Received: 2 December 2022 Revised: 21 April 2023 Accepted: 2 May 2023

DOI: 10.1002/prot.26510

RESEARCH ARTICLE

The structural analysis of the periplasmic domain of

Sinorhizobium meliloti chemoreceptor McpZ reveals
a novel fold and suggests a complex mechanism of
transmembrane signaling

Safoura Salar 1 | Nicolas E. Ball 2 | Hiba Baaziz 1 | Jay C. Nix 3 |

Richard C. Sobe 1 | K. Karl Compton 1 | Igor B. Zhulin 4 | Anne M. Brown 2 |
Birgit E. Scharf 1 | Florian D. Schubot 1

1
Department of Biological Sciences, Virginia
Polytechnic Institute and State University, Abstract
Blacksburg, Virginia, USA
Chemotaxis is a fundamental process whereby bacteria seek out nutrient sources and
2
Department of Biochemistry, Virginia
Polytechnic Institute and State University,
avoid harmful chemicals. For the symbiotic soil bacterium Sinorhizobium meliloti, the
Blacksburg, Virginia, USA chemotaxis system also plays an essential role in the interaction with its legume host.
3
Advanced Light Source, Lawrence Berkeley The chemotactic signaling cascade is initiated through interactions of an attractant or
National Laboratory, Berkeley, California, USA
4
Department of Microbiology and
repellent compound with chemoreceptors or methyl-accepting chemotaxis proteins
Translational Data Analytics Institute, The (MCPs). S. meliloti possesses eight chemoreceptors to mediate chemotaxis. Six of these
Ohio State University, Columbus, Ohio, USA
receptors are transmembrane proteins with periplasmic ligand-binding domains (LBDs).
Correspondence The specific functions of McpW and McpZ are still unknown. Here, we report the crys-
Florian D. Schubot, Virginia Polytechnic
tal structure of the periplasmic domain of McpZ (McpZPD) at 2.7 Å resolution.
Institute and State University, Department of
Biological Sciences, 5002 Derring Hall, McpZPD assumes a novel fold consisting of three concatenated four-helix bundle mod-
926 West Campus Dr, Blacksburg, Virginia
ules. Through phylogenetic analyses, we discovered that this helical tri-modular domain
24061, USA.
Email: fschubot@vt.edu fold arose within the Rhizobiaceae family and is still evolving rapidly. The structure,

Funding information
offering a rare view of a ligand-free dimeric MCP-LBD, reveals a novel dimerization
National Science Foundation; NIH Office of interface. Molecular dynamics calculations suggest ligand binding will induce conforma-
the Director
tional changes that result in large horizontal helix movements within the membrane-
proximal domains of the McpZPD dimer that are accompanied by a 5 Å vertical shift of
the terminal helix toward the inner cell membrane. These results suggest a mechanism
of transmembrane signaling for this family of MCPs that entails both piston-type and
scissoring movements. The predicted movements terminate in a conformation that
closely mirrors those observed in related ligand-bound MCP-LBDs.

KEYWORDS
chemotaxis, helical tri-modular sensor domain, ligand-binding domain, methyl-accepting
chemotaxis protein, piston, scissoring, transmembrane signaling

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any
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© 2023 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.

1394 wileyonlinelibrary.com/journal/prot Proteins. 2023;91:1394–1406.


SALAR ET AL.
1 | I N T RO DU CT I O N
Chemotaxis is the process used by bacterial cells to seek out
nutrient sources and eukaryotic hosts. 1–5 In addition, chemotaxis
signaling pathways facilitate bacterial escape from environmental
pollutants and other harmful chemicals, known as repellents.6,7
Escherichia coli serves as the classical model system for elucidat-
ing the underlying molecular mechanisms of the chemotaxis sys-
tem. 6,8 Universally, signaling is initiated by chemoreceptors or
methyl-accepting chemotaxis proteins (MCPs), a group of bacte-
rial receptors that detect environmental changes through special-
ized receiver domains. 6,9 Canonical MCPs are multidomain
proteins containing a variable periplasmic sensory domain
coupled to two conserved transmembrane helices and a large
4,8,9
equally conserved cytoplasmic region. Ligand binding regu-
lates the autophosphorylation activity of the MCP-associated
kinase CheA, which, in turn, controls phosphorylation of its cog-
nate response regulator CheY. 1 Binding of CheY-P to the cyto-
plasmic base of the flagellar motor causes the peritrichous
flagella to rotate in a clockwise (CW) direction ultimately result-
ing in a tumbling motion of the bacterial cells. Presence of an
attractant causes CheA inhibition, reversal to counterclockwise
(CCW) flagellar rotation, and smooth swimming toward the nutri-
ent source. A complex adaptive system ensures that receptors do
not become saturated as the bacterial cell migrates up the nutri-
ent gradient. 4,10–12 Due to the complexity of chemotaxis systems
and a scarcity of direct structural information for the transmem-
brane regions, many questions remain regarding the mechanism
of signal transduction.
Bacterial chemotaxis also plays an important role in establish-
ing the symbiotic relationship between the soil bacterium Sinorhi-
zobium meliloti and its legume host alfalfa.13,14 S. meliloti
possesses eight chemoreceptors, sequentially named McpT to
McpZ and IcpA (Internal Chemotaxis Protein A).15–17 McpZ is pre-
dicted to have an extraordinarily large and structurally distinctive
periplasmic domain for which the ligand is unknown. To identify
regions critical for ligand recognition and signal transduction, we
characterized the crystal structure of the periplasmic domain of
McpZ (McpZPD). The structure of McpZ PD, solved at 2.7 Å resolu-
tion, revealed a dimeric protein with a novel helical trimodular
(HTM) fold. Phylogenetic analysis suggests that this new receptor
class emerged relatively recently in the Rhizobiaceae family. The
HTM fold appears to have arisen through either internal gene
duplication within a gene encoding a helical bimodular (HBM)
receptor18 or the insertion of an unrelated gene fragment into an
HBM encoding sequence.
The distinctive dimeric structure of ligand-free McpZPD served as
a starting point for molecular dynamics (MD) calculations to explore
the conformational space of the protein and gain insights into the
potential signaling mechanism. Intriguingly, the observed move-
ments suggest McpZ seamlessly integrates elements of the two
prevailing models for MCP cross-membrane signaling.
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1395
2 | M A T E R I A L S A N D M ET H O D S
2.1 | Expression and purification of the McpZPD
The mcpZ PD coding sequence, encompassing the codons for amino
acids 39–424 of the original mcpZ gene, was expressed from plas-
mid pTYB11 in E. coli ER2566 to produce a fusion protein contain-
ing a self-cleaving N-terminal intein-chitin binding domain (intein-
CBD) tag. Four liters of cell culture were grown at 37  C in LB con-
taining 100 μg of ampicillin/mL until they reached an OD600 of
0.8. Gene expression was induced by adding 0.6 mM isopropyl-
β- D-thiogalactopyranoside (IPTG) and continuing growth at 16 C
for an additional 16 h. Cultures were then centrifuged, and cell
pellets were collected. Cells were suspended in buffer A (20 mM
Tris/HCl, 500 mM NaCl, 1 mM EDTA, and 10% glycerol [pH 8.0])
supplemented with 10 mg/mL of DNase, 1 mM PMSF, and 1
Halt™ Protease Inhibitor Cocktail (Thermo Fischer Scientific). Cells
were lysed with a French pressure cell (SLM Aminco, Silver Spring,
MD) at 16 000 lbs/inch,2 prior to clearing the lysate via centrifu-
gation at 56 000g for 1 h at 4 C. The clear supernatant was fil-
tered and loaded onto a chitin (New England BioLabs) affinity
column. On-column cleavage of the intein-CBD-tag was per-
formed by incubating the bound sample in cleavage buffer (buffer
A supplemented with 50 mM DTT) for 72 h at 4 C. The protein
was eluted with buffer A and concentrated using a 50 mL Amicon
stirred cell (Millipore, Bedford, MA, USA) with a 3 kDa MWCO
regenerated cellulose membrane. The concentrated protein was
further purified by loading the sample onto a HiPrep 26/60 Sepha-
cryl S-200 HR column (GE Healthcare Life Sciences) that was pre-
equilibrated in buffer B (100 mM HEPES, 100 mM NaCl, pH 7.0)
or in buffer C (100 mM tricine, 150 mM NaCl, 1 mM EDTA,
pH 8.0) for DSF and SEC-MALS experiments. Purity of the eluted
protein was assessed via SDS-PAGE. Fractions containing only
McpZ PD were pooled and concentrated to 120 μM via a 50-mL
Amicon stirred cell (Millipore, Bedford, MA, USA) with a 3 kDa
MWCO regenerated cellulose membrane and stored at 80  C.
The chromatogram for the elution from HiPrep 26/60 Sephacryl
S-200 HR column and an SDS-PAGE of the elution peak are pro-
vided in Figure S1A.
2.2 | Differential scanning fluorimetry
For the differential scanning fluorimetry (DSF) assay, a 30-μL solution
containing 50 μM McpZPD protein in buffer C and 2X SYPRO orange
was pipetted into a 96-well optical plate. Thermal denaturation was
performed in an ABI 7300 real-time PCR thermocycler (BioRad) by
increasing the temperature from 10 to 95 C with a 30 s equilibration at
each half degree celsius. The melting temperature (Tm) was calculated
by identifying the minimum of the negative first derivative of fluores-
cence intensity values. The described melting curve is provided in
Figure S1B.
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1396 SALAR ET AL.

2.3 | Multiangle light scattering analysis TABLE 1 X-ray diffraction data collection and refinement
statistics.

Size exclusion chromatography coupled with multiangle light scatter- Space group I 41 2 2 (#98)
ing (SEC-MALS) was employed to determine the oligomeric state of Unit cell: a, b, c (Å) 184.3, 184.3, 113.4
McpZPD in solution. To this end, 40 μM McpZPD protein in buffer C 
Unit cell: α, β, γ ( ) 90, 90, 90
was applied as a 100-μL injection to a Superdex 200 Increase 10/300 Resolution range (Å) 46.6–2.7 (2.8–2.7)a
GL column (Cytiva) preequilibrated in the same buffer at a flow rate of
Total reflections 794 884 (78 182)a
0.3 mL/min by an AKTA pure FPLC (Cytiva). The eluate was passed
Unique reflections 27 023 (2669)a
through an inline miniDAWN light scattering detector and Optilab dif-
Multiplicity 29.4 (27.3)a
ferential refractive index (dRI) detector (Wyatt Technology Corpora-
Completeness (%) 99.9 (99.9)a
tion). Calculations of the molecular mass from the intensity of
I/σ(I) 22 (1.5)a
scattered light and dRI measurements were performed using ASTRA
8.1 (Wyatt Technology Corporation). The SEC-MALS data are summa- Rmerge 0.14 (3.64)a

rized in Figure S1C. CC1/2 value 1.0 (0.7)a

Refinement statistics
Resolution (Å) 46.6–2.7
2.4 | Crystallization, x-ray diffraction data Rwork/Rfree 0.244/0.260
collection, structure determination, model building, Root mean square deviation bonds (Å) 0.010
and refinement Root mean square deviation angles ( ) 1.2
2
Mean B factor (Å ) 104
Crystals of McpZPD were obtained through high-throughput screening
Ramachandran plot
of commercially available crystallization conditions using a sitting drop
Favored region (%) 94.5
format. The hexagonal-shaped crystals used for diffraction data col-
Outliers (%) 0
lection were obtained directly from the Morpheus II screen (Molecular
a
Dimensions). The crystallization drop contained a 1:1 mixture of Outer shell statistics are provided in parentheses.

0.12 mM McpZPD solution in buffer B and a crystallization screen con-

dition composed of 2 mM lanthanides (0.5 mM each Yttrium[III] chlo-
ride hexahydrate, Erbium[III] chloride hexahydrate, Terbium[III]
chloride hexahydrate, and Ytterbium[III] chloride hexahydrate), 0.1 M
MOPSO, Bis-Tris pH 6.5, and 31% (v/v) Precipitant Mix 8 (10%, w/v
PEG 20000, 50%, w/v trimethylpropane, 2%, w/v nondetergent
sulfobetaine-195). Crystals grew over a 10-day period at room tem-
perature. The crystals were transferred into a 2 μL drop of cryo-
protectant obtained by mixing 70 μL mother liquor with 30 μL of a
solution containing 20% ethylene glycol and 2 M NDSB-201. Crystals
were rapidly mounted and flash-frozen in liquid nitrogen.
Diffraction data collection was performed at the Advanced Light
Source Beamline 4.2.2 at Lawrence Berkeley National Laboratory.
Because the crystallization condition contains a mixture of Lantha-
nides several x-ray wavelengths were tested to optimize a potential
anomalous signal to facilitate single anomalous dispersion (SAD) phas-
ing. Ultimately, a wavelength of 1.0 Å was chosen to collect a 2.7 Å
diffraction data set. Ten ordered heavy atoms in the crystal, all mod-
eled as Yb(III), gave a strong anomalous signal that was used to gener-
ate a high-quality SAD-phased electron density map with the AutoSol
routine of the PHENIX software package.19 The excellent quality of
the initial electron density map permitted the building of almost the
entire protein backbone using the AutoBuild routine of PHENIX. Itera-
20
tive cycles of model building in WINCOOT and automated refine-
ment with PHENIX were subsequently employed to construct and
refine the final model. Diffraction data and refinement statistics are
provided in Table 1. The final model has been deposited in the protein
data bank with the PDB code 8F7N.
2.5 | Molecular dynamics simulations
The ligand-free crystal structure of McpZPD lacked a complete side-
chain for E189 which was replaced with a complete amino acid using
PyMOL 2.5.4.21 Fifteen rotamers were calculated with the best having
an RMSD of 0.024 (4–4 atoms), and a strain of 22.07.
The GROMACS 2020.4 software suite22,23 was used for all MD
simulations. The CHARMM36m forcefield and the CHARMM-
modified TIP3P water model24–26 were applied. The system was built
in a cubic box (4882 nm3) with a minimum solute-box distance of
1 nm. The system contained 150 mM KCl (445 K atoms and 441 CL
atoms) to achieve a net neutral charge and to mimic physiological con-
ditions. Residues and peptide termini were protonated according to
their canonical states at pH 7.
To remove structural constraints imposed by crystal packing con-
tacts, energy minimization was performed with the steepest descent
algorithm with a maximum force constraint of 1000 kJ/mol nm. Three
replicate systems were individually equilibrated at constant volume
and temperature (NVT), then constant pressure and temperature
(NPT), while restraining heavy atoms. NVT equilibration used the
modified Berendsen temperature coupling method27 at a temperature
of 298 K for 100 ps. Next, isothermal-isobaric conditions of 298 K
and 1 bar were applied for 100 ps using the Berendsen pressure cou-
pling method.
Atom restraints were removed, and periodic boundary conditions
were applied to all MD simulations. A short-range cutoff of 1.2 nm

SALAR ET AL.
was applied to all nonbonded interactions. Long-range interactions
were calculated using the particle mesh Ewald (PME) method28,29
using cubic interpolation and a Fourier grid spacing of 0.16 nm. An
integration timestep of 2 fs was used along with the fourth order P-
LINCS algorithm30 to constrain all bonds. The modified Berendsen
temperature coupling method, Parrinello–Rahman pressure coupling
31,32
method, at 298 K and 1 bar respectively, and the Verlet cutoff
scheme were used for all production MD simulations. Periodic bound-
ary conditions were employed. Van der Waals forces were computed
with the Lennard–Jones equation and smoothly switched to zero from
1.0–1.2 nm. After the initial production MD simulation, the three rep-
licates were each extended to 500 ns, for a total of 1.5 μs of simula-
tion time. Initial starting structures, dominant morphology structure
files from simulation, and parameter files can be found on Open Sci-
ence Framework (https://osf.io/82n73/) in Research Projects. Trajec-
tories were analyzed with root-mean-square deviation (RMSD), root-
mean-square fluctuation (RMSF), protein secondary structure (DSSP),
and a principal component analysis (PCA), by using the GROMACS
22
analysis package and in-house scripts. A clustering analysis was con-
ducted over the last 300 ns of the simulation using the Gromos algo-
rithm with a rms cutoff of 0.2 nm. Quantification of protein
movement was calculated from the trajectory of the protein and posi-
tional data. Displacement, pitch angle, and roll angle measurements
were obtained for each helix, each four-helix bundle, and each four-
helix bundle pair. Calculations were made using in-house python
scripts and python scripts from https://github.com/speleo3/pymol-
psico/blob/master/psico/orientation.py. The results of the MD simu-
lations are summarized in Figures S2–S4, Tables S1–S3, and two ani-
mations Movies S1 and S2.
2.6 | Bioinformatics
BLAST searches33 against NCBI sequence databases (RefSeq and
Clustered nr) were performed with default parameters. Multiple
sequence alignment was constructed using the MAFFT v7 L-INS-i
algorithm.34 HHpred search35 with default parameters was performed
against profile models from Pfam and PDB databases. Taxonomy
36
information was retrieved from the Genome Taxonomy Database,
release 202, and genome trees were generated with AnnoTree.37
3 | RESULTS
3.1 | McpZPD assumes a novel helical tri-
modular fold
The crystal structure of McpZPD was determined via single-
wavelength anomalous dispersion (SAD) phasing using the strong
anomalous signal generated by heavy metal ions contained in the
crystallization solution that crystallized with the protein. The asym-
metric unit of the crystal was composed of a single McpZPD molecule.
The final refined model of this molecule encompasses amino acids
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1397
48 to 410 of McpZ (Figure 1). The 9 amino-terminal and 14 carboxy-
terminal residues of the crystallized construct did not yield interpret-
able electron density and are presumed to be structurally flexible.
McpZPD is nonglobular in shape, with a long dimension of about
137 Å. The protein is entirely helical, consisting of eight alpha helices,
which form three concatenated FHBs. The membrane-proximal and
the central FHB are vertically aligned. Yet, the central FHB is rotated
by about 152 in the clockwise direction relative to the membrane-
proximal FHB. Helices H1, H2, H3, and H8 form the membrane-
proximal FHB. Helices H3, and H8 form the rigid linker to the central
FHB where they pair with helices H6, and H7. Leading out to the
membrane-distal FHB, helices H3 and H6 are strongly kinked at resi-
dues N168 and L291, respectively. The distal bundle is completed by
the short helices H4 and H5 with the latter assuming a notably curved
shape (Figure 1A.). Overall, the central and membrane-distal FHBs are
arranged at an approximate 57 angle. Helix H8 forms part of the
proximal and distal FHBs, while the 90-residue long helix H3 provides
the rigid conduit that links all three subdomains. We performed a
search of the Protein Data Bank (PDB) for structurally related proteins
and discovered that there are a number of proteins that align well
with the combined membrane-proximal and central subdomains.
However, no other known structure contains a third FHB. Therefore,
the presence of a helical tri-modular (HTM) domain places McpZ into
a new receptor family.
3.2 | Phylogenetic evidence for Rhizobial origins of
the HTM domain
A BLAST search with default parameters against the NCBI Reference
proteins (RefSeq) database was performed using a sequence corre-
sponding to the periplasmic domain of McpZ (RefSeq accession
WP_014529036.1) flanked by predicted transmembrane regions (resi-
dues 16–448). A total of 1245 unique sequences were identified
(Dataset S1: all similar sequences) and of those 1169 matched the
query sequence for more than 90% of its length (Dataset S2: all similar
sequences of similar length), indicating the presence of a homologous
domain of the same size. To generate a taxonomically balanced, repre-
sentative dataset of these sequences, we performed a search using
the same query against the NCBI nr clustered database, where
sequences are clustered at 90% identity and 90% length using the
MMseqs2 algorithm.38 A total of 238 clusters were identified and
181 of these clusters contained sequences matching the query
sequence over more than 90% of its length. Sequences representing
each cluster (automatically generated by the nr clustered database)
were downloaded and used as Dataset S3: representative similar
sequences of similar length.
Phyletic distribution of HTM domains was first analyzed by asses-
sing NCBI taxonomy of BLAST hits identified in searches against the
RefSeq database. This analysis showed that essentially all hits in Data-
set S1 were from alphaproteobacteria, specifically members of the
orders Rhizobiales and Hyphomicrobiales. Then, we used a more robust
genome phylogeny based taxonomic scheme36 with a substantially
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1398 SALAR ET AL.

F I G U R E 1 Structure of McpZPD. (A) Cartoon depiction of McpZPD. A single McpZPD molecule forms the asymmetric unit of the crystal. The
three four-helix bundle subdomains are labeled according to their relative positions to the inner membrane of the cell. The length of the molecule
and the angle between the central and distal FHBs are displayed. The figure was generated using Pymol.62 (B) Topology diagram for McpZPD.
(C) Correlation between primary and secondary structure of McpZPD.

revised taxonomy for alphaproteobacteria. This analysis showed that
all identified HTM domains originated from a single branch of a single
bacterial family—Rhizobiaceae—in the order Rhizobiales (Figure 2A).
The only exception was one sequence (RefSeq accession
WP_226920380.1) from a betaproteobacterium Kinneretia sp. XES5
from the order Burkholderiales, which is likely indicative of a horizontal
gene transfer event. Thus, we conclude that the HTM domain family
originated in the common ancestor of the evolutionarily youngest (far-
thest from root of the genome tree: Figure 2A.) group of the family
Rhizobiaceae.
To identify a protein domain ancestral to HTM, we performed
two types of analyses. First, the sequence corresponding to McpZPD
(residues 38–426) was used in a HHpred search against profile models
of the Pfam and PDB databases. The best matches were to the HBM,
Helical bimodular sensor domain from Pfam (Pfam accession number
PF16591.8; probability = 98.56%) and to the structure of the ligand-
binding domain of McpS chemoreceptor from Pseudomonas putida
(PDB ID: 2YFA; probability = 98.51%), which is a founding sequence
for the HBM domain family. Both best hits aligned to the N-terminal
half of the McpZ periplasmic domain (Figure S5). The same models
also match to the C-terminal half of the McpZ periplasmic domain, but
with slightly lower probability scores (96.21% and 95.94%,
correspondingly) and N- and C-terminal matching profiles overlapped
significantly (Figure S6). This suggests that (i) the HBM was a likely
ancestor of the HTM and (ii) an internal duplication of HBM or inser-
tion of an unrelated FHB region into HBM gave birth to the HTM. To
further verify this, we aligned HBM sequences with closest BLAST
hits that had less than 90% length match to the query (found in Data-
set S1, but not in Dataset S2). These closely related sequences had
much shorter periplasmic regions (260 compared to nearly
400 amino acids of McpZPD), while all HTM sequences had a large
insertion in the middle of the periplasmic region, corresponding to the
membrane-distal FHB in the HTM (Figure S7). To determine the
domain family of the shorter (260 aa) periplasmic regions identified
in the original BLAST search, we performed a HHpred search against
Pfam and PDB profiles. The closest such sequence, methyl-accepting
chemotaxis protein from Rhizobium sp. PP-WC-1G-195 (RefSeq
accession WP_245423295.1; 45.89% identity to McpZ in the peri-
plasmic region, E value = 2e 27), matched closely to Pfam HBM
(probability = 99.61%) and to sensor kinase TorS from Vibrio parahae-
molyticus (PDB code 3O1I; probability = 99.58%) over the entire
length of its predicted periplasmic region. This strongly suggests that
the HBM domain is the closest relative and therefore the likely ances-
tor of the newly discovered HTM family.
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1399

Legend on next page.

FIGURE 2
SALAR ET AL.
 10970134, 2023, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/prot.26510 by CAPES, Wiley Online Library on [24/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1400 SALAR ET AL.

3.3 | Crystal packing, sequence conservation dimer in solution, and as such this dimer is likely the principal building
patterns, SEC-MALS, and structural homology point block of the crystal (Figure S1C).
toward a symmetric McpZPD dimer

Canonical MCPs are dimeric, but the isolated periplasmic domains
often only dimerize in the presence of their cognate ligand. However,
even though there is only a single molecule in the asymmetric unit of
the crystal, our packing analysis suggested the presence of a symmet-
ric McpZPD dimer. Application of the twofold axis generates an inter-
twined McpZPD dimer with an extensive interface. Using AreaIMOL
39
from the CCP4 program suite, we computed a combined buried sur-
face area of 6300 Å2 at the dimer interface. Examination of the
sequence conservation patterns obtained from the multiple sequence
alignments of McpZ homologs (Figure S7) revealed two highly con-
served regions at the predicted dimerization interface. The first region
is located within the membrane-proximal FHB module. The second
area containing numerous conserved surface-exposed residues maps
to the connecting region between the central and distal modules
(Figures 2D and S8). The notion that the observed dimer constitutes a
biologically relevant unit is further supported by the comparison of
McpZPD with its closest known structural homologs, the periplasmic
domains of the MCP McpS from Pseudomonas putida KT2440,
40
McpSPD, (PDB code 3O1I) and of the signaling histidine kinase TorS
from Vibrio parahaemolyticus, TorSPD, (PDB code 2YFA).41 The alpha-
carbons of 219 residues of McpZPD and McpSPD could be superim-
posed with an RMSD of 2.8 Å (Figure 3A and S9), while the superposi-
tion of a McpZPD monomer with a TorSPD molecule yielded an RMSD
value of 3.5 Å for the 254 aligned alpha-carbons (Figures 3B and
S10).42 Notably, both McpSPD and TorSPD form dimers in their crystal
structures with very similar interfaces within the membrane-proximal
FHB modules, where each protein uses a pair of helices to create an
extensive interface (Figure 5A). As observed in the former two struc-
tures, the membrane-proximal and central FHBs of McpZPD form a
similar interface in the symmetry-generated dimer. However, the
McpZPD dimer shows important differences to the former two struc-
tures in the alignment of the membrane-proximal FHBs (Figure 5B,C).
Dimerization is solely mediated by the two H1 helices to form a com-
paratively small interface. A large number of the residues in H1 is con-
served, some of these mediate dimerization, such as N49, L52, S56,
and K59. However, this conformation also leaves the conserved
hydrophobic residues L55 and Y62 from H1 as well as W401 and
A408 from H8 either partially or completely solvent exposed
(Figure S11). Using SEC-MALS, we confirmed that McpZPD forms a
3.4 | Small ligand binding pockets and a putative
docking site for periplasmic binding proteins are
partially conserved in McpZPD
A well-established DSF assay using Biolog MicroPlates™ (PM1,
PM2A, PM3B, PM5 and PM6) containing an array of carbon sources,
nitrogen sources, nutrient supplements, and peptides failed to reveal
any ligands for McpZPD.43–45 We also tested the polyamines cadaver-
ine, putrescine, spermine and spermidine, because a cluster of genes
encoding a spermidine/putrescine ABC transporter is located down-
stream of the mcpZ gene but no binding has been observed (data not
shown). Using the crystal structure, we therefore sought to identify
potential ligand binding sites in the McpZPD through comparative
analysis. The membrane-proximal and central FHBs of McpZPD align
well with the two modules of McpSPD (Figure 3A). McpSPD has two
ligand binding pockets, one within each FHB.40 The membrane-
proximal FHB binds succinate and malate, whereas acetate is recog-
nized by the second FHB. Superposition of the two McpS FHBs dem-
onstrated that the same regions are associated with ligand binding in
the two modules.40 The succinate/malate binding site contains three
arginine and several other polar amino acids but only a small number
of hydrophobic residues. The structurally equivalent region of
McpZPD revealed a number of conserved residues; however these are
nonpolar such as M65 and L69. The well-preserved residues Y62 and
I397 also line the pocket, while two moderately preserved asparagine
residues, N66 and N398, lend some polarity to the putative ligand
binding site (Figure 3A and S6). Overall, if this pocket is indeed
involved in ligand binding, the electrostatic properties of the ligand
should be very different from succinate. The acetate binding pocket in
the second FHB of McpSPD is also characterized by the presence of
three arginines that facilitate acetate binding (Figure S9). Only one of
these arginines, R125, is conserved in the central FHB of McpZPD.
R139 reaches into the putative ligand binding pocket, however, this
residue is not conserved within the central FHBs of McpZ homologs.
In addition to binding to ligands directly, MCPs are known to
detect various nutrient sources indirectly through the mediation of
ligand bound periplasmic binding proteins (PBPs). There is no known
crystal structure of an MCP-PBP complex in the HBM family of recep-
tors; however, the histidine kinase TorS has been co-crystallized with

F I G U R E 2 Phylogeny and sequence conservation of McpZ. (A) Distribution of HTM-containing chemoreceptors in the family Rhizobiaceae.
The presence of HTM is marked by red circles on terminal branches of the Rhizobiaceae family genome tree. Branches shown in blue identify
genera where chemotaxis systems are present. The likely origin of the HTM domain is marked by a red circle next to the longest internal branch.
Numbers in brackets show the number of genomes in each clade. Asterisk indicates that only one genome is available in a given clade. These
symbols are automatically generated by AnnoTree. (B) McpZPD colored according to sequence conservation. (C) Packing of a single layer of
McpZPD molecules inside the crystal. A single symmetric dimer is highlighted. (D) Cartoon depiction of a McpZPD dimer. Molecules are colored
according to sequence conservation using the same approach as in (B). The large highly conserved patch between the central and membrane-
distal FHBs is now buried at the dimer interface. (B) and (D) were generated with the ConSurf server63 using the subset of McpZ homolog
sequences also used in (A). The conservation scores were generated using the default Bayesian method.
 10970134, 2023, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/prot.26510 by CAPES, Wiley Online Library on [24/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SALAR ET AL. 1401

F I G U R E 3 Structural comparison of McpZPD with McpSPD and TorSPD. (A). Left panel. Superposition of McpZPD and McpSPD. Right panel.
Conservation-colored surface depiction of the region in McpZPD, which is equivalent to the succinate binding pocket in the membrane-proximal
FHB of McpSPD. Conserved residues are labeled and their side chains are displayed. The succinate is faintly shown to mark the putative ligand
binding pocket. (B). Left panel. Superposition of McpZPD and TorSPD. Right panel. TorT molecule (gray) modeled with a McpZPD dimer (blue and
green). The model was created by superimposing a 1:1 TorS-TorT complex onto the McpZPD dimer. The TorSPD molecules were then hidden to
mark potential binding sites for periplasmic binding proteins on McpZPD.

its cognate trimethylamine-N-oxide (TMAO) bound PBP TorT.41 The
TorT binding site is located at the far end of the membrane-distal
FHB and extends across the TorS dimer. The PBP binding site is highly
conserved among TorS homologs.41 When the TorSPD and McpZPD
structures are superimposed through their distal and central FHBs,
respectively, TorT is positioned at the top of the central McpZPD near
the kink that leads to the third FHB (Figures 3B and S10). The PBP
appears to fit just underneath the membrane-distal FHB suggesting a
potential role of this region in PBP recruitment. However, overall
sequence conservation of the putative PBP binding pocket is low
among McpZ homologs, whereas the equivalent sections are highly
conserved in TorS. Thus, there is no strong supporting evidence for
the presence of a PBP docking site at this location. Collectively, the
comparative analysis of McpZPD with McpSPD and TorSPD in conjunc-
tion with sequence conservation patterns only supports the presence
of a ligand binding pocket in the membrane-proximal FHB of McpZPD.
3.5 | Molecular dynamics simulations of McpZPD
suggest vertical and horizontal displacements
throughout the dimer
Because the structure offered a rare view of a ligand-free dimeric
MCP receptor domain, we sought to explore the conformational space
the protein could sample by using molecular dynamics
(MD) simulations. Initially, we attempted to create a larger MCP
receptor model that encompassed the transmembrane helices using
both AlphaFold46 and Robetta.47 Individual chains of these models
were extremely similar to each other but had a root-mean-square
deviation (RMSD) of more than 5 Å from the crystal structure. Super-
position of the McpZPD dimer with an AlphaFold-generated dimer
gave an RMSD of more than 7.9 Å, indicating a lack of confidence in
the predicted dimer interface (not shown). Therefore, we decided,
instead, to pursue MD simulations utilizing the experimentally
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1402 SALAR ET AL.

determined structure in order to probe structural shifts that might
occur at the dimer interface. This work is based on the observation
from single molecule studies that ligand-free proteins dynamically
cycle through many conformations including those that represent
ligand-bound states, albeit at lower frequency. Three independent
MD simulations runs were performed for 500 ns each, for a total sam-
pling time of 1.5 μs RMSD calculations of the protein backbone indi-
cated that the structure was mostly unchanging after initial
equilibration (Figure S2) and clustering algorithms applied to the last
300 ns of simulation time provided dominant morphologies that
represented 85%–88% of simulation time for each replicate
(Figure 4A). Secondary structure was also unchanged for the duration
of the simulation as compared to the starting structure (Figure S3),
suggesting that the resolved structure had a favorable secondary and
tertiary structure arrangement that was maintained when simulated in
solvent. The three replicates converged on very similar clusters RMSD
values between 1.5 and 2.2 Å. The observed trends were the same
for all clusters but the degree whereby certain motions occurred did
vary. The comparison of the starting structure with the dominant mor-
phologies from simulations is represented in Figure 4A to highlight
movements. During simulation, conformational changes are initiated
at the hinge region between the central and distal FHBs. The change,

signified by a straightening of the bend angle of H3 from 120 to

130 (Figure 4B), causes a downward shift of H7 in the central FHB.
This motion, in turn, forces a straightening of helix H8 and a piston-
type vertical motion of the C-terminus of H8 toward the cell mem-
brane (Figure 5C). In addition to this vertical shift, we also observed a
horizontal twisting motion within the membrane-proximal FHB, which
leads to a compaction of the McpZPD dimerization interface
(Figure 5B). Rotation and displacement of FHB cross-chain pairs are
generally coupled and symmetric (Tables S1–S3). The large displace-
ments of the FHBs are driven by the rotation of individual helices
rather than their displacement (Tables S1 and S2). The shorter
displacement of the central FHBs in the dimer indicates that they rotate
about their center of mass during the hinging process (Table S2).
The bending angles of H3 and H8 in the starting and end structures are
shown Figure 4B. Two animations provided with the supplementary
material visualize the overall predicted motion trajectories for the
McpZPD dimer (Movie S1) and the horizontal movements predicted to
occur within the membrane proximal FHB (Movie S2). These structural
changes make intriguing predictions about a possible mechanism of
transmembrane signaling which are explored further in Section 4.
4 | DI SCU SSION
E. coli, the classical model system for studying chemotaxis, utilizes
only five chemoreceptors,6 however, genome sequence data suggest
that many bacteria have vastly larger MCP repertoires at their dis-
posal. Pseudomonas species, for example, may encode up to 37 MCPs.9
Based on their molecular architecture MCPs may be classified into
several families, with the overwhelming majority falling into the super-
families PAS, CACHE, or FHB.9 Receptors in the FHB family have so
far been classed into four subcategories. More than 70% of FHB
receptors contain just a single FHB, while the remainder falls into the
TarH, CHASE3, and HBM families.9,12 Our structural and phylogenetic
analyses have revealed that the periplasmic domain of McpZ
(McpZPD) belongs to the new HTM family within the FHB superfamily
that contains over 1100 identifiable members. The HTM family genes
are still rapidly evolving, especially within the central and membrane-
distal FHBs, perhaps suggesting that many of the receptors contained
within this family have yet to settle on distinctive functions for these
two submodules.
HBM-type receptors such as P. putida McpS and McpQ are most
closely related to McpZ. Although McpS has been shown to utilize
both FHB modules for signal sensing, its primary response is mediated

F I G U R E 4 Results of MD calculations. (A). Superposition of the most prevalent conformations obtained the MD calculations with the original
structure of the McpZPD dimer. RMSD values and percent of simulation time the dimers are observed in these prevalent conformations are
indicated below. The final conformations of the three replicate runs are very similar with RMSD values ranging between 1.5 and 2.2 Å. (B). Kink
angles of helices 1 and 8 of McpZ. Comparison of initial structure (left) and the MD endpoint conformations (right). The hinge angle between the
central of distal FHBs straightens, which in turn causes a straightening of the kinked H8 helix. The net results are a  55 clockwise rotation of
the membrane proximal FHBs around and a  5 Å downward shift of H8.
 10970134, 2023, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/prot.26510 by CAPES, Wiley Online Library on [24/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SALAR ET AL. 1403

F I G U R E 5 Mechanistic implications of the distinctive dimerization interface of McpZPD in the membrane-proximal FHB region.
(A) Dimerization interfaces of the ligand bound McpSPD, PcaYPD (PDB code: 6S3B), and TorSPD viewed from the perspective of the inner
membrane. N- and C-terminal helices, which connect to the transmembrane helices in the full-length proteins are color-coded blue and red,
respectively. Numbers indicate the distances between the helices in Å. (B). MD-predicted scissoring motion. Left, N-terminal and C-terminal
helices of the McpZPD dimer viewed from the perspective of the inner membrane. Right, MD calculations predict significant horizontal helical
shifts of N- and C-terminal helices of the McpZPD dimer. We propose that the shifts occur upon ligand binding. Numbers indicate the distances
between the helices in Å. (C) MD-predicted piston-type motion. Left, side view of the membrane-proximal FHB of the McpZPD dimer. The
indicated dihedral angle measures the H8-H1-H1-H8 angle between the termini of these helices. Right, MD calculations predict up to a 5.6 Å
vertical shift of H8 toward the membrane of the McpZPD dimer, whereas H1 remains in the same plane. We propose that the shift occurs upon
ligand binding. The vertical motion is also manifested in a nearly 60 shift of the H8-H1-H1-H8 dihedral angle between the termini of these
helices. Angle values are averaged across the three replicates.

by the membrane-proximal module.40 McpS recognizes dicarboxy-
lates intermediates of the tricarboxylic acid (TCA) cycle such as
succinate and malate using a binding pocket that involves residues
from both protomers within the McpSPD dimer.40 The McpS para-
log McpQ preferentially binds citrate and citrate-metal-ion com-
48
plexes using a comparable mechanism. Reflecting the ionic
nature of the ligand, the associated binding pocket of McpS con-
tain three arginine residues and two additional polar amino acids.
In contrast, although the structurally equivalent region is enriched
in conserved residues among McpZ homologs, the pocket is much
more hydrophobic; suggesting that a TCA cycle intermediate is not
an McpZ ligand, a notion that is supported by our unsuccessful
ligand screening effort, which included these compounds. Because
TorS objectively constitutes the closest structural relative of
McpZPD in the PDB, we also strongly considered the possibility
that McpZ signal sensing is indirect through the mediation of a
PBP, which has been observed for numerous other MCPs.49 There
is experimental evidence that the HBM containing CtpL from
P. aeruginosa senses inorganic phosphate through interactions with
the PBP PstS, however, the location of their interface is not
known.50 Therefore, we used the superposition of McpZPD with
TorSPD in a TorSPD-TorT complex to identify a putative PBP dock-
ing site in McpZPD. Indeed, this comparison suggests that a PBP
could bind just between the central and membrane-distal FHBs,
however, while the TorS-TorT interface is strongly conserved in
TorS, no such conservation pattern was observed for McpZPD.
There are few examples where both dimeric apo- and holo-forms
of MCPs have been structurally characterized to reveal conforma-
tional changes associated with ligand binding. The classic paradigm for
how force is generated through ligand binding was established using
E. coli Tar and Tsr chemoreceptors as model systems.51,52 The overall
consensus is that chemoreceptors use a universal signaling mechanism
characterized by a subtle piston-like motion exerted by the periplas-
mic domain onto the second transmembrane helix.53–55 Within the
periplasmic domains, the structural changes associated with ligand
binding are small conformational shifts within each protomer of the
dimer. This model fits well with the observation that chemoreceptor
arrays are fairly static in the absence and presence of a stimulant.6,56
However, two recent studies suggest that alternative means of force
generation are possible. The periplasmic domain of the chemorecep-
tor MCP2201 was shown to be dimeric in the absence of a ligand but
forms trimers in the presence of citrate.57 Ligand binding is also asso-
ciated with conformational changes, but the dimer-trimer transition
appears to be the more significant signaling event.58 Recently, the
FHB receptor PcaYPD from P. putida was crystallized both in the
absence and presence of several ligands.59 Ligand binding caused a
shift of the protomers parallel to the dimerization interface, which
results in a scissoring motion rather than a piston-type movement. In

1404
addition, although the ligand-bound dimer of PcaYPD is overall more
symmetric than the apo-protein, ligand binding consistently caused
the partial unfolding of the very C-terminal end of H4 in one of the
protomers. This relaxation may also have significance for signal trans-
duction but has yet to be explored.
Interestingly, independent of their starting points, the ligand-
bound structures of McpSPD, TorSPD and PcaYPD all display similar
arrangements of their N-terminal and C-terminal helices (Figure 5A),
suggesting related signaling mechanisms. All three exhibit a four-
helix interface with very similar spacing (Figure 5A). The present
structure of McpZPD offers another example of a ligand-free dimeric
ligand binding domain of an MCP. In this context, the striking differ-
ences between the dimerization interfaces of McpZPD on one hand
and those observed in TorSPD, McpSPD, and PcaYPD on the other
hand may have mechanistic implications. These differences are par-
ticularly pronounced within the membrane-proximal FHBs, where all
contacts between the two McpZPD protomers are mediated by the
two H1-helices. Three residues pair directly with their symmetry-
related counterparts, namely N49, L52, and S56, while two
hydrogen-bonded K59-N60 pairs complete the interface between
the two protomers. N49, L52, S56, and K59 are part of a cluster of
well-conserved residues in helix H1, spanning from S49 to L69
(Figure S11). The C-terminal H8 helix of McpZPD, is stacked against
H1 but, unlike in TorSPD, McpSPD, and PcaYPD, does not participate
in dimer interactions (Figures 5B and S11). The trajectories of our
molecular dynamics simulations converge on a second conformation
for the McpZPD dimer that closely resembles those of the ligand-
bound conformations observed in the other three receptors. In this
conformation, H1 and H8 from the two protomers are evenly
spaced forming a four-helix interface. This structural rearrangement
requires a large horizontal movement of the four helices, which sup-
ports a scissoring-type motion at the membrane. Remarkably, the
collective movements also suggest a vertical shift of the H8 helices
toward the cell membrane by up to 5.6 Å. H1 on the other hand
undergoes no vertical movement (Figure 5C). This observation is
consistent with the piston-type movements that have been classi-
cally associated with MCP signaling. Therefore, the overall findings
of the present study suggest a mechanism of transmembrane signal-
ing for FHB MCPs that seamlessly integrates the two prevailing
models for transmembrane signaling. Although we cannot dismiss
the possibility that the observed changes are exaggerated due to
the absence of the transmembrane helices in the studied structure,
such integrated mechanism has been previously reported for two-
component signaling histidine kinases NarX and NarQ.60,61 In these
systems the amplitudes of the ligand-induced changes are smaller
but the overall trends are the same.
5 | C O N CL U S I O N
In summary, the reported crystal structure of the periplasmic domain
of S. meliloti McpZ represents the first example of a new receptor sub-
family with a helical tri-modular periplasmic domain. Sequence
10970134, 2023, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/prot.26510 by CAPES, Wiley Online Library on [24/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SALAR ET AL.
conservation patterns identified strong conservation only at the
dimerization interface and within the membrane-proximal FHB, which
suggests that McpZ homologs are still rapidly evolving. The ligand-
free McpZPD structure revealed a novel dimerization interface. Sup-
ported by MD calculations, we propose that this conformation of the
dimer is broadly representative of the ligand-free state for multiple
members of this receptor family. We further hypothesize that ligand
binding triggers signal transduction by inducing both scissoring and
piston-type motions in the membrane-proximal FHB.
AUTHOR CONTRIBU TIONS
Safoura Salar: Conceptualization; investigation; methodology; formal
analysis; data curation; writing – review and editing; writing – original
draft; validation; visualization. Nicolas E. Ball: Investigation; validation;
visualization; data curation; formal analysis. Hiba Baaziz: Investigation;
writing – review and editing; methodology. Jay C. Nix: Investigation.
Richard C. Sobe: Investigation. K. Karl Compton: Methodology; inves-
tigation. Igor B. Zhulin: Investigation; methodology; writing – review
and editing; formal analysis. Anne M. Brown: Investigation; validation;
visualization; formal analysis; supervision; data curation. Birgit
E. Scharf: Conceptualization; funding acquisition; writing – review and
editing; supervision; project administration; resources. Florian
D. Schubot: Conceptualization; investigation; funding acquisition;
writing – original draft; writing – review and editing; formal analysis;
supervision; resources; data curation; project administration;
validation.
ACKNOWLEDG MENTS
The present study was supported by National Science Foundation
grant MCB-1817652 to B.E.S and F.D.S. National Institutes of Health
grant R35GM131760 to I.B.Z. Use of the Advanced Photon Source
was supported by the U.S. Department of Energy, Office of Science,
Office of Basic Energy Sciences, under Contract No. W-
31-109-Eng-38.
PE ER RE VIEW
The peer review history for this article is available at https://www.
webofscience.com/api/gateway/wos/peer-review/10.1002/prot.
26510.
DATA AVAILABILITY STAT EMEN T
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
OR CID
Florian D. Schubot https://orcid.org/0000-0002-7403-4735
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How to cite this article: Salar S, Ball NE, Baaziz H, et al. The
structural analysis of the periplasmic domain of Sinorhizobium
meliloti chemoreceptor McpZ reveals a novel fold and suggests
a complex mechanism of transmembrane signaling. Proteins.
2023;91(10):1394‐1406. doi:10.1002/prot.26510