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Article

Isolation and Preliminary X-Ray Crystallographic Characterisation of the Periplasmic Ligand-Binding Domain of the Chemoreceptor Tlp3 from Campylobacter hepaticus

by
Diana Kovaleva
1,†,
Yue Xin
1,†,
Mohammad F. Khan
1,†,
Yu H. Chin
1 and
Anna Roujeinikova
1,2,*
1
Department of Microbiology, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia
2
Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Crystals 2025, 15(6), 542; https://doi.org/10.3390/cryst15060542
Submission received: 15 April 2025 / Revised: 30 May 2025 / Accepted: 2 June 2025 / Published: 6 June 2025
(This article belongs to the Section Biomolecular Crystals)
Browse Figures
Versions Notes

Abstract

The Campylobacter genus includes many pathogenic species, with Campylobacter hepaticus primarily implicated in spotty liver disease in poultry. Chemotaxis is one of the well-established mechanisms of pathogenesis of Campylobacter. The chemoreceptor Tlp3, previously studied in C. jejuni, mediates responses to diverse ligands. Differences between the ligand-binding pockets of Tlp3s in C. hepaticus and C. jejuni may influence ligand specificity and niche adaptation. Here, we report a method for production of the ligand-binding domain of C. hepaticus Tlp3 (Ch Tlp3-LBD) in Escherichia coli inclusion bodies that yields crystallisable protein. Size-exclusion chromatography analysis showed Ch Tlp3-LBD is a monomer in solution. Ch Tlp3-LBD was crystallised using PEG 6000 and LiCl as the precipitants. The crystal lattice symmetry was P2221, with unit cell geometry of a = 82.0, b = 137.7, c = 56.1 Å, and α = β = γ = 90°. X-ray diffraction data have been acquired to 1.6 Å resolution using synchrotron radiation. Estimation of the Matthews coefficient (VM = 2.8 Å3 Da−1) and the outcome of molecular replacement suggested the asymmetric unit is composed of two protein molecules. This work lays the foundation for studies towards understanding the structural basis of ligand recognition by C. hepaticus Tlp3 and its role in pathogenesis.

1. Introduction

The Campylobacter genus includes several pathogenic species, with Campylobacter jejuni being the most well-studied human pathogen, largely because it is a leading cause of human bacterial gastroenteritis [1,2,3]. Campylobacter hepaticus, a Gram-negative, S-shaped bacterium with bipolar flagella [4], is another member of this genus primarily associated with spotty liver disease (SLD) in poultry [5]. The disease manifests as multiple grey/white spots (necrotic lesions) on the liver, reduced production of eggs, and elevated mortality rates in chickens [5].
C. hepaticus was first isolated and identified as a novel strain during disease outbreaks in England (2015) and Australia (2016), and its role as the causative agent of SLD was subsequently confirmed through fulfilment of Koch’s postulates [4,5]. Unlike C. jejuni, which spreads to humans through contaminated meat and dairy products and untreated water [1,2], C. hepaticus has not been confirmed as a zoonotic pathogen [6]. However, its close taxonomic relationship to C. jejuni (with approximately 83% average nucleotide identity) raises concerns about potential future risks to human health [6,7]. Furthermore, C. hepaticus presents a major economic challenge to the poultry industry, resulting in a decline in egg production by up to as much as 25% and a rise in mortality rates of free-range flocks by up to 10% [4].
C. hepaticus is present in the liver and bile of affected birds [8]. This indicates that bacterial trafficking from the gastrointestinal tract to colonisation sites via chemotaxis is a crucial step in disease pathogenesis [8]. Indeed, approximately 60 motility and chemotaxis genes have been identified in the C. hepaticus genome [9].
Chemotaxis is a complex signalling system present in many bacteria. It facilitates stimuli-directed movement, playing a key role in host colonisation and invasion by bacteria [10]. Flagellar-driven motility can be guided by external gradients of chemoattractants or chemorepellents [11,12]. These molecules are recognised by the ligand-binding domains of methyl-accepting chemotaxis protein (MCP) receptors, also known as transducer-like proteins (Tlps). Upon signal detection, chemoreceptors activate intracellular signalling cascades via their cytoplasmic signalling domains, which controls the behaviour of the flagellar motors. This enables bacteria to navigate towards favourable environments and away from harmful conditions, thereby promoting survival [13,14,15,16].
One of the key Tlps associated with chemotaxis in Campylobacter genus is Tlp3, otherwise referred to as Campylobacter chemoreceptor for multiple ligands (CcmL) [17]. First identified in C. jejuni, Tlp3/CcmL facilitates the chemotaxis response to a diverse range of chemoattractants and chemorepellents, including hydrophobic amino acids, purine, organic acids and glycans [18,19,20]. Loss of Tlp3 function in C. jejuni resulted in reduced chemotaxis, increased autoagglutination and biofilm formation, and impaired ability to adhere to and invade epithelial cells in vitro [17]. These findings highlighted the contribution of C. jejuni Tlp3 in bacterial pathogenesis and adaptation to the host environment.
The Tlp3 chemoreceptor comprises a periplasmic ligand-binding domain (LBD), a membrane-spanning moiety, and a conserved signalling domain in the cytoplasm. Analysis of the crystal structure of Tlp3 from C. jejuni LBD showed that it has a dCache (double calcium channel and chemotaxis receptor) fold [19,20,21] and revealed how this receptor recognises hydrophobic amino acids at a structural level. The structural basis of recognition of other reported ligands (purine, organic acids, glycans) by Tlp3 remains to be established.
Sequence alignment (Figure A1) shows that Tlp3 LBDs from C. hepaticus and C. jejuni share 64% amino acid sequence identity. However, there are notable differences in the amino acid residues lining their ligand-binding pockets. To investigate how these differences affect ligand specificity and influence colonisation of different niches (liver versus caeca), we initiated structural studies on Tlp3 LBD from C. hepaticus. Here, we report overexpression, purification by refolding from inclusion bodies, and preliminary crystallographic characterisation of this protein. The findings of this study and their further applications may help reduce C. hepaticus colonisation in poultry and limit economic losses from SLD. In the long term, this structural knowledge could support surveillance efforts or inform risk assessments regarding zoonotic potential.

2. Materials and Methods

2.1. Gene Cloning and Overexpression

The two transmembrane helices of Tlp3 from C. hepaticus strain HV10 (Genbank ID AXP08895.1) were analysed using the DeepTMHMM deep learning model version 1.0.42 [22] and predicted to comprise residues 8–28 and 282–302 (Figure 1). The periplasmic ligand-binding domain (Ch Tlp3-LBD, amino acid residues 32–280) coding sequence was subjected to codon optimisation for efficient expression in Escherichia coli, synthesised, and then inserted by Genscript into the pET151/D-TOPO vector (Invitrogen, Carlsbad, CA, USA). E. coli BL21 DE3 cells were transformed with the resulting plasmid, which included a His6 tag and a cleavage site for the tobacco etch virus (TEV) protease at the N-terminus. Cell culture was grown at 310 K in Luria–Bertani broth supplemented with 50 mg/L ampicillin with shaking at 180 rpm until OD600 reached 0.8. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (1 mM) was then used to induce expression of Ch Tlp3-LBD. After 4 h of further growth, centrifugation at 6000× g for 15 min at 277 K was used to harvest the cells. The resulting protein used for crystallisation consisted of residues 32–280, including an N-terminal artefact GIDPFT sequence integrated as part of the engineered TEV cleavage site.

2.2. Solubilisation of Inclusion Bodies

Following resuspension in buffer A (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM MgCl2, 25 U/mL PierceTM Universal Nuclease for Cell Lysis (Thermo Fisher Scientific, Waltham, MA, USA) and 0.2 mM phenylmethanesulfonyl fluoride (PMSF)), cells were lysed by sonication. The sample was subjected to centrifugation at 10,000× g for 30 min at 277 K to clear the lysate. Pellet and supernatant fractions were analysed using SDS-PAGE, which revealed that Ch Tlp3-LBD was primarily located within pelleted inclusion bodies (IBs). The IBs were then solubilised using a modification of the procedure outlined in [23]. IBs were subjected to 3 washes with buffer B (0.2 mM PMSF, 10 mM Tris-HCl pH 8.0, and 1% w/v Triton X-100), and 2 washes with buffer C (0.2 mM PMSF, 10 mM Tris-HCl pH 8.0). After each wash, the IBs were collected by 30 min centrifugation at 10,000× g. To solubilise IBs, they were incubated overnight in buffer D (8 M urea, 10 mM Tris-HCl pH 8.0, 0.2 mM PMSF and 10 mM dithiothreitol (DTT)) at 277 K with axial rotation. The denatured protein solution was clarified by 30 min centrifugation at 20,000× g. The protein concentration was measured using the Bradford assay [24]. For storage at 193 K, the protein solution was supplemented with 20% (v/v) glycerol and snap-frozen in liquid nitrogen in aliquots.

2.3. Refolding and Purification

Denatured Ch Tlp3-LBD (60 mg) was subjected to rapid dilution into 250 mL of pre-chilled buffer E (0.4 M L-arginine monohydrochloride, 4 M urea, 0.5 mM oxidised L-glutathione, 5 mM reduced L-glutathione, 100 mM Tris-HCl pH 8.0) to initiate refolding. The mixture was incubated at 277 K for 16 h with stirring. Subsequently, the sample was dialysed for 24 h against buffer F (10 mM Tris-HCl pH 8.0), with buffer changes every six hours. Following dialysis, the buffer was supplemented with NaCl, imidazole and Tris–HCl pH 8.0 to final concentrations of 500, 15 and 20 mM, respectively. The sample was then applied to a 5 mL Ni-NTA affinity column (GE Healthcare) pre-equilibrated with buffer G (15 mM imidazole, 500 mM NaCl and 20 mM Tris–HCl pH 8.0). Four column volumes of the same buffer were used to wash the affinity column, and buffer H (20 mM Tris–HCl pH 8.0, 500 mM NaCl and 500 mM imidazole) was used to elute Ch Tlp3-LBD from the column. The His6 tag was removed by overnight incubation with His6-TEV (Invitrogen, Carlsbad, CA, USA) while dialysing against buffer I (1% (v/v) glycerol, 0.5 mM DTT, 150 mM NaCl, 10 mM Tris-HCl pH 8.0) at 277 K. The sample was then dialysed for 2 h against buffer I without DTT, supplemented with imidazole and NaCl (final concentrations 15 and 500 mM, respectively) and passed again through the Ni-NTA column to capture His6-TEV, cleaved His6 tag, and any remaining uncleaved protein. The flow-through fractions, containing tag-free Ch Tlp3-LBD, were combined and concentrated using an Amicon Ultracel centrifugal filter with a 10 kDa cutoff. Finally, Ch Tlp3-LBD was passed through the Superdex 75 HiLoad 26/60 size-exclusion column (GE Healthcare, Chicago, IL, USA) in buffer J (150 mM NaCl, 10 mM Tris-HCl pH 8.0). Peak fractions were collected and pooled, and the purity of Ch Tlp3-LBD was assessed using SDS-PAGE. We used the equation Vretention (mL) = 631.3–104.3 × log MW [25] to estimate the oligomeric state of the protein in solution based on its retention volume during size-exclusion chromatography.

2.4. Thermal Shift Assay

We performed a thermal shift assay (TSA) using a real-time PCR cycler Rotor-Gene Q (Qiagen, Venlo, The Netherlands) to assess the folding state of the purified Ch Tlp3-LBD protein. Each reaction was set up in a final volume of 25 μL, comprising 10 μM protein, 150 mM NaCl, 100 mM Tris-HCl (pH 8.0), and 10× SYPRO Orange dye (Sigma-Aldrich catalogue number S5692, 5000× stock). The temperature range was 25 °C to 80 °C; the ramp rate was 0.5 °C min−1. We tracked protein unfolding by following changes in the SYPRO orange fluorescence (excitation at 530 nm, emission at 555 nm) which occurs when the dye interacts with the newly exposed hydrophobic regions of the protein. Each measurement was repeated three times.

2.5. Crystallisation

We concentrated Ch Tlp3-LBD to 19 mg/mL and clarified the solution by 30 min centrifugation at 17,000× g at 277 K. Crystallisation screening was carried out using the vapour diffusion sitting-drop technique. The 96-condition screens BCS (Molecular Dimensions), JCSG+ Suite (Qiagen), JBS HTS1 and 2 (Jena Bioscience), and PEG/Ion HT (Hampton Research) and the Phoenix crystallisation robot (Art Robbins Instruments) were used. During high-throughput robotic trials, crystallisation drops were prepared by mixing 100 nL of the protein solution with the same volume of the reservoir solution and equilibrating against 50 μL of the reservoir solution. Protein crystals appeared after 2 days in condition C10 of the BCS screen, B8 and C2 (JCSG+), and C3 and F2 (JBS HTS1). Optimisation was performed manually using a hanging-drop diffusion technique. The hanging drops were formed by mixing 2 μL of reservoir solution with 2 μL of protein solution, suspended over 500 μL of the reservoir solution. Well-diffracting single crystals were obtained using a protein concentration of 18 mg/mL and a reservoir solution comprising 14% w/v PEG 6000, 100 mM Na citrate pH 5.0 and 1 M LiCl.

2.6. Data Acquisition and Initial Crystallographic Analysis

X-ray diffraction data were acquired for a single cryo-cooled crystal at 1.6 Å resolution. Data collection was performed at the Australian Synchrotron beamline MX1, using a Dectris EIGER2 9M photon-counting detector (Dectris, Baden, Switzerland). Data integration and scaling was performed using the XDS package [26] and the AIMLESS program [27] within the CCP4 suite [28]. The solvent content was analysed by calculating the Matthews coefficient with the MATTHEWS_COEF tool [29] from the CCP4 package version 7.0.078. The summary of key data collection statistics is presented in Table 1. We conducted molecular replacement with Phaser [30] using the structure predicted by AlphaFold3 [31] as the search model. The first round of refinement was undertaken using Phenix [32].

3. Results

3.1. Cloning, Overproduction in Inclusion Bodies, Refolding, Purification and Thermal Shift Assay

The nucleotide sequence of the gene encoding the periplasmic region (the ligand-binding domain) of Tlp3 from C. hepaticus (Ch Tlp3-LBD) was subjected to codon optimisation for efficient expression in E. coli. An expression vector for Ch Tlp3-LBD with a cleavable His6 tag at the N-terminus was generated by inserting the synthetic gene into the plasmid pET151/D-TOPO. Ch Tlp3-LBD was expressed in E. coli BL21 DE3 by inducing T7 polymerase with IPTG, which resulted in accumulation of protein in the form of inclusion bodies (IBs). To refold the recombinant Ch Tlp3-LBD from IBs, it was denatured and diluted into a buffer containing 4 M urea, 100 mM Tris-HCl pH 8.0, 0.4 M L-arginine monohydrochloride, 0.5 mM oxidised L-glutathione and 5 mM reduced L-glutathione. This approach yielded approximately 10 mg of protein starting with 60 mg of IBs.
Following affinity chromatography, cleavage of the tag and gel-filtration chromatography, staining of the SDS–PAGE gel using Coomassie Blue confirmed that the final Ch Tlp3-LBD had a purity of over 95% (Figure 2a). The resulting tag-free protein comprised residues 32–280 of C. hepaticus Tlp3 and the additional GIDPFT residues at the N-terminus from introducing the recognition site for TEV. Estimation of the protein molecular weight (MW) using SDS-PAGE produced a value of approximately 25 kDa (Figure 2a), which is close to the theoretical MW derived from the amino acid sequence (28.6 kDa). To verify the folded state of purified Ch Tlp3-LBD, we carried out a thermal denaturation profiling experiment (thermal shift assay), where we followed fluorescence changes of SYPRO Orange dye upon its binding to hydrophobic regions exposed during protein unfolding. Ch Tlp3-LBD exhibited a sharp, temperature-dependent transition (Figure 2b), characteristic of proteins that are folded prior to thermal denaturation, and that undergo cooperative unfolding upon temperature increase. The size-exclusion chromatography elution profile on the pre-calibrated column contained only one significant, symmetrical peak corresponding to the elution volume of 163 mL (Figure 2c), matching to a calculated approximate MW of 31 kDa. Comparison with the MW derived from the sequence indicates that, under the tested biochemical conditions, Ch Tlp3-LBD behaves as a monomer.

3.2. Crystallisation and Intitial Crystallographic Assessment

As the first step towards understanding the function of Ch Tlp3-LBD, we performed crystallisation trials using commercially sourced screens. Promising initial conditions were then optimised manually to yield single crystals that diffracted well. The best conditions used PEG 6000 and LiCl as the precipitating agents and a protein concentration of 18 mg/mL. Crystal formation was generally observed within 48 h (Figure 2d). A single cryo-cooled crystal was used to collect a complete X-ray diffraction dataset to a resolution of 1.6 Å, using the MX1 station of the Australian Synchrotron (Figure A2). Data indexing with XDS suggested a primitive orthorhombic space group, with unit cell geometry of a = 82.0, b = 137.7, c = 56.1 Å, and α = β = γ = 90°. The average I/(I) value was 18.8 (46.29–1.60 Å resolution range) and 1.6 in the shell with the highest resolution (1.63–1.60 Å). The dataset comprised 344,796 measurements of 81,116 unique reflections with an Rmerge value of 0.023 (0.359 in the 1.63–1.60 Å resolution shell). Overall completeness was 96%, with 86% in the outer resolution shell (Table 1). Assessment with Phenix Xtriage [33] presented no evidence of twinning. The assumption of two protein subunits per asymmetric unit produced a Matthews coefficient value of 2.8 Å3 Da−1, consistent with typical values observed for protein crystals [34]. In line with this, molecular replacement yielded a solution containing two subunits.
Analysis of the Phaser output showed that the best molecular replacement solution was achieved when the structure predicted using AlphaFold3 was used as the search model. This solution was characterised by a rotation function Z-score (RFZ) of 7.0, translation function Z-score (TFZ) of 9.6, and a maximum log-likelihood gain (LLG) of 99. The electron density map calculated for the top molecular replacement solution at 6 Å resolution clearly showed distinct protein globules separated by solvent channels (Figure 2e), confirming that the molecular replacement solution is correct. In contrast, using the crystal structure of C. jejuni Tlp3-LBD as a search model produced a solution with a significantly lower LLG (35) and a featureless map. This difference suggests that the AlphaFold3-predicted model of Ch Tlp3-LBD better approximates the native structure compared to the C. jejuni Tlp3 LBD crystal structure.
A preliminary round of xyz, B refinement of the best molecular replacement solution resulted in the reduction in R and Rfree vales to 28.9% and 31.8%, respectively. The resulting electron density map showed excellent fit with this initial model (Figure 2f) and revealed density for nearly the entire polypeptide chain, spanning from the remnant of the TEV-cleavage site at the N-terminus to residue 270. This confirms the identity and structural integrity of the crystallised protein, as well as the successful cleavage of the affinity tag. Iterative model building and refinement is currently underway.

4. Discussion

Cloning, overexpression, isolation, crystallisation and initial crystallographic characterisation of the periplasmic ligand-binding domain (LBD) of Campylobacter hepaticus Tlp3 (Ch Tlp3-LBD) represent a significant step towards elucidating how this chemoreceptor recognises its ligands at a structural level. In addition, this work lays the foundation for studies towards understanding the role of this chemosensing in the development of spotty liver disease (SLD) in poultry. Deposition of Ch Tlp3-LBD in E. coli inclusion bodies necessitated refolding, which is often challenging for proteins containing cysteine residues (Ch Tlp3-LBD has two). The inclusion of L-arginine and a redox system (reduced and oxidised glutathione) in the refolding buffer likely promoted protein refolding by minimising aggregation and preventing formation of incorrect disulfide bonds. The folded state of Ch Tlp3-LBD was confirmed by a thermal shift assay, which demonstrated a clear transition from the folded to the unfolded state upon thermal denaturation. The observed monomeric state Ch Tlp3-LBD in solution, inferred from size-exclusion chromatography, is in agreement with the reports on dCache domains from different chemoreceptors similarly exhibiting monomeric behaviour [19,23,35]. The production of highly pure recombinant Ch Tlp3-LBD now enables systematic exploration of its ligand specificity by, e.g., high-throughput library screening to find small molecules that interact with the receptor. These ligands could act as natural attractants or repellents with roles in the host colonisation and evasion of host immune defences. Hits from the screens can be subjected to co-crystallisation with Ch Tlp3-LBD to facilitate structural analysis of receptor–ligand interactions at atomic resolution. To investigate the physiological relevance of these ligands, binding studies can be complemented by chemotaxis motility assays, thereby linking in vitro binding affinity to functional behavioural responses.
To further confirm the biological function of Ch Tlp3, mutagenesis experiments targeting tlp3 may be carried out in conjunction with soft agar motility assays. These experiments could provide insights into the role of Tlp3 in chemotactic direction and colonisation of host tissues and whether inactivation of this receptor impairs liver targeting. Such evidence would offer functional validation for the role of Ch Tlp3 during early infection, and for the use of Ch Tlp3 as a therapeutic target.
The high-resolution (1.6 Å) diffraction dataset obtained for Ch Tlp3-LBD crystals provides a robust foundation for solving its atomic structure. This achievement is also significant because well-ordered crystals will facilitate detailed analysis of ligand-binding interactions through future co-crystallisation studies. In addition, determination of the atomic structure of Ch Tlp3-LBD will allow a comparison of oligomeric state in the crystal based on the arrangement of protein molecules in the crystal lattice with the monomeric state of Ch Tlp3-LBD in solution. Furthermore, determination of the structure of Ch Tlp3-LBD in free and ligand-bound forms would allow comparisons that, at this resolution, may reveal conformational changes that transmit the signal across the membrane. This could, in the future, lead to the identification of key residues involved in the propagation of chemotactic signals within the C. hepaticus species.
Finally, the high-resolution data collected in this study will allow experimental determination the Ch Tlp3-LBD structure with high accuracy, paving the way for future molecular dynamics simulation studies to understand the intramolecular movements that are critical for the protein function, as well as high-throughput computational docking studies to identify candidate binders. The latter approach would have the potential to identify natural ligands of the receptor, and to find small molecules that can function as agonists and antagonists, modulating the function of this receptor.
In the longer term, Ch Tlp3 has the potential to become a target for antimicrobial intervention strategies. Determination of the three-dimensional structure of the ligand-binding pocket and understanding the conformational changes involved in signal transduction can guide the rational development of small-molecule inhibitors of receptor function. These molecules can be used as chemotaxis antagonists to block the migration of C. hepaticus towards the hepatic niche and reduce the burden of spotty liver disease in poultry production systems.
Notably, sequence alignment highlighted small but significant differences between the ligand-binding pockets of Tlp3s from C. jejuni and C. hepaticus (Figure A1). These variations likely underpin divergent ligand specificities enabling the two species to adapt to distinct niches with the host: C. jejuni targets the caeca, whereas C. hepaticus colonises the liver. Future structural analysis of Ch Tlp3-LBD, particularly in complex with ligands, will provide insights into how sequence variations influence ligand recognition. Such insights could inform the rational design of chemotaxis inhibitors to disrupt C. hepaticus colonisation, offering a novel strategy to mitigate spotty liver disease, safeguard poultry health, and reduce economic losses in the industry.
Beyond host–pathogen interaction studies, this receptor may find more general biotechnological uses. For instance, Tlp3 LBD may potentially be engineered for application in biosensing devices that screen for particular hydrophobic ligands or peptides in mixed samples as novel tools for environmental monitoring or diagnostics. Understanding the sensory mechanisms of Ch Tlp3 could also inform broader efforts to manipulate chemotaxis in related pathogens, or support the design of biosensors based on bacterial receptor domains.

5. Conclusions

This study establishes a methodology for producing the crystallisable ligand-binding domain of the chemoreceptor Tlp3 from the important animal pathogen C. hepaticus and lays the foundation for resolving its atomic structure. The outcomes of this study pave the way for future studies aimed at determining the ligand specificity of Ch Tlp3-LBD and its structural basis, as well as elucidating its role in the pathogenesis of spotty liver disease.

Author Contributions

A.R. conceived and coordinated this study; Y.X., M.F.K. and A.R. designed the experiments; Y.X., M.F.K. and Y.H.C. performed the experiments; D.K., Y.X., M.F.K., Y.H.C. and A.R. analysed the data; D.K., Y.X., M.F.K., Y.H.C. and A.R. wrote and/or edited the paper. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Australian Research Council (ARC DP180101807 grant to AR).

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

We thank Geoffrey Kwai Wai Kong (Monash Macromolecular Crystallisation Facility) for his assistance with robotic crystallisation screens. We also thank the Australian Synchrotron staff for their assistance with data collection.

Conflicts of Interest

The authors declare that they have no competing interests.

Abbreviations

The following abbreviations are used in this manuscript:
Tlp3Transducer-like protein 3
SLDSpotty liver disease
LBDLigand-binding domain
CcmLCampylobacter chemoreceptor for multiple ligands
dCacheDouble calcium channels and chemotaxis receptors
TEVTobacco etch virus
PMSFPhenylmethanesulfonyl fluoride
DTTDithiothreitol
IBsInclusion bodies

Appendix A

Crystals 15 00542 g0a1
Figure A1. Alignment of the amino acid sequences of the ligand-binding domains of Tlp3s from C. jejuni and C. hepaticus. Residues that stabilise the amino acid ligand in C. jejuni Tlp3 are marked with an arrow (red for conserved residues, black for conservative substitutions). Conserved residues are shown using a red background. The alignment was carried out using ClustalW version 2.1 [36]. The figure was prepared using ESPript version 3.0 [37].
Figure A1. Alignment of the amino acid sequences of the ligand-binding domains of Tlp3s from C. jejuni and C. hepaticus. Residues that stabilise the amino acid ligand in C. jejuni Tlp3 are marked with an arrow (red for conserved residues, black for conservative substitutions). Conserved residues are shown using a red background. The alignment was carried out using ClustalW version 2.1 [36]. The figure was prepared using ESPript version 3.0 [37].
Crystals 15 00542 g0a1
Crystals 15 00542 g0a2
Figure A2. A representative diffraction image.
Figure A2. A representative diffraction image.
Crystals 15 00542 g0a2

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Crystals 15 00542 g001
Figure 1. The schematics of the membrane topology of Ch Tlp3 delineating the ligand-binding domain, signalling domain and two transmembrane helices.
Figure 1. The schematics of the membrane topology of Ch Tlp3 delineating the ligand-binding domain, signalling domain and two transmembrane helices.
Crystals 15 00542 g001
Crystals 15 00542 g002
Figure 2. Purification and crystallographic characterisation of purified recombinant Ch Tlp3-LBD. (a) Reduced 16.5% SDS-PAGE gel of purified Ch Tlp3-LBD stained with Coomassie Blue. Molecular-weight markers are shown in kDa. (b) Thermal shift assay results (from an experiment performed in triplicate) indicating a temperature-dependent transition of purified Ch Tlp3-LBD from the folded to the unfolded state. (c) Elution profile of purified Ch Tlp3-LBD subjected to size-exclusion chromatography (Superdex 75 HiLoad 26/60). The small amount of protein eluting at the void volume (~100 mL) represents non-specific aggregates. (d) Crystals of Ch Tlp3-LBD. (e) An electron density map generated from the molecular replacement solution at a resolution of 6 Å. (f) Representative segment of the electron density map calculated for the initial model subjected to the first round of refinement. The map is contoured at 1 σ threshold.
Figure 2. Purification and crystallographic characterisation of purified recombinant Ch Tlp3-LBD. (a) Reduced 16.5% SDS-PAGE gel of purified Ch Tlp3-LBD stained with Coomassie Blue. Molecular-weight markers are shown in kDa. (b) Thermal shift assay results (from an experiment performed in triplicate) indicating a temperature-dependent transition of purified Ch Tlp3-LBD from the folded to the unfolded state. (c) Elution profile of purified Ch Tlp3-LBD subjected to size-exclusion chromatography (Superdex 75 HiLoad 26/60). The small amount of protein eluting at the void volume (~100 mL) represents non-specific aggregates. (d) Crystals of Ch Tlp3-LBD. (e) An electron density map generated from the molecular replacement solution at a resolution of 6 Å. (f) Representative segment of the electron density map calculated for the initial model subjected to the first round of refinement. The map is contoured at 1 σ threshold.
Crystals 15 00542 g002
Table 1. Data collection and processing statistics. Values in parentheses are for the highest-resolution shell.
Table 1. Data collection and processing statistics. Values in parentheses are for the highest-resolution shell.
Diffraction sourceMX1 beamline, Australian Synchrotron
Wavelength (Å)0.95
DetectorEIGER2 9M
Total oscillation span (°)120
Temperature (K)100
Mosaicity (°)0.09
Resolution range (Å)46.29–1.60 (1.63–1.60)
Space groupP21212
Unit cell parameters
a, b, c (Å)56.1, 137.7, 82.0
α, β, γ (°)90, 90, 90
Mean I/σ(I)18.8 (1.6)
Multiplicity4.3 (2.3)
Completeness (%)96 (86)
Observed reflections344,796 (7878)
Unique reflections81,116 (3462)
Rmerge0.023 (0.359)
Rmeas0.026 (0.445)
CC(1/2) (%)100 (83)
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MDPI and ACS Style

Kovaleva, D.; Xin, Y.; Khan, M.F.; Chin, Y.H.; Roujeinikova, A. Isolation and Preliminary X-Ray Crystallographic Characterisation of the Periplasmic Ligand-Binding Domain of the Chemoreceptor Tlp3 from Campylobacter hepaticus. Crystals 2025, 15, 542. https://doi.org/10.3390/cryst15060542

AMA Style

Kovaleva D, Xin Y, Khan MF, Chin YH, Roujeinikova A. Isolation and Preliminary X-Ray Crystallographic Characterisation of the Periplasmic Ligand-Binding Domain of the Chemoreceptor Tlp3 from Campylobacter hepaticus. Crystals. 2025; 15(6):542. https://doi.org/10.3390/cryst15060542

Chicago/Turabian Style

Kovaleva, Diana, Yue Xin, Mohammad F. Khan, Yu H. Chin, and Anna Roujeinikova. 2025. "Isolation and Preliminary X-Ray Crystallographic Characterisation of the Periplasmic Ligand-Binding Domain of the Chemoreceptor Tlp3 from Campylobacter hepaticus" Crystals 15, no. 6: 542. https://doi.org/10.3390/cryst15060542

APA Style

Kovaleva, D., Xin, Y., Khan, M. F., Chin, Y. H., & Roujeinikova, A. (2025). Isolation and Preliminary X-Ray Crystallographic Characterisation of the Periplasmic Ligand-Binding Domain of the Chemoreceptor Tlp3 from Campylobacter hepaticus. Crystals, 15(6), 542. https://doi.org/10.3390/cryst15060542

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