistance enzyme from Moraxella
2
catarrhalis
3 4 5
Peter J. Stogios1, 2, Georgina Cox3,*, Haley L. Zubyk3, Elena Evdokimova1, 2, Zdzislaw
6
Wawrzak4, Gerard D. Wright3, Alexei Savchenko1,2,5,#
7 8
1
9
Ontario, Canada M5G 1L6
Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto,
10
2
Center for Structural Genomics of Infectious Diseases (CSGID)
11
3
Department of Biochemistry and Biomedical Sciences, Michael G. DeGroote Institute for
12
Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada L8S 4K1
13
4
14
United States
15
5
16
Health Research Innovation Centre, 3330 Hospital Drive NW, HRIC-2C66, Calgary, Alberta,
17
T2N 4N1
LS-CAT, Synchrotron Research Center, Northwestern University, Argonne, Illinois 60439,
Department of Microbiology, Immunology and Infectious Diseases, University of Calgary,
18 19 20
*Current address: Department of Molecular and Cellular Biology, University of Guelph,
21
Guelph, Ontario, Canada N1G 2W1
22
#
23
210-7980.
Corresponding author: Alexei Savchenko. Email: alexei.savchenko@ucalgary.ca Tel: (403)
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24
Abstract
25
Lipid A phosphoethanolamine (PEtN) transferases render bacteria resistant to the last resort
26
antibiotic colistin. The recent discoveries of pathogenic bacteria harboring plasmid-borne PEtN
27
transferases (mcr) genes have illustrated the serious potential for wide dissemination of these
28
resistance elements.
29
environmental niches with Enterobacteriaceae. Here, we describe the crystal structure of the
30
catalytic domain of the chromosomally-encoded colistin resistance PEtN transferase, ICRMc (for
31
intrinsic colistin resistance) of Moraxella catarrhalis. The ICRMc structure in complex with
32
PEtN reveals key molecular details including specific residues involved in catalysis and PEtN
33
binding. It also demonstrates that ICRMc catalytic domain dimerization is required for substrate
34
binding. Our structure-guided phylogenetic analysis provides sequence signatures defining
35
potentially colistin-active representatives in this enzyme family. Combined, these results advance
36
the molecular and mechanistic understanding of PEtN transferases and illuminate their origins.
The origin of mcr-1 is traced to Moraxella species co-occupying
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37
ACS Chemical Biology
Introduction
38
The polymyxin antibiotic colistin is classified as an antibiotic of critical importance by
39
the World Health Organization due to its activity against multi-drug resistant Gram-negative
40
pathogens 1. Colistin is utilized as a treatment of last resort for recalcitrant infections such as
41
those caused by carbapenem-resistant Enterobacteriaceae. Even though colistin is nephrotoxic,
42
the seriousness of such infections and the dearth of alternative treatments has increasingly
43
secured a place for this antibiotic in the infectious disease treatment armory in the absence of
44
new antibiotics 2.
45
Many species are intrinsically-resistant to polymyxins, including Neisseria meningitidis, 3,4
46
Helicobacter pylori, Burkholderia cepacia and Moraxella catarrhalis
47
resistance harbored by these bacteria include expression of efflux pumps, shedding of capsule
48
polysaccharide and modification of lipopolysaccharide (LPS), the latter of which appears the
49
most prevalent (reviewed in 5). Diverse covalent modifications of LPS, which include among
50
others the addition of phosphoethanolamine (PEtN) groups (reviewed in 6) are thought to alter
51
the physical properties of the outer membrane, resulting in polymyxin resistance. The increased
52
clinical and agricultural use of colistin has been linked to the mobilization and transfer of colistin
53
resistance elements to human pathogenic bacteria such as Escherichia coli, Klebsiella
54
pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii, representing serious
55
threats to the effectiveness of polymyxins against these species 3.
. The mechanisms of
56
Well-characterized representatives of PEtN transferases include EptA from E. coli, V.
57
cholerae and H. pylori, EptA (originally named LptA) from N. meningitidis, PmrC from A.
58
baumannii and CptA from Campylobacter jejuni (reviewed in 6). These chromosomally-encoded
59
enzymes catalyze the transfer of PEtN from phosphatidylethanolamine (PE) onto the lipid A
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moiety of LPS. E. coli EptB is another type of PEtN transferase which specifically catalyzes the
61
modification of the outer 3-deoxy-D-manno-octulosonic acid (Kdo) residue of LPS 7.
62
The recent discovery of the PEtN transferase encoded by the mcr-1 gene 8 mobilized on a
63
plasmid raised the possibility of accelerated dissemination and acquisition of colistin resistance
64
among pathogens and/or its collection into multi-drug resistance cassettes 9. Indeed, this gene
65
has since been detected in other Enterobacteriaceae species around the world, in additional
66
patient isolates and in retrospective searches of culture collections
67
found on mobile genetic elements containing up to nine other antibiotic resistance genes,
68
including extended-spectrum β-lactamases (ESBLs)
69
mobilization of colistin resistance, additional genes encoding PEtN transferases including mcr-2,
70
which shares 81% sequence identity with mcr-1, plus the more sequence-distinct mcr-3, mcr-4
71
and mcr-5 have been recently discovered on Enterobacteriaceae plasmids
72
transferases have mobilized multiple times and thus are becoming worrisome resistance
73
determinants that may have serious and long-lasting clinical implications.
10,14,18
.
10-17
. mcr-1 has also been
As further evidence of the
19-21
. Thus, PEtN
74
Resistance genes manifesting in the clinic can often be traced to environmental and other
75
bacteria which represent a global reservoir of resistance genes that can be transferred between
76
diverse genera via mobile genetic elements (reviewed in
77
clinical levels of antibiotic resistance such as qnrA and blaCTX-M were subsequently traced to
78
aquatic bacteria (reviewed in
79
suggested for mcr-1 and similar PEtN transferase genes. Sequence similarity searches identify
80
close MCR-1 and MCR-2 orthologs (61-65% identity of the full-length protein sequences) in the
81
Moraxellaceae family, specifically in the human pathogen Moraxella catarrhalis, the nematode
82
symbiont Moraxella osloensis, and the environmental bacteria Enhydrobacter aerosaccus and
24
).
22,23
). For example, genes conferring
Along the same lines, environmental paralogues were
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83
Psychrobacter spp. 8. Based on these observations, it was suggested that Moraxella species
84
occupying the same ecological niche as E. coli encode the ancestral gene of mcr-1 and mcr-2 19;
85
a subsequent study indeed detected mcr-1 orthologous genes in numerous Moraxella species
86
Thus, Moraxella or related species may represent an ecological reservoir for mobilization of
87
PEtN transferases to other pathogens; however, the molecular and functional characterization of
88
these ancestral MCR-1 proteins has been lacking.
25
.
89
According to sequence analysis, PEtN transferases are comprised of an N-terminal
90
transmembrane region and a periplasm-facing C-terminal hydrolase domain belonging to the
91
alkaline phosphatase superfamily. The periplasmic catalytic domains of MCR-1, MCR-2, EptA
92
and EptC have been recently structurally characterized. Some of the revealed common molecular
93
features observed in the active site of these enzymes included a conserved catalytic threonine
94
(often covalently linked to phosphate) and multiple Zn2+ binding sites
95
structures of PEtN catalytic domains also featured disulfide bridges between spatially co-
96
localized cysteine residues. These structural data provided the molecular framework for
97
understanding of PEtN transferases, however, the molecular information concerning substrate
98
recognition is still lacking. Furthermore, significant sequence divergence between EptA and
99
EptC and MCR-1 (EptA and EptC both share only 42% of sequence identity to MCR-1 in the
100
catalytic domain) necessitates further structural and functional studies into closer representatives
101
of the latter enzyme to define the possible role of these enzymes in development of colistin
102
resistance.
26-33
.
The crystal
103
In this study, we characterized the crystal structure of the catalytic domain of the PEtN
104
transferase from M. catarrhalis (which we designate as ICRMc), the protein that represents the
105
closest known ortholog to MCR-1 and MCR-2 and is responsible for intrinsic colistin resistance
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of this bacterium. Our crystal structures also provide insights into the molecular basis of
107
recognition of the PEtN substrate and identify a putative role of dimerization for resistance
108
activity.
109
characteristics in their catalytic domains, supporting the hypothesis that Moraxellaceae are the
110
environmental reservoir of PEtN transferases.
Our findings show that MCR-1 and ICRMc share key structural and functional
111 112
Results and Discussion
113
icrMc confers polymyxin resistance
114
To test the ability of ICRMc to confer the resistance we expressed icrMc in E. coli
115
BW25113 and compared its ability to grow in the presence of colistin and polymyxin B with that
116
of the same E. coli strain expressing mcr-1. Both the icrMc and mcr-1 expressing strains showed
117
4 to 16 fold (depending on promoter strength/expression level
118
antibiotics compared to the E. coli strain with no expression vector (Table 1). Next we tested
119
whether the expression of the icrMc or mcr-1 fragments corresponding to the PEtN transferase
120
periplasmic catalytic domain (designed icrMccat and mcr-1cat) in the same E. coli strain is
121
sufficient to confer polymyxin resistance. The results showed that E. coli harboring icrMccat or
122
mcr-1cat did not show a polymyxin resistance phenotype. These results demonstrated that
123
expression of icrMc confers polymyxin resistance comparable to that of mcr-1 and that both
124
enzymes require their N-terminal transmembrane regions to confer drug resistance in the cell.
34
) higher resistance to these
125 126
MCR-1, MCR-2 and ICRMc define a distinct group in the broader PEtN transferase family
127
To clarify the phylogenetic relationship between ICRMc and other PEtN transferases, we
128
performed a phylogenetic reconstruction of the PEtN transferase family, including 89 known
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MCR and related proteins (Figure 1). This analysis revealed five clades which segregate along
130
their substrate specificity. One of the clades is represented by MCR-3, MCR-4 and EptA, known
131
to catalyze transfer PEtN from PE to lipid A. The second one features orthologs of MCR-5 found
132
in Legionella species. The third clade is represented by EptB, which acts on Kdo, while the
133
fourth clade includes EptC/CptA, which target lipid A. The fifth clade contains orthologs of
134
MCR-1 and MCR-2, which we termed as MCR-1-like.
135
divergence reflected in low amino acid sequence similarity between representatives of different
136
clades. Specifically, EptB and MCR-3 shared only 25% and 32% of sequence identity with
137
MCR-1. Within the MCR-3/MCR-4/EptA clade, the MCR-3 and MCR-4 enzymes appear to be
138
distinct from the rest of their orthologs, suggesting separate evolutionary paths or ancestral
139
origins. These observations are consistent with multiple incidents of mobilization of intrinsic
140
PEtN ancestral genes leading to diversification among members of this family and reflecting
141
different functional properties. Consistent with previous reports
142
MCR-1-like
clade
contains
a
8,25
The five clades show significant
, our phylogenetic reconstruction recapitulated that
143
the
closely
associated
144
Gammaproteobacteria that share between 57 and 98% of sequence identity with MCR-1,
145
including MCR-1, MCR-2 and members in Moraxellaceae species: ICRMc, ICRM.
146
ICRE .aerosaccus. Beyond this group, the MCR-1-like clade contained a group of uncharacterized
147
proteins, including representatives from Sulfurospirillum spp., Arcobacter spp., Campylobacter
148
sputurum and Marinobacter subterrani and these share between 41 and 79% identity with MCR-
149
1. Notably, this group also shares significant similarity with the EptA clade. These observations
150
indicate that the ability to confer colistin resistance established for MCR-1 and ICRMc may be
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group
of
sequences
from
osloensis
and
7
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shared by the wider sequence-related group of PEtN transferases, which forms a distinct clade in
152
the PEtN transferase superfamily.
153
Alignment of PEtN transferase sequences (Figure S1) revealed higher degree of sequence
154
conservation in the C-terminal region and (i.e. 68% sequence identity between ICRMc and MCR-
155
1 or MCR-2). Combined with the comparable resistance phenotype conferred by expression of
156
these enzymes, this points to strong conservation of functional and structural features between
157
the ICRMc and MCR-1/MCR-2 enzymes.
158 159
Crystal structure of the catalytic domain of ICRMc
160
Next, we purified and crystallized the catalytic domain of ICRMc (ICRMccat) spanning
161
residues 235 to 578 (Figure 2A). The crystal structure of this domain was determined to 2.61 Å
162
by Molecular Replacement using the structure of N. meningitis EptA/LptA 26 as a model. The x-
163
ray crystallographic statistics are summarized in Table 2.
164
The crystal structure contains one copy of the ICRMccat domain in the asymmetric unit.
165
Residues 244 through 578 could be traced in the electron density, while residues 235-243 appear
166
disordered.
167
characterized by a central seven-stranded β-sheet mainly parallel in topology, with the edge
168
strand β12 oriented in an anti-parallel orientation (Figure 2A).
The resolved ICRMccat residues adopted the common sulfatase/hydrolase fold,
169
The structure of ICRMccat reveals two unambiguous disulfide bonds (Cys390 to Cys398,
170
Cys448 to Cys456). Another pair of cysteine residues, Cys311 and Cys321 appear reduced,
171
however the proximity of their side chains suggests the possibility of formation of an additional
172
disulfide bond (Figure 2A). We interpreted additional difference electron density in the structure
173
as six sulfate and one phosphate ions. Unexpectedly, the ICRMccat crystal structure did not
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contain additional electron density corresponding to the expected Zn2+ ion required for PEtN
175
transferases
176
ICRMccat purification, we soaked ICRMccat protein crystals in zinc chloride-supplemented buffer
177
(see Methods for details). We detected significant anomalous diffraction at the Zn2+ dispersion
178
absorption peak for such crystals and identified additional electron density corresponding to a
179
single Zn2+ atom bound to ICRMccat active site (Figure 2A, Figure 3).
26,27,29-33
. Interpreting this as possible depletion of Zn2+ ions in the course of
180
The active site region of the ICRMccat localized to a small (~200 Å3) pocket at the “top”
181
of the central β-sheet (as shown in Figure 2B) lined by residues from converging loops. This
182
pocket was on the large, flat face of the ICRMccat domain close to its N-terminus. Apart from the
183
Zn2+, the active site contains a phosphate ion at 3.7 Å distance from the Zn2+, which is anchored
184
by a network of interactions with the active site residues (Figure 3). The Zn2+ ion is coordinated
185
primarily by the side chains of Glu273, Thr315, Asp498 and His499. The phosphate forms
186
interactions with the sidechain hydroxyl and backbone amide of Thr315, the sidechain of His511
187
and two ordered water molecules, one of which also interacted with the Zn2+ ion. The Zn2+ and
188
phosphate-binding residues are in turn oriented by a network of interactions with other active site
189
residues including Asn359, Ser360 and His424. Notably, Thr315 in the ICRMccat active site
190
occupies the position corresponding to the phosphate-accepting nucleophilic residue in the other
191
PEtN transferases26,27,29-32. Analysis of ICRMccat residues interacting with both Zn2+ and the
192
phosphate ions also highlights His429 as a functionally important residue, although its
193
interaction with the Zn2+ was mediated through a hydrogen bond with the coordinated water
194
molecule. In addition to the Zn2+ and phosphate ions, the ICRMccat active site contained a sulfate
195
ion positioned at 11 Å distance from the phosphate ion described above. The sulfate forms close
196
interactions with Arg436, which in turn forms a salt bridge with Glu397. In total, our analysis
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197
suggested that at least eighteen ICRMc active site residues participate in interactions with the
198
Zn2+, phosphate and/or sulfate ligands.
199 200
Substrate recognition requires the dimerization of the ICRMccat domain
201
To characterize the molecular basis of substrate recognition by ICRMccat, we crystallized
202
Thr315Ala ICRMccat variant in the presence of the disaccharide head-group of lipid A or PEtN, a
203
component of the lipid substrate phosphatidylethanolamine.
204
nucleophilic residue that attacks the substrate phosphate group. Consistent with this prediction,
205
mutation to Ala abolishes antibiotic resitance activity (Table 1). This crystal structure was
206
determined to 1.64 Å (Table 2) and revealed a different unit cell and packing compared to the
207
wild type ICRMccat. In contrast to the ICRMccat apo structure, the ICRMccatThr315Ala•PEtN
208
complex structure features two ICRMccatThr315Ala molecules associated as a homodimer with
209
~1190 Å2 buried at the dimerization interface. The ICRMccat protein chains in the PEtN complex
210
structure associate in a symmetrical “front-to-front” orientation, with the two active sites facing
211
each other (Figure 4A, 4B). Dimerization involves interactions between 38 residues in each
212
monomer and impacts both the shape and accessibility of the active sites. In the ICRMccat dimer
213
the entrance to the catalytic centers is buried along the dimerization interface with access through
214
deep clefts formed by this interface (Figure 4C). Consistent with this observed association of
215
ICRMccat domains, we also detected the formation of a dimer in solution by size exclusion
216
chromatography, which is more prominent in the presence of Zn2+ ions (Figure S2).
Thr315 is predicted to be the
217
Well-defined additional electron density allowed unambiguous positioning of one PEtN
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molecule in each active site of the ICRMccatThr315Ala dimer in the complex structure (Figure 5).
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The phosphate of PEtN molecule forms interactions with the Zn2+ ion and the side chain of
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His511, as well with the backbone of Ala315 and three water molecules. Intriguingly, the Tyr338
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from the partner monomer chain also contributes to coordination of the substrate molecule
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(Figure 5). Notably, the position of the phosphate group of PEtN molecule in the complex
223
structure corresponds to the position of the phosphate ion in the apo-structure. Orientation of the
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PEtN molecule in the ICRMccat active site is consistent with the predicted catalytic mechanism of
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this family whereby a threonine residue (Thr315 in ICRMc) performs nucleophilic attack on the
226
phosphate atom of the phosphatidylethanolamine substrate to generate a phosphoethanolamine –
227
Thr intermediate species. We did not observe any interactions between the ethanolamine moiety
228
of PEtN and the enzyme. Taken together, the structural analysis of the ICRMccatThr315Ala• PEtN
229
complex suggests that dimerization of this domain may facilitate the coordination of the
230
substrate providing an additional interaction partner (Tyr338) from adjacent monomer that may
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be required for optimal positioning of the phosphatidylethanolamine substrate for catalysis.
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One of the tested ICRMccatThr315Ala crystals obtained in the presence of lipid A head-group
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lacked the additional density corresponding to the substrate molecule but featured a second Zn2+
234
ion (Zn2 in Figure S3) in the active sites of the ICRMccat dimer.
235
corresponds to that of the phosphate of PEtN in complex structure. In this structure, the Zn2 is
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coordinated by side chains of residues Glu273, His429 and His511 and the backbone amide of
237
Ala315. In this interaction, the His429 underwent a rotamer change relative to its position in the
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ICRMc apo-structure. Thus there could be an interplay between the binding of a second Zn2+
239
atom and the phosphate groups of the enzyme’s substrates. Furthermore, the active sites of
240
ICRMccatThr315Ala•di-Zn2+ structure contain elongated difference electron density corresponding to
241
a polyethylene glycol molecule present in the crystallization solution (Figure S3). The position of
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this molecule in the active site approaches the catalytic center of the enzyme and forms a packing
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interaction against His429 and hydrophobic interactions with Met426 and the aliphatic region of
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the Lys378 sidechain. Accordingly, we propose that the position of the polyethylene glycol
245
molecule may be indicative of the position of the lipid A head-group in the ICRMccat active site.
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Guided by our structural analysis, we tested the roles of ICRMccat residues implicated in
247
interactions with Zn2+ and PEtN by site-directed mutagenesis. Residues Thr315, His429, Arg436
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and Tyr338 were individually mutated to Ala in the context of the full-length ICRMc protein. The
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E. coli strains expressing the corresponding ICRMc variants completely lost resistance to colistin
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and polymyxin B compared to the strain carrying the wild type enzyme, confirming the
251
important role of these residues for ICRMc activity (Table 1). Based on these results we postulate
252
that coordination of Zn2+ binding by Thr315, His429, Tyr338 residues and unexpectedly,
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interactions of residues Tyr338 and Arg436 with substrate molecules as facilitated by ICRMccat
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domain dimerization, may be essential for the catalytic activity of ICRMc.
255 256
Comparative structural analysis highlights signature active site features of the PEtN
257
transferase family
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We conducted a comparative analysis of this structure versus those of recently
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characterized MCR-1 and MCR-2 resistance enzymes as well as the structures of previously
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characterized EptA and EptC to clarify the shared and distinct features of these PEtN
261
transferases.
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The ICRMc structure superimposes with that of MCR-1 and MCR-2 with RMSD of 0.5 Å
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and 0.5 Å over 251 and 270 matching Cα atoms, respectively (Figure 6). This superimposition
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reveals only minor structural differences primarily in conformations of the β5-η4 and β6-η5
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loops. More importantly, all 18 residues of the active site (Figure 6) are absolutely conserved
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across these three enzymes. Furthermore, these 18 amino acids, which includes the histidine
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(His429) residue undergoing rotation between binding to PEtN or Zn2+ ion in ICRMc structures,
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are universally conserved across the MCR-1-like PEtN transferases (Figure S1). Interestingly,
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the Tyr338 residue, whose role in PEtN binding is facilitated by catalytic domain dimerization, is
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also conserved in the orthologs of ICRMc, MCR-1 and MCR-2. This observation prompted us to
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suggest that the active site architecture, binding mode of PEtN, and coordination of substrates
272
across a dimer interface as visualized in the ICRMc cat-PEtN complex structure is conserved
273
across the MCR-1-like clade of PEtN transferases defined above.
274
Expanding our structure-based sequence analysis to the recently-identified MCR-3,
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MCR-4 and MCR-5 enzymes revealed conservation of 12 out of 18 active site residues
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invariable in MCR-1-like enzymes (Figure S1). These include residues involved in coordination
277
of the Zn2+ ion and in catalysis. Notably, we were not able to identify the equivalent of ICRMccat
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residue Tyr338 in MCR-3 or MCR-4. These differences may be indicative of deviation in
279
substrate-binding mode between MCR-1-like PEtN transferases and the MCR-3, MCR-4 and
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MCR-5 enzymes.
281
Detailed comparative analysis of ICRMc and EptA and EptC revealed differences in the
282
conformations of the β5-η4 and β6-η5 loops and in active site composition (Figure S4). Of the
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18 active site residues in MCR-1-like enzymes described above, only 11 are conserved in EptA
284
and EptC (Figures S1 and S4). Once again, the conserved residues in EptA and EptC active sites
285
cluster to the Zn2+ binding sites, consistent with a common catalytic mechanism shared across
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the entire PEtN transferase family. In contrast, the four non-conserved residues corresponding to
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ICRMc residues Asn359, Ser360, Lys378 and Met426 cluster together and away from the Zn2+
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binding site. Identification of these subtle differences may be reflective of EptA and EptC
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deviating in substrate preference from MCR-1-like enzymes.
290
Our observation that the dimeric state of ICRMccatThr315Ala forms a solvent-occluded
291
pocket containing a PEG molecule suggests that only in the dimer state of the catalytic domain,
292
or in the context of the full-length protein containing the transmembrane region, is the protein
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capable of forming an active site structure that can recognize the larger lipid A substrate. Indeed,
294
analysis of the crystal structure of EptA, the MCR-1 ortholog from N. meningitidis, suggested
295
that the membrane-spanning portion and the linker to the catalytic domain are involved in
296
mediating substrate interactions35. The detergent (DDM) molecule trapped in the membrane-
297
spanning portion of EptA is oriented toward the active site of the catalytic domain suggesting it
298
mimicks the position of enzyme’s substrate (Figure 7). With our ICRMccatThr315Ala•PEtN crystal
299
structure in hand, we are able to posit a model of the ternary complex with a lipid positioned to
300
attack the phosphoethanolamine-threonine intermediate (Figure 7). This model was developed
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through a superposition of the structures of EptA•DDM and ICRMccatThr315Ala•PEtN which
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showed that the position of these two ligands do not overlap: more specifically, the O3B of
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DDM and O3 of PEtN approached each other in this model and thus this overlay can be used to
304
interpret the position of a phosphorylated lipid molecule in the active site (Figure 7). This model
305
also validates the prediction that EptA Glu114 interacts with the amine group of PEtN 35 as this
306
residue and its equivalent in ICRMc (E113) is positioned within bonding distance to this
307
functional group. Consistent with its functional importance, this glutamate residue is conserved
308
throughout the PEtN transferase family (except for the EptB subgroup), suggesting it plays a role
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in discrimination for PE. Further structural and enzymatic studies into PEtN transferase and
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their interactions with native substrates are required to further validate this model and reveal
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additional key details.
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The binding of substrates may require the ICRMccat domain’s dimerization which
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according to our structural analysis provides additional interaction partners for the substrate
314
molecules in both actives sites of the dimer. A molecular dynamics simulation of EptA
315
showed that the soluble domain can adopt multiple conformations and “roll” over the simulated
316
membrane surface, indicating that the dimerization interface we observed in ICRMc complex
317
structure may be accommodated in such a fluid situation.
35
318
Our demonstration of the conservation of the molecular details of the ICRMc, MCR-1 and
319
MCR-2 active sites supports the notion that Moraxella species form the reservoir of at least this
320
group of PEtN transferases. This stresses that structural and functional studies of intrinsic
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resistance enzymes in species co-habitating environmental niches as human pathogens are key
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components of developing a better understanding of the origins and mechanisms of resistance
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impacting clinical management of pathogens. As has been evidenced in numerous examples,
324
and even in the ongoing discovery of new mcr genes, the source of clinical antibiotic resistance
325
is the environment. Progress towards developing a complete understanding of the broader
326
resistome, such as our characterization of ICRMc, is a key component of resistance surveillance
327
and anticipation of future mobilization events.
328
Finally, our structure-based phylogenetic analysis provides a foundational organization of
329
PEtN transferases that clearly shows that these enzymes are diverse in sequence and each
330
discovery of novel mcr genes identifies founding members of subclasses of PEtN transferases.
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This phenomenon parallels the expansion of β-lactamase families over the course of decades of
332
β-lactam clinical deployment
36
and underscores the extent of the dissemination of polymyxin
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resistance. As with β-lactamase family-specific properties, functional insights into individual
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PEtN transferases may not be conserved across the subgroups.
335
structural and molecular diversity of MCR enzymes is critical for drug discovery efforts against
336
these enzymes.
Thus, appreciation of the
337 338
Acknowledgements
339
We thank R. Di Leo for cloning. The structures presented were solved by the Center for
340
Structural Genomics of Infectious Diseases (CSGID, http://csgid.org); this project has been
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funded in whole or in part with U.S. Federal funds from the National Institute of Allergy and
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Infectious Diseases, National Institutes of Health, Department of Health and Human Services,
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under Contract Numbers HHSN272201200026C and HHSN272201700060C. This research
344
was supported by the Ontario Research Fund Research Excellence (ORF-RE) grant #RE07-048
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(to G.D. Wright and A. Savchenko). This research was also supported by a Canadian Institutes of
346
Health Research grant (FRN-148463) and by a Canada Research Chair in Antibiotic
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Biochemistry (to G.D. Wright).
348 349
References
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489
Figure Legends
490 491
Figure 1. Phylogenetic relationships of PEtN transferases. The four major clades are
492
independently colored and labeled. Nodes are labeled by Bayesian probability. Arrow labels
493
ICRMc sequence. Protein sequences with an assigned name are labeled with the name plus
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originating species in superscript initials, those proteins identified in mobile elements are labeled
495
with the protein name only. Protein sequences without an assigned name are indicated only with
496
the species name.
497
498
Figure 2. Structure of ICRMc•mono-Zn2+ complex. A) Domain architecture, with termini of
499
the TM and catalytic domains indicated. B) Overall structure. Secondary structure elements are
500
labeled, active site sulfate, phosphate molecules and amino acids are shown in sticks, Zn2+ ion as
501
a black sphere, and cysteines / disulfide bonds with yellow sticks. C) Electrostatic surface
502
representation. Scale of colors is from red = -10 kB/T, white = 0, blue = +10. N-terminus of this
503
domain (residue 243) is labeled.
504
molecules. Dashed black line indicates active site cleft.
Sticks are shown for active site phosphate and sulfate
505 506
Figure 3. Active site of ICRMc•mono-Zn2+ complex. The Zn2+ ion is shown as a grey sphere
507
with purple electron density (anomalous difference map contoured at 20 σ), sulfate electron
508
density is Fo-Fc difference map contoured at 5 σ. Dashes indicate hydrogen bonds or salt bridge
509
interactions. Water molecules are shown as red spheres. The distance between the Zn2+ ion and
510
sulfate ion is indicated.
511 512
Figure 4. Structure of dimeric ICRMccatThr315Ala•mono-Zn2+•PEtN complex. A) Two chains
513
are independently coloured. Zn2+ shown in grey spheres, PEtN shown in green ball-and-stick. B)
514
Surface representation. Two entry paths to active sites are labeled. White shading indicates
515
residues in active site clefts. Bottom view is rotated 90° from the top view in the plane of the
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page and cut-away for visualization into the solvent-occluded active sites.
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ACS Chemical Biology
517 518
Figure 5. Active site of dimeric ICRMccatThr315Ala•mono-Zn2+•PEtN complex. A) Same view
519
is shown as in Figure 3. Residues coloured light green are from the partner chain in the dimer.
520
The Zn2+ ion is shown as a grey sphere with anomalous difference density contoured at 20 σ.
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PEtN is shown in ball-and-stick with Fo-Fc difference density contoured at 3.0 σ. Dashes indicate
522
hydrogen bonds or salt bridge interactions. Water molecules are shown as red spheres. B)
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Schematic of Zn2+ (shown as Zn1) and PEtN recognition by ICRMccatThr315Ala.
524 525
Figure 6. Comparison of structures of ICRMc, MCR-1 and MCR-2 catalytic domains. Left =
526
overall superposition of the three structures, ICRMc shown in pink, MCR-1 in light blue, MCR-2
527
in orange. Zoom = active site residues. Parenthesis indicate mutation A315 in ICRMc , asterisks
528
indicate Y308 from MCR-1 and MCR-2 are not shown as their crystal structures were not
529
dimeric. PEtN as bound to ICRMccatThr315Ala is shown in green sticks.
530 531
Figure 7. Model of full-length ICRMc•mono-Zn2+•PEtN complex based on the structure of
532
N. meningitidis EptA.
533
Left = overall structures, with ICRMc shown in dark red and EptA in shades of green. DDM as
534
bound to EptA and PEtN as bound to ICRMc are shown in black and green ball-and-stick,
535
respectively. Dashed lines = putative plane of inner membrane. Right = zoom of active site.
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Residues interacting with Zn2+, DDM or PEtN are shown in sticks and labeled. Double-ended
537
arrow indicates close approach of E114/E143 with amine group of PEtN. Box indicates overlap
538
in spatial position of DDM O3B and PEtN O3 atoms. BH = base helix; PH2 and PH2’ =
539
periplasmic helices 2 and 2’ (nomenclature from 35).
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Page 30 of 35
540 541
Tables
542
Table 1. Heterologous polymyxin resistance conferred by icrMc and mcr-1.
543 Gene None
icrMc
mcr-1
Construct None None pGDP1::icrMc pGDP2::icrMc pGDP1::icrMcThr315Ala pGDP1::icrMcTyr338Arg pGDP1::icrMcHis429Ala pGDP1::icrMcArg436Ala pGDP1::icrMccat pGDP2::icrMccat pGDP1::icrMc pGDP2::icrMc pGDP1::icrMcThr315Ala pGDP1::icrMcTyr338Arg pGDP1::icrMcHis429Ala pGDP1::icrMcArg436Ala pGDP1::icrMccat pGDP2::icrMccat pGDP1::mcr-1 pGDP2::mcr-1 pGDP1::mcr-1cat pGDP2::mcr-1cat pGDP1::mcr-1 pGDP2::mcr-1 pGDP1::mcr-1cat pGDP2::mcr-1cat
Antibiotic Colistin Polymyxin B
Colistin
Polymyxin B
Colistin
Polymyxin B
MIC (µg/mL) 0.25 0.25 2 1 0.25 0.25 0.25 0.25 0.25 0.25 2 1 0.25 0.25 0.25 0.25 0.25 0.25 4 4 N.D. 0.25 4 4 N.D. 0.5
544
Susceptibility testing was performed with E. coli BW25113; pGDP1 = Pbla promoter; pGDP2 =
545
Plac promoter. N.D. = no data.
546 547
Table 2. X-ray diffraction data collection and refinement statistics
548 Structure
ICRMc•PO4 complex
ICRMc•mono-Zn2+ complex
ICRMc Thr315Ala•diZn2+•PEG complex
PDB Code
6BNE
6BNF
6BNC
ICRMc Thr315Ala•monoZn2+•PEtN complex 6BND
Data collection Space group
P4132
P4132
P21212
P21212
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549 550 551 552 553 554 555
ACS Chemical Biology
Structure
ICRMc•PO4 complex
ICRMc•mono-Zn2+ complex
ICRMc Thr315Ala•diZn2+•PEG complex
PDB Code Cell dimensions a=b=c (Å) Resolution, Å Rmergea Rpim CC1/2 I / σ(I) Completeness, % Redundancy
6BNE
6BNF
6BNC
ICRMc Thr315Ala•monoZn2+•PEtN complex 6BND
147.52 104.32 – 2.61 0.181 (3.313)b 0.028 (0.515) 0.840 24.7 (2.6) 100 (100) 77.3 (79.5)
148.27 104.84 – 2.33 0.161 (3.941) 0.025 (0.672) 0.841 24.0 (2.0) 100 (100) 76.5 (66.5)
74.05, 154.25, 66.39 30.00 – 1.50 0.088 (0.595) 0.026 (0.203) 0.982 30.68 (4.1) 99.9 (100) 11.9 (8.8)
74.52, 155.24, 66.41 31.80 – 1.64 0.108 (1.950) 0.044 (0.827) 0.594 14.5 (1.3) 100 (100) 13.4 (12.6)
Refinement Resolution, Å 60.23 – 2.61 46.88 – 2.33 29.82 – 1.50 31.81 – 1.64 No. of unique 16528, 830 23489, 2163 121223, 10848 94685, 3840 reflections: working, test R-factor/free R23.6/27.1 (48.0/48.7) 20.9/24.8 (40.8/46.4) 13.2/15.3 (19.8/23.4) 15.1/19.2 (34.2/32.7) factorc No. of refined atoms, molecules 5293 5255 Protein 2602 2607 2 Zinc N/A 1 4 Phosphate* 5 5 N/A N/A 5 5 N/A N/A Sulfate* N/A N/A 16 PEtN N/A 37 20 42 103 Other solvent 209 1093 1128 Water 116 B-factors Protein 79.1 67.8 17.3 26.9 Zinc N/A 63.8 11.2 26.9 Phosphate* 108.0 82.0 N/A N/A Sulfate* 87.6 78.6 N/A N/A PEtN N/A N/A N/A 29.1 Other solvent 116.2 95.0 29.3 22.9 Water 65.44 66.6 36.2 46.2 r.m.s.d. Bond lengths, Å 0.006 0.002 0.017 0.006 1.380 1.274 1.468 1.319 Bond angles, ° a Rmerge = ΣhΣi|Ii(h) - 〈I(h)〉/ΣhΣiIi(h), where Ii(h) and 〈I(h)〉 are the ith and mean measurement of the intensity of reflection h. b Figures in parentheses indicate the values for the highest resolution shells of the data. c R-factor = Σ|Fpobs – Fpcalc|/ΣFpobs, where Fpobs and Fpcalc are the observed and calculated structure factor amplitudes, respectively. * = molecules in the active site cleft.
556
Methods
557
MIC determination
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Page 32 of 35
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Full-length icrMc and its catalytic domain (icrMccat; residues 235-578) plus full-length E.
559
coli mcr-1 and its catalytic domain (mcr-1cat; residues 208-541) were ligated into the pGDP1
560
and pGDP2 vectors (in house derivatives of the pBR322 vector; New England BioLabs). These
561
plasmids utilize constitutive promoters of differing strength; the stronger β-lactamase Pbla
562
promoter (pGDP1) and the weaker Plac promoter (pGDP2). Amino acid substitutions in icrMc
563
were performed using Phusion High-Fidelity DNA Polymerase (Thermo Scientific) and the
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pGDP1:icrMC construct. Constructs were confirmed by sequencing (Central MOBIX facility,
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McMaster University). Susceptibility testing (in triplicate, with two biological replicates) was
566
performed with E. coli BW25113 cultured in Mueller-Hinton II broth (cation adjusted) (Becton,
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Dickinson and Co, Franklin Lakes NJ) using the broth microdilution method according to CLSI
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guidelines. Inoculum was prepared using the colony resuspension method and plates were
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incubated with aeration at 37°C for 18 h.
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Protein expression and purification
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cDNA of icrMc from Moraxella catarrhalis strain BC1 (Genbank accession EGE18576)
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coding for residues 235 to 578 of ICRMc was synthesized by Integrated DNA Technologies and
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subcloned into the pMCSG53 expression vector that codes for a N-terminal His6-tag and a TEV
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protease cleavage site. icrMc was expressed in E. coli BL21(DE3) Gold cells, grown to an OD600
576
of 0.6 at 37 °C, chilled to 16 °C and induced overnight with 500 µM isopropyl β-D-
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thiogalactopyranoside.
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resuspended in binding buffer [50 mM Hepes (pH 7.5), 100-300 mM sodium chloride, 10 mM
579
imidazole and 2% glycerol (v/v)], lysed by sonication, and cell debris removed via centrifugation
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at 30,000 x g. Cleared lysate was loaded onto a 5-mL Ni-NTA column (QIAGEN) pre-
Cells were harvested via centrifugation at 5000 x g and pellets
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ACS Chemical Biology
581
equilibrated with binding buffer, extensively washed with binding buffer containing 30 mM
582
imidazole, and protein was eluted using the above buffer with 250 mM imidazole. Fractions
583
containing ICRMc were identified by SDS-polyacrylamide gel electrophoresis and further purified
584
via gel filtration on a HiLoad 16/60 Superdex75 prep-grade column [10 mM Hepes (pH 7.5) and
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50 mM potassium chloride].
586 587
Crystallization and structure determination
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All crystallization was performed at room temperature using the sitting drop method and 2 µL
589
protein or protein:ligand mixture plus 2 µL reservoir solution. Zn2+-free ICRMc was crystallized
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with reservoir solution 0.1 M sodium acetate pH 4.6 and 1.2 M ammonium sulfate and the
591
crystal was cryoprotected in 8% glycerol, 8% ethylene glycol and 8% sucrose. Crystals of Zn2+-
592
bound mono-Zn2+•ICRMc were obtained by soaking apo crystals with 2.5 mM zinc chloride for
593
30 minutes followed by cryoprotection in the above cryosolution.
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ICRMccatThr315Ala•mono-Zn2+•PetN were obtained from reservoir solution 0.1 M MES pH 6, 22%
595
(w/v) PEG 400, 2.5 mM zinc chloride and 5 mM PetN and the crystal was cryoprotected in
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paratone oil. Crystals of ICRMccatThr315Ala•di-Zn2+•PEG were obtained from reservoir solution
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0.1 M Tris pH 8, 28% (w/v) PEG 4k, 3 mM zinc chloride and 2 mM lipid A head-group
598
(Glycobiotech GmbH) and the crystal was cryoprotected in paratone oil. All X-ray diffraction
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data at 100 K were collected at the Advanced Photon Source, Argonne National Laboratory, Life
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Sciences Collaborative Access Team beamline 21-ID-D at the following wavelengths
601
corresponding to the measured zinc absorption peak: ICRMc•PO4 – 1.27424 Å / 9730 keV;
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ICRMc•mono-Zn2+ – 1.27424 Å / 9730 keV; ICRMccatThr315Ala•mono-Zn2+•PEtN – 1.27687 Å /
603
9710 keV; ICRMccatThr315Ala•di-Zn2+•PEG - 1.27815 Å / 9700 keV. X-ray data were reduced with
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Page 34 of 35
604
XDS 37 and CCP4 Aimless 38 or HKL-3000 39 for the di-Zn2+•PEG structure. The WT structures
605
were solved by Molecular Replacement using the ICRMc sequence-substituted structure of
606
Neisseria meningitis EptA/LptA (PDB id 4kay
607
ICRMccatThr315Ala structures were phased using the anomalous signal from Zn2. Refinement was
608
completed with Phenix.refine and Coot41. All B-factors were refined as isotropic with TLS
609
parameterization. Zn2+, sulfate ions and the positions of the six cysteine residues implicated in
610
disulfide bond formation were verified by the presence of peaks in an anomalous difference map.
611
In the WT ICRMc structure, the peak in the anomalous difference electron density peak was 32 σ /
612
0.148 e-/A3. In the ICRMccatThr315Ala•di-Zn2+ structure, the anomalous difference density peak
613
heights were 91 σ/0.896 e-/A3 and 82 σ/0.827 e-/A3 in one active site, 85 σ/0.839 e-/A3 and 84
614
σ/0.828 e-/A3 in the second active site. In the ICRMccatThr315Ala•mono-Zn2+•PEtN complex, the
615
anomalous different density peak heights were 95.0σ/0.96 e-/A3 and 81.6σ/0.86 e-/A3 in one
616
active site and 85.3 σ/0.90 e-/A3 and 84.3 σ/0.89 e-/A3 in the second active site. All geometry
617
was verified using the Phenix and the wwPDB server.
26
) using Phenix.phaser
40
while the
618 619
Sequence and structural analysis
620
Sequence orthologs of ICRMc, MCR-1, MCR-2, MCR-3, MCR-4, EptA, EptB and EptC were
621
identified in Genbank using BLAST. Sequence alignment was performed using Jalview
622
verified by eye and inspection of structural features of ICRMc.
623
produced by ESPript43. Phylogenetic reconstruction was conducted using the aligned full-length
624
PEtN transferase sequences and the program Mr. Bayes44. Structure similarity searches were
625
performed using the Dali server
626
Model of full-length ICRMc onto EptA was constructing using Phyre2
45
42
and
Sequence alignment was
. Active site volume was calculated by the CastP server
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46
.
. Dimerization interface
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ACS Chemical Biology
627
burial in the ICRMccatThr315Ala crystal structure was calculated using the PDBePISA server 48 and
628
experimentally tested in solution using a Superdex S200 size exclusion column.
629 630
Supporting information
631
The Supporting Information is available free of charge at the ACS Publications website.
632
Sequence alignment of PEtN transferases, size exclusion chromatography of ICRMc,
633
structure of the active site of the dimeric ICRMccatThr315Ala•di-Zn2+•PEG complex, and
634
comparison of the structures of ICRMc with EptA and EptC (PDF).
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