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: [email protected] 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

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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.

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SALAR ET AL. 1395

1 | I N T RO DU CT I O N 2 | M A T E R I A L S A N D M ET H O D S

Chemotaxis is the process used by bacterial cells to seek out 2.1 | Expression and purification of the McpZPD
nutrient sources and eukaryotic hosts. 1–5 In addition, chemotaxis
signaling pathways facilitate bacterial escape from environmental The mcpZ PD coding sequence, encompassing the codons for amino
pollutants and other harmful chemicals, known as repellents.6,7 acids 39–424 of the original mcpZ gene, was expressed from plas-
Escherichia coli serves as the classical model system for elucidat- mid pTYB11 in E. coli ER2566 to produce a fusion protein contain-
ing the underlying molecular mechanisms of the chemotaxis sys- ing a self-cleaving N-terminal intein-chitin binding domain (intein-
tem. 6,8 Universally, signaling is initiated by chemoreceptors or CBD) tag. Four liters of cell culture were grown at 37  C in LB con-
methyl-accepting chemotaxis proteins (MCPs), a group of bacte- taining 100 μg of ampicillin/mL until they reached an OD600 of
rial receptors that detect environmental changes through special- 0.8. Gene expression was induced by adding 0.6 mM isopropyl-
ized receiver domains. 6,9 Canonical MCPs are multidomain β- D-thiogalactopyranoside (IPTG) and continuing growth at 16 C
proteins containing a variable periplasmic sensory domain for an additional 16 h. Cultures were then centrifuged, and cell
coupled to two conserved transmembrane helices and a large pellets were collected. Cells were suspended in buffer A (20 mM
4,8,9
equally conserved cytoplasmic region. Ligand binding regu- Tris/HCl, 500 mM NaCl, 1 mM EDTA, and 10% glycerol [pH 8.0])
lates the autophosphorylation activity of the MCP-associated supplemented with 10 mg/mL of DNase, 1 mM PMSF, and 1
kinase CheA, which, in turn, controls phosphorylation of its cog- Halt™ Protease Inhibitor Cocktail (Thermo Fischer Scientific). Cells
nate response regulator CheY. 1 Binding of CheY-P to the cyto- were lysed with a French pressure cell (SLM Aminco, Silver Spring,
plasmic base of the flagellar motor causes the peritrichous MD) at 16 000 lbs/inch,2 prior to clearing the lysate via centrifu-
flagella to rotate in a clockwise (CW) direction ultimately result- gation at 56 000g for 1 h at 4 C. The clear supernatant was fil-
ing in a tumbling motion of the bacterial cells. Presence of an tered and loaded onto a chitin (New England BioLabs) affinity
attractant causes CheA inhibition, reversal to counterclockwise column. On-column cleavage of the intein-CBD-tag was per-
(CCW) flagellar rotation, and smooth swimming toward the nutri- formed by incubating the bound sample in cleavage buffer (buffer
ent source. A complex adaptive system ensures that receptors do A supplemented with 50 mM DTT) for 72 h at 4 C. The protein
not become saturated as the bacterial cell migrates up the nutri- was eluted with buffer A and concentrated using a 50 mL Amicon
ent gradient. 4,10–12 Due to the complexity of chemotaxis systems stirred cell (Millipore, Bedford, MA, USA) with a 3 kDa MWCO
and a scarcity of direct structural information for the transmem- regenerated cellulose membrane. The concentrated protein was
brane regions, many questions remain regarding the mechanism further purified by loading the sample onto a HiPrep 26/60 Sepha-
of signal transduction. cryl S-200 HR column (GE Healthcare Life Sciences) that was pre-
Bacterial chemotaxis also plays an important role in establish- equilibrated in buffer B (100 mM HEPES, 100 mM NaCl, pH 7.0)
ing the symbiotic relationship between the soil bacterium Sinorhi- or in buffer C (100 mM tricine, 150 mM NaCl, 1 mM EDTA,
zobium meliloti and its legume host alfalfa.13,14 S. meliloti pH 8.0) for DSF and SEC-MALS experiments. Purity of the eluted
possesses eight chemoreceptors, sequentially named McpT to protein was assessed via SDS-PAGE. Fractions containing only
McpZ and IcpA (Internal Chemotaxis Protein A).15–17 McpZ is pre- McpZ PD were pooled and concentrated to 120 μM via a 50-mL
dicted to have an extraordinarily large and structurally distinctive Amicon stirred cell (Millipore, Bedford, MA, USA) with a 3 kDa
periplasmic domain for which the ligand is unknown. To identify MWCO regenerated cellulose membrane and stored at 80  C.
regions critical for ligand recognition and signal transduction, we The chromatogram for the elution from HiPrep 26/60 Sephacryl
characterized the crystal structure of the periplasmic domain of S-200 HR column and an SDS-PAGE of the elution peak are pro-
McpZ (McpZPD). The structure of McpZ PD, solved at 2.7 Å resolu- vided in Figure S1A.
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 2.2 | Differential scanning fluorimetry
HTM fold appears to have arisen through either internal gene
duplication within a gene encoding a helical bimodular (HBM) For the differential scanning fluorimetry (DSF) assay, a 30-μL solution
receptor18 or the insertion of an unrelated gene fragment into an containing 50 μM McpZPD protein in buffer C and 2X SYPRO orange
HBM encoding sequence. was pipetted into a 96-well optical plate. Thermal denaturation was
The distinctive dimeric structure of ligand-free McpZPD served as performed in an ABI 7300 real-time PCR thermocycler (BioRad) by
a starting point for molecular dynamics (MD) calculations to explore increasing the temperature from 10 to 95 C with a 30 s equilibration at
the conformational space of the protein and gain insights into the each half degree celsius. The melting temperature (Tm) was calculated
potential signaling mechanism. Intriguingly, the observed move- by identifying the minimum of the negative first derivative of fluores-
ments suggest McpZ seamlessly integrates elements of the two cence intensity values. The described melting curve is provided in
prevailing models for MCP cross-membrane signaling. 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] 2.5 | Molecular dynamics simulations
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 The ligand-free crystal structure of McpZPD lacked a complete side-
PEG 20000, 50%, w/v trimethylpropane, 2%, w/v nondetergent chain for E189 which was replaced with a complete amino acid using
sulfobetaine-195). Crystals grew over a 10-day period at room tem- PyMOL 2.5.4.21 Fifteen rotamers were calculated with the best having
perature. The crystals were transferred into a 2 μL drop of cryo- an RMSD of 0.024 (4–4 atoms), and a strain of 22.07.
protectant obtained by mixing 70 μL mother liquor with 30 μL of a The GROMACS 2020.4 software suite22,23 was used for all MD
solution containing 20% ethylene glycol and 2 M NDSB-201. Crystals simulations. The CHARMM36m forcefield and the CHARMM-
were rapidly mounted and flash-frozen in liquid nitrogen. modified TIP3P water model24–26 were applied. The system was built
Diffraction data collection was performed at the Advanced Light in a cubic box (4882 nm3) with a minimum solute-box distance of
Source Beamline 4.2.2 at Lawrence Berkeley National Laboratory. 1 nm. The system contained 150 mM KCl (445 K atoms and 441 CL
Because the crystallization condition contains a mixture of Lantha- atoms) to achieve a net neutral charge and to mimic physiological con-
nides several x-ray wavelengths were tested to optimize a potential ditions. Residues and peptide termini were protonated according to
anomalous signal to facilitate single anomalous dispersion (SAD) phas- their canonical states at pH 7.
ing. Ultimately, a wavelength of 1.0 Å was chosen to collect a 2.7 Å To remove structural constraints imposed by crystal packing con-
diffraction data set. Ten ordered heavy atoms in the crystal, all mod- tacts, energy minimization was performed with the steepest descent
eled as Yb(III), gave a strong anomalous signal that was used to gener- algorithm with a maximum force constraint of 1000 kJ/mol nm. Three
ate a high-quality SAD-phased electron density map with the AutoSol replicate systems were individually equilibrated at constant volume
routine of the PHENIX software package.19 The excellent quality of and temperature (NVT), then constant pressure and temperature
the initial electron density map permitted the building of almost the (NPT), while restraining heavy atoms. NVT equilibration used the
entire protein backbone using the AutoBuild routine of PHENIX. Itera- modified Berendsen temperature coupling method27 at a temperature
20
tive cycles of model building in WINCOOT and automated refine- of 298 K for 100 ps. Next, isothermal-isobaric conditions of 298 K
ment with PHENIX were subsequently employed to construct and and 1 bar were applied for 100 ps using the Berendsen pressure cou-
refine the final model. Diffraction data and refinement statistics are pling method.
provided in Table 1. The final model has been deposited in the protein Atom restraints were removed, and periodic boundary conditions
data bank with the PDB code 8F7N. were applied to all MD simulations. A short-range cutoff of 1.2 nm
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SALAR ET AL. 1397

was applied to all nonbonded interactions. Long-range interactions 48 to 410 of McpZ (Figure 1). The 9 amino-terminal and 14 carboxy-
were calculated using the particle mesh Ewald (PME) method28,29 terminal residues of the crystallized construct did not yield interpret-
using cubic interpolation and a Fourier grid spacing of 0.16 nm. An able electron density and are presumed to be structurally flexible.
integration timestep of 2 fs was used along with the fourth order P- McpZPD is nonglobular in shape, with a long dimension of about
LINCS algorithm30 to constrain all bonds. The modified Berendsen 137 Å. The protein is entirely helical, consisting of eight alpha helices,
temperature coupling method, Parrinello–Rahman pressure coupling which form three concatenated FHBs. The membrane-proximal and
31,32
method, at 298 K and 1 bar respectively, and the Verlet cutoff the central FHB are vertically aligned. Yet, the central FHB is rotated
scheme were used for all production MD simulations. Periodic bound- by about 152 in the clockwise direction relative to the membrane-
ary conditions were employed. Van der Waals forces were computed proximal FHB. Helices H1, H2, H3, and H8 form the membrane-
with the Lennard–Jones equation and smoothly switched to zero from proximal FHB. Helices H3, and H8 form the rigid linker to the central
1.0–1.2 nm. After the initial production MD simulation, the three rep- FHB where they pair with helices H6, and H7. Leading out to the
licates were each extended to 500 ns, for a total of 1.5 μs of simula- membrane-distal FHB, helices H3 and H6 are strongly kinked at resi-
tion time. Initial starting structures, dominant morphology structure dues N168 and L291, respectively. The distal bundle is completed by
files from simulation, and parameter files can be found on Open Sci- the short helices H4 and H5 with the latter assuming a notably curved
ence Framework (https://osf.io/82n73/) in Research Projects. Trajec- shape (Figure 1A.). Overall, the central and membrane-distal FHBs are
tories were analyzed with root-mean-square deviation (RMSD), root- arranged at an approximate 57 angle. Helix H8 forms part of the
mean-square fluctuation (RMSF), protein secondary structure (DSSP), proximal and distal FHBs, while the 90-residue long helix H3 provides
and a principal component analysis (PCA), by using the GROMACS the rigid conduit that links all three subdomains. We performed a
22
analysis package and in-house scripts. A clustering analysis was con- search of the Protein Data Bank (PDB) for structurally related proteins
ducted over the last 300 ns of the simulation using the Gromos algo- and discovered that there are a number of proteins that align well
rithm with a rms cutoff of 0.2 nm. Quantification of protein with the combined membrane-proximal and central subdomains.
movement was calculated from the trajectory of the protein and posi- However, no other known structure contains a third FHB. Therefore,
tional data. Displacement, pitch angle, and roll angle measurements the presence of a helical tri-modular (HTM) domain places McpZ into
were obtained for each helix, each four-helix bundle, and each four- a new receptor family.
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- 3.2 | Phylogenetic evidence for Rhizobial origins of
lations are summarized in Figures S2–S4, Tables S1–S3, and two ani- the HTM domain
mations Movies S1 and S2.
A BLAST search with default parameters against the NCBI Reference
proteins (RefSeq) database was performed using a sequence corre-
2.6 | Bioinformatics sponding to the periplasmic domain of McpZ (RefSeq accession
WP_014529036.1) flanked by predicted transmembrane regions (resi-
BLAST searches33 against NCBI sequence databases (RefSeq and dues 16–448). A total of 1245 unique sequences were identified
Clustered nr) were performed with default parameters. Multiple (Dataset S1: all similar sequences) and of those 1169 matched the
sequence alignment was constructed using the MAFFT v7 L-INS-i query sequence for more than 90% of its length (Dataset S2: all similar
algorithm.34 HHpred search35 with default parameters was performed sequences of similar length), indicating the presence of a homologous
against profile models from Pfam and PDB databases. Taxonomy domain of the same size. To generate a taxonomically balanced, repre-
36
information was retrieved from the Genome Taxonomy Database, sentative dataset of these sequences, we performed a search using
release 202, and genome trees were generated with AnnoTree.37 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
3 | RESULTS 181 of these clusters contained sequences matching the query
sequence over more than 90% of its length. Sequences representing
3.1 | McpZPD assumes a novel helical tri- each cluster (automatically generated by the nr clustered database)
modular fold were downloaded and used as Dataset S3: representative similar
sequences of similar length.
The crystal structure of McpZPD was determined via single- Phyletic distribution of HTM domains was first analyzed by asses-
wavelength anomalous dispersion (SAD) phasing using the strong sing NCBI taxonomy of BLAST hits identified in searches against the
anomalous signal generated by heavy metal ions contained in the RefSeq database. This analysis showed that essentially all hits in Data-
crystallization solution that crystallized with the protein. The asym- set S1 were from alphaproteobacteria, specifically members of the
metric unit of the crystal was composed of a single McpZPD molecule. orders Rhizobiales and Hyphomicrobiales. Then, we used a more robust
The final refined model of this molecule encompasses amino acids genome phylogeny based taxonomic scheme36 with a substantially
 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
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 correspondingly) and N- and C-terminal matching profiles overlapped
all identified HTM domains originated from a single branch of a single significantly (Figure S6). This suggests that (i) the HBM was a likely
bacterial family—Rhizobiaceae—in the order Rhizobiales (Figure 2A). ancestor of the HTM and (ii) an internal duplication of HBM or inser-
The only exception was one sequence (RefSeq accession tion of an unrelated FHB region into HBM gave birth to the HTM. To
WP_226920380.1) from a betaproteobacterium Kinneretia sp. XES5 further verify this, we aligned HBM sequences with closest BLAST
from the order Burkholderiales, which is likely indicative of a horizontal hits that had less than 90% length match to the query (found in Data-
gene transfer event. Thus, we conclude that the HTM domain family set S1, but not in Dataset S2). These closely related sequences had
originated in the common ancestor of the evolutionarily youngest (far- much shorter periplasmic regions (260 compared to nearly
thest from root of the genome tree: Figure 2A.) group of the family 400 amino acids of McpZPD), while all HTM sequences had a large
Rhizobiaceae. insertion in the middle of the periplasmic region, corresponding to the
To identify a protein domain ancestral to HTM, we performed membrane-distal FHB in the HTM (Figure S7). To determine the
two types of analyses. First, the sequence corresponding to McpZPD domain family of the shorter (260 aa) periplasmic regions identified
(residues 38–426) was used in a HHpred search against profile models in the original BLAST search, we performed a HHpred search against
of the Pfam and PDB databases. The best matches were to the HBM, Pfam and PDB profiles. The closest such sequence, methyl-accepting
Helical bimodular sensor domain from Pfam (Pfam accession number chemotaxis protein from Rhizobium sp. PP-WC-1G-195 (RefSeq
PF16591.8; probability = 98.56%) and to the structure of the ligand- accession WP_245423295.1; 45.89% identity to McpZ in the peri-
binding domain of McpS chemoreceptor from Pseudomonas putida plasmic region, E value = 2e 27), matched closely to Pfam HBM
(PDB ID: 2YFA; probability = 98.51%), which is a founding sequence (probability = 99.61%) and to sensor kinase TorS from Vibrio parahae-
for the HBM domain family. Both best hits aligned to the N-terminal molyticus (PDB code 3O1I; probability = 99.58%) over the entire
half of the McpZ periplasmic domain (Figure S5). The same models length of its predicted periplasmic region. This strongly suggests that
also match to the C-terminal half of the McpZ periplasmic domain, but the HBM domain is the closest relative and therefore the likely ances-
with slightly lower probability scores (96.21% and 95.94%, tor of the newly discovered HTM family.
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
1399

Legend on next page.

FIGURE 2
SALAR ET AL.
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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 3.4 | Small ligand binding pockets and a putative
often only dimerize in the presence of their cognate ligand. However, docking site for periplasmic binding proteins are
even though there is only a single molecule in the asymmetric unit of partially conserved in McpZPD
the crystal, our packing analysis suggested the presence of a symmet-
ric McpZPD dimer. Application of the twofold axis generates an inter- A well-established DSF assay using Biolog MicroPlates™ (PM1,
twined McpZPD dimer with an extensive interface. Using AreaIMOL PM2A, PM3B, PM5 and PM6) containing an array of carbon sources,
39
from the CCP4 program suite, we computed a combined buried sur- nitrogen sources, nutrient supplements, and peptides failed to reveal
face area of 6300 Å2 at the dimer interface. Examination of the any ligands for McpZPD.43–45 We also tested the polyamines cadaver-
sequence conservation patterns obtained from the multiple sequence ine, putrescine, spermine and spermidine, because a cluster of genes
alignments of McpZ homologs (Figure S7) revealed two highly con- encoding a spermidine/putrescine ABC transporter is located down-
served regions at the predicted dimerization interface. The first region stream of the mcpZ gene but no binding has been observed (data not
is located within the membrane-proximal FHB module. The second shown). Using the crystal structure, we therefore sought to identify
area containing numerous conserved surface-exposed residues maps potential ligand binding sites in the McpZPD through comparative
to the connecting region between the central and distal modules analysis. The membrane-proximal and central FHBs of McpZPD align
(Figures 2D and S8). The notion that the observed dimer constitutes a well with the two modules of McpSPD (Figure 3A). McpSPD has two
biologically relevant unit is further supported by the comparison of ligand binding pockets, one within each FHB.40 The membrane-
McpZPD with its closest known structural homologs, the periplasmic proximal FHB binds succinate and malate, whereas acetate is recog-
domains of the MCP McpS from Pseudomonas putida KT2440, nized by the second FHB. Superposition of the two McpS FHBs dem-
40
McpSPD, (PDB code 3O1I) and of the signaling histidine kinase TorS onstrated that the same regions are associated with ligand binding in
from Vibrio parahaemolyticus, TorSPD, (PDB code 2YFA).41 The alpha- the two modules.40 The succinate/malate binding site contains three
carbons of 219 residues of McpZPD and McpSPD could be superim- arginine and several other polar amino acids but only a small number
posed with an RMSD of 2.8 Å (Figure 3A and S9), while the superposi- of hydrophobic residues. The structurally equivalent region of
tion of a McpZPD monomer with a TorSPD molecule yielded an RMSD McpZPD revealed a number of conserved residues; however these are
value of 3.5 Å for the 254 aligned alpha-carbons (Figures 3B and nonpolar such as M65 and L69. The well-preserved residues Y62 and
S10).42 Notably, both McpSPD and TorSPD form dimers in their crystal I397 also line the pocket, while two moderately preserved asparagine
structures with very similar interfaces within the membrane-proximal residues, N66 and N398, lend some polarity to the putative ligand
FHB modules, where each protein uses a pair of helices to create an binding site (Figure 3A and S6). Overall, if this pocket is indeed
extensive interface (Figure 5A). As observed in the former two struc- involved in ligand binding, the electrostatic properties of the ligand
tures, the membrane-proximal and central FHBs of McpZPD form a should be very different from succinate. The acetate binding pocket in
similar interface in the symmetry-generated dimer. However, the the second FHB of McpSPD is also characterized by the presence of
McpZPD dimer shows important differences to the former two struc- three arginines that facilitate acetate binding (Figure S9). Only one of
tures in the alignment of the membrane-proximal FHBs (Figure 5B,C). these arginines, R125, is conserved in the central FHB of McpZPD.
Dimerization is solely mediated by the two H1 helices to form a com- R139 reaches into the putative ligand binding pocket, however, this
paratively small interface. A large number of the residues in H1 is con- residue is not conserved within the central FHBs of McpZ homologs.
served, some of these mediate dimerization, such as N49, L52, S56, In addition to binding to ligands directly, MCPs are known to
and K59. However, this conformation also leaves the conserved detect various nutrient sources indirectly through the mediation of
hydrophobic residues L55 and Y62 from H1 as well as W401 and ligand bound periplasmic binding proteins (PBPs). There is no known
A408 from H8 either partially or completely solvent exposed crystal structure of an MCP-PBP complex in the HBM family of recep-
(Figure S11). Using SEC-MALS, we confirmed that McpZPD forms a 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 3.5 | Molecular dynamics simulations of McpZPD
TorT binding site is located at the far end of the membrane-distal suggest vertical and horizontal displacements
FHB and extends across the TorS dimer. The PBP binding site is highly throughout the dimer
conserved among TorS homologs.41 When the TorSPD and McpZPD
structures are superimposed through their distal and central FHBs, Because the structure offered a rare view of a ligand-free dimeric
respectively, TorT is positioned at the top of the central McpZPD near MCP receptor domain, we sought to explore the conformational space
the kink that leads to the third FHB (Figures 3B and S10). The PBP the protein could sample by using molecular dynamics
appears to fit just underneath the membrane-distal FHB suggesting a (MD) simulations. Initially, we attempted to create a larger MCP
potential role of this region in PBP recruitment. However, overall receptor model that encompassed the transmembrane helices using
sequence conservation of the putative PBP binding pocket is low both AlphaFold46 and Robetta.47 Individual chains of these models
among McpZ homologs, whereas the equivalent sections are highly were extremely similar to each other but had a root-mean-square
conserved in TorS. Thus, there is no strong supporting evidence for deviation (RMSD) of more than 5 Å from the crystal structure. Super-
the presence of a PBP docking site at this location. Collectively, the position of the McpZPD dimer with an AlphaFold-generated dimer
comparative analysis of McpZPD with McpSPD and TorSPD in conjunc- gave an RMSD of more than 7.9 Å, indicating a lack of confidence in
tion with sequence conservation patterns only supports the presence the predicted dimer interface (not shown). Therefore, we decided,
of a ligand binding pocket in the membrane-proximal FHB of McpZPD. instead, to pursue MD simulations utilizing the experimentally
 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
1402 SALAR ET AL.

determined structure in order to probe structural shifts that might displacement of the central FHBs in the dimer indicates that they rotate
occur at the dimer interface. This work is based on the observation about their center of mass during the hinging process (Table S2).
from single molecule studies that ligand-free proteins dynamically The bending angles of H3 and H8 in the starting and end structures are
cycle through many conformations including those that represent shown Figure 4B. Two animations provided with the supplementary
ligand-bound states, albeit at lower frequency. Three independent material visualize the overall predicted motion trajectories for the
MD simulations runs were performed for 500 ns each, for a total sam- McpZPD dimer (Movie S1) and the horizontal movements predicted to
pling time of 1.5 μs RMSD calculations of the protein backbone indi- occur within the membrane proximal FHB (Movie S2). These structural
cated that the structure was mostly unchanging after initial changes make intriguing predictions about a possible mechanism of
equilibration (Figure S2) and clustering algorithms applied to the last transmembrane signaling which are explored further in Section 4.
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 4 | DI SCU SSION
of the simulation as compared to the starting structure (Figure S3),
suggesting that the resolved structure had a favorable secondary and E. coli, the classical model system for studying chemotaxis, utilizes
tertiary structure arrangement that was maintained when simulated in only five chemoreceptors,6 however, genome sequence data suggest
solvent. The three replicates converged on very similar clusters RMSD that many bacteria have vastly larger MCP repertoires at their dis-
values between 1.5 and 2.2 Å. The observed trends were the same posal. Pseudomonas species, for example, may encode up to 37 MCPs.9
for all clusters but the degree whereby certain motions occurred did Based on their molecular architecture MCPs may be classified into
vary. The comparison of the starting structure with the dominant mor- several families, with the overwhelming majority falling into the super-
phologies from simulations is represented in Figure 4A to highlight families PAS, CACHE, or FHB.9 Receptors in the FHB family have so
movements. During simulation, conformational changes are initiated far been classed into four subcategories. More than 70% of FHB
at the hinge region between the central and distal FHBs. The change, receptors contain just a single FHB, while the remainder falls into the

signified by a straightening of the bend angle of H3 from 120 to TarH, CHASE3, and HBM families.9,12 Our structural and phylogenetic

130 (Figure 4B), causes a downward shift of H7 in the central FHB. analyses have revealed that the periplasmic domain of McpZ
This motion, in turn, forces a straightening of helix H8 and a piston- (McpZPD) belongs to the new HTM family within the FHB superfamily
type vertical motion of the C-terminus of H8 toward the cell mem- that contains over 1100 identifiable members. The HTM family genes
brane (Figure 5C). In addition to this vertical shift, we also observed a are still rapidly evolving, especially within the central and membrane-
horizontal twisting motion within the membrane-proximal FHB, which distal FHBs, perhaps suggesting that many of the receptors contained
leads to a compaction of the McpZPD dimerization interface within this family have yet to settle on distinctive functions for these
(Figure 5B). Rotation and displacement of FHB cross-chain pairs are two submodules.
generally coupled and symmetric (Tables S1–S3). The large displace- HBM-type receptors such as P. putida McpS and McpQ are most
ments of the FHBs are driven by the rotation of individual helices closely related to McpZ. Although McpS has been shown to utilize
rather than their displacement (Tables S1 and S2). The shorter 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- however, while the TorS-TorT interface is strongly conserved in
lates intermediates of the tricarboxylic acid (TCA) cycle such as TorS, no such conservation pattern was observed for McpZPD.
succinate and malate using a binding pocket that involves residues There are few examples where both dimeric apo- and holo-forms
from both protomers within the McpSPD dimer.40 The McpS para- of MCPs have been structurally characterized to reveal conforma-
log McpQ preferentially binds citrate and citrate-metal-ion com- tional changes associated with ligand binding. The classic paradigm for
48
plexes using a comparable mechanism. Reflecting the ionic how force is generated through ligand binding was established using
nature of the ligand, the associated binding pocket of McpS con- E. coli Tar and Tsr chemoreceptors as model systems.51,52 The overall
tain three arginine residues and two additional polar amino acids. consensus is that chemoreceptors use a universal signaling mechanism
In contrast, although the structurally equivalent region is enriched characterized by a subtle piston-like motion exerted by the periplas-
in conserved residues among McpZ homologs, the pocket is much mic domain onto the second transmembrane helix.53–55 Within the
more hydrophobic; suggesting that a TCA cycle intermediate is not periplasmic domains, the structural changes associated with ligand
an McpZ ligand, a notion that is supported by our unsuccessful binding are small conformational shifts within each protomer of the
ligand screening effort, which included these compounds. Because dimer. This model fits well with the observation that chemoreceptor
TorS objectively constitutes the closest structural relative of arrays are fairly static in the absence and presence of a stimulant.6,56
McpZPD in the PDB, we also strongly considered the possibility However, two recent studies suggest that alternative means of force
that McpZ signal sensing is indirect through the mediation of a generation are possible. The periplasmic domain of the chemorecep-
PBP, which has been observed for numerous other MCPs.49 There tor MCP2201 was shown to be dimeric in the absence of a ligand but
is experimental evidence that the HBM containing CtpL from forms trimers in the presence of citrate.57 Ligand binding is also asso-
P. aeruginosa senses inorganic phosphate through interactions with ciated with conformational changes, but the dimer-trimer transition
the PBP PstS, however, the location of their interface is not appears to be the more significant signaling event.58 Recently, the
known.50 Therefore, we used the superposition of McpZPD with FHB receptor PcaYPD from P. putida was crystallized both in the
TorSPD in a TorSPD-TorT complex to identify a putative PBP dock- absence and presence of several ligands.59 Ligand binding caused a
ing site in McpZPD. Indeed, this comparison suggests that a PBP shift of the protomers parallel to the dimerization interface, which
could bind just between the central and membrane-distal FHBs, results in a scissoring motion rather than a piston-type movement. In
 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
1404 SALAR ET AL.

addition, although the ligand-bound dimer of PcaYPD is overall more conservation patterns identified strong conservation only at the
symmetric than the apo-protein, ligand binding consistently caused dimerization interface and within the membrane-proximal FHB, which
the partial unfolding of the very C-terminal end of H4 in one of the suggests that McpZ homologs are still rapidly evolving. The ligand-
protomers. This relaxation may also have significance for signal trans- free McpZPD structure revealed a novel dimerization interface. Sup-
duction but has yet to be explored. ported by MD calculations, we propose that this conformation of the
Interestingly, independent of their starting points, the ligand- dimer is broadly representative of the ligand-free state for multiple
bound structures of McpSPD, TorSPD and PcaYPD all display similar members of this receptor family. We further hypothesize that ligand
arrangements of their N-terminal and C-terminal helices (Figure 5A), binding triggers signal transduction by inducing both scissoring and
suggesting related signaling mechanisms. All three exhibit a four- piston-type motions in the membrane-proximal FHB.
helix interface with very similar spacing (Figure 5A). The present
structure of McpZPD offers another example of a ligand-free dimeric AUTHOR CONTRIBU TIONS
ligand binding domain of an MCP. In this context, the striking differ- Safoura Salar: Conceptualization; investigation; methodology; formal
ences between the dimerization interfaces of McpZPD on one hand analysis; data curation; writing – review and editing; writing – original
and those observed in TorSPD, McpSPD, and PcaYPD on the other draft; validation; visualization. Nicolas E. Ball: Investigation; validation;
hand may have mechanistic implications. These differences are par- visualization; data curation; formal analysis. Hiba Baaziz: Investigation;
ticularly pronounced within the membrane-proximal FHBs, where all writing – review and editing; methodology. Jay C. Nix: Investigation.
contacts between the two McpZPD protomers are mediated by the Richard C. Sobe: Investigation. K. Karl Compton: Methodology; inves-
two H1-helices. Three residues pair directly with their symmetry- tigation. Igor B. Zhulin: Investigation; methodology; writing – review
related counterparts, namely N49, L52, and S56, while two and editing; formal analysis. Anne M. Brown: Investigation; validation;
hydrogen-bonded K59-N60 pairs complete the interface between visualization; formal analysis; supervision; data curation. Birgit
the two protomers. N49, L52, S56, and K59 are part of a cluster of E. Scharf: Conceptualization; funding acquisition; writing – review and
well-conserved residues in helix H1, spanning from S49 to L69 editing; supervision; project administration; resources. Florian
(Figure S11). The C-terminal H8 helix of McpZPD, is stacked against D. Schubot: Conceptualization; investigation; funding acquisition;
H1 but, unlike in TorSPD, McpSPD, and PcaYPD, does not participate writing – original draft; writing – review and editing; formal analysis;
in dimer interactions (Figures 5B and S11). The trajectories of our supervision; resources; data curation; project administration;
molecular dynamics simulations converge on a second conformation validation.
for the McpZPD dimer that closely resembles those of the ligand-
bound conformations observed in the other three receptors. In this ACKNOWLEDG MENTS
conformation, H1 and H8 from the two protomers are evenly The present study was supported by National Science Foundation
spaced forming a four-helix interface. This structural rearrangement grant MCB-1817652 to B.E.S and F.D.S. National Institutes of Health
requires a large horizontal movement of the four helices, which sup- grant R35GM131760 to I.B.Z. Use of the Advanced Photon Source
ports a scissoring-type motion at the membrane. Remarkably, the was supported by the U.S. Department of Energy, Office of Science,
collective movements also suggest a vertical shift of the H8 helices Office of Basic Energy Sciences, under Contract No. W-
toward the cell membrane by up to 5.6 Å. H1 on the other hand 31-109-Eng-38.
undergoes no vertical movement (Figure 5C). This observation is
consistent with the piston-type movements that have been classi- PE ER RE VIEW
cally associated with MCP signaling. Therefore, the overall findings The peer review history for this article is available at https://www.
of the present study suggest a mechanism of transmembrane signal- webofscience.com/api/gateway/wos/peer-review/10.1002/prot.
ing for FHB MCPs that seamlessly integrates the two prevailing 26510.
models for transmembrane signaling. Although we cannot dismiss
the possibility that the observed changes are exaggerated due to DATA AVAILABILITY STAT EMEN T
the absence of the transmembrane helices in the studied structure, The data that support the findings of this study are available from the
such integrated mechanism has been previously reported for two- corresponding author upon reasonable request.
component signaling histidine kinases NarX and NarQ.60,61 In these
systems the amplitudes of the ligand-induced changes are smaller OR CID
but the overall trends are the same. Florian D. Schubot https://orcid.org/0000-0002-7403-4735

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