Structure and conformational dynamics of the Pseudomonas CbrA transceptor

Abstract

The CbrA protein is a central regulator of carbon metabolism, biofilm formation, and virulence in Pseudomonas species, but the molecular mechanisms by which CbrA links nutrient sensing to downstream signaling has remained unclear. CbrA is a rare “transceptor” that combines membrane transporter and histidine kinase domains into a single functional polypeptide. The structural basis for histidine recognition and membrane transport, as well as signaling through intracellular histidine kinase domains has remained elusive. Here we determined a cryo‐EM structure of CbrA which provides key molecular details of the SLC5‐STAC domains in this unusual system. Unexpectedly, the small peptide CbrX encoded upstream of CbrA formed a stable complex with the SLC5 transporter domain, but was not essential for growth of Pseudomonas putida on histidine as a sole carbon source. The cryo‐EM structure reveals how histidine binds within the transporter, and molecular dynamics simulations provide insight into proton gradient driven conformational changes that enable histidine transport. These findings define the molecular architecture of key CbrA functional domains, and lay the foundation for developing a comprehensive understanding of coupling between membrane transport and downstream signaling pathways that guide essential physiological traits in Pseudomonas.

1. INTRODUCTION

Pseudomonadaceae are metabolically versatile bacteria that inhabit diverse environmental niches, with Pseudomonas aeruginosa in particular acting as an opportunistic pathogen that can cause serious life‐threatening infections (Letizia et al. 2025; Qin et al. ²⁰²²). The ecological fitness and clinical significance of Pseudomonas aeruginosa is underpinned by the organism's ability to rapidly adapt to varying nutrient conditions (Crone et al. 2020), form biofilms (Cendra and Torrents 2021), and increasingly develop resistance to multiple antimicrobial agents (Letizia et al. 2025; Qin et al. ²⁰²²). A central mediator in the adaptability of Pseudomonaceae is the CbrA/B regulatory network (Monteagudo‐Cascales et al. ²⁰²²). The CbrA/B system integrates environmental cues to coordinate carbon and nitrogen metabolism (Li and Lu 2007; Nishijyo et al. ²⁰⁰¹; Valentini et al. ²⁰¹⁴), biofilm formation (Chen et al. 2024), and other cellular processes critical for survival and virulence (Yeung et al. 2011; Yeung et al. ²⁰¹⁴). CbrA functions as a global regulatory hub by coordinating the detection of environmental cues like nutrient availability with phosphorylation and activation of the σ⁵⁴‐dependent transcription factor CbrB (Figure 1a). Activated CbrB has been shown to directly control the expression of at least 61 different genes (Barroso et al. 2018), including those involved in amino acid biosynthesis and metabolism such as the histidine utilization (hut) operon (Figure 1a) (Amador et al. 2010; Li and Lu ²⁰⁰⁷; Nishijyo et al. ²⁰⁰¹; Zhang and Rainey ²⁰⁰⁸). In addition, activated CbrB has been shown to regulate production of the iron scavengers pyoverdine and pyochelin (Yeung et al. 2011), as well as the small RNAs crcZ and crcY which sequester the Crc‐Hfq complex to modulate carbon catabolite repression (Figure 1a) (Linares et al. 2010; Moreno et al. ²⁰¹²; Sonnleitner et al. ²⁰⁰⁹; Sonnleitner and Haas ²⁰¹¹). Disruption of CbrA markedly alters Pseudomonas growth and biofilm formation under certain conditions (Chen et al. 2024), and significantly attenuates virulence and microbial burden in murine infection models (Yeung et al. 2014). Given its central role in Pseudomonas physiology and pathogenicity, the CbrA/B network represents a compelling target for future therapeutic development.

FIGURE 1.

Topology and genomic organization of CbrXA. (a) Schematic showing the overall domain organization of CbrA (blue) and CbrB (red). CbrA is composed of an N‐terminal SLC5 transporter domain, followed by cytosolic STAC, PAS, DHp, and catalytic (CA) domains. The schematic shows CbrA as a homodimer, based on classical models of histidine kinase structure/function. CbrB (red) is composed of receiver (REC), AAA⁺ ATPase, and helix‐turn‐helix (HTH) subdomains. Phosphoryl group transfer between a conserved histidine in the DHp domain of CbrA and a conserved aspartate in the REC domain of CbrB serves to activate CbrB as a σ‐54 transcription factor. (b) Genomic organization of cbrXA–cbrB in P. putida KT2440. CbrX and cbrA are translationally coupled behind the cbrA promoter. The response regulator cbrB is under control of an independent promoter. The inset in the dashed outline shows genomic overlap of cbrX (cyan highlight, black letters) and cbrA (blue letters). The stop codon for cbrX is shown in red underlined letters. (c) AlphaFold predicted secondary structure of cbrX, showing two alpha helices separated by a flexible linker.

CbrA is a rare example of a “transceptor” protein that combines membrane transporter and histidine kinase domains into a single polypeptide (Figure 1a). The N‐terminus of CbrA consists of a solute carrier family 5 (SLC5) domain with 13 transmembrane (TM) helices embedded within the inner membrane. Previous studies have demonstrated that the SLC5 domain is competent to mediate uptake of histidine from the extracellular environment in Pseudomonas fluorescens (Zhang et al. 2015) and P. putida (Wirtz et al. 2020), and that a functional copy of CbrA is required for growth on histidine as a sole carbon/nitrogen source (Monteagudo‐Cascales et al. ²⁰¹⁹; Zhang et al. ²⁰¹⁵). Furthermore, Wirtz et al. demonstrated that histidine transport through the SLC5 domain is dependent on the proton motive force (Wirtz et al. 2020), which is consistent with other SLC family transporters that operate through a sodium‐independent transport mechanism (Jungnickel et al. 2018; Shaffer et al. ²⁰⁰⁹; Wirtz et al. ²⁰²¹). Although previous evidence demonstrates that the SLC5 domain of CbrA forms a functional histidine transporter (Wirtz et al. 2020; Zhang et al. ²⁰¹⁵), a structural and mechanistic understanding of this histidine transport process remains unclear.

In addition to the N‐terminal SLC5 transporter domain, CbrA also contains domains that extend into the cytoplasm (Figure 1a). Immediately following the SLC5 domain is a distinctive SLC and two‐component system associated component (STAC) domain. A previous crystal structure of an isolated STAC domain from Archaeoglobus fulgidus revealed a small four‐helical bundle (Korycinski et al. 2015). While the precise role of STAC domains remains unclear, their presence in various transceptor‐like proteins suggests they may serve as regulatory linkers, coupling conformational changes between the membrane‐embedded SLC5 domain and downstream regions of transceptors. Located C‐terminally to the STAC domain are regions of CbrA that are classically found in histidine kinases (Bhate et al. 2015), including Per‐Arnt‐Sim (PAS), Dimerization and Histidine phosphotransfer (DHp), and catalytic (CA) domains that extend away from the membrane (Figure 1a). Thermal denaturation based binding studies have previously suggested that the PAS domain in CbrA is capable of binding histidine (Bhate et al. 2015), indicating that transport of histidine through the SLC5 domain may serve as a trigger for further ligand binding and/or conformational changes to activate the cytosolic histidine kinase domains. Such activation is thought to trigger phosphorylation of a conserved histidine in the DHp domain, which would then serve as a docking point for phosphotransfer to a conserved aspartate in CbrB (Figure 1a) (Monteagudo‐Cascales et al. ²⁰²²).

Despite extensive biochemical and microbiological characterization of the CbrA/B system in various Pseudomonas species, the signal that triggers CbrA autophosphorylation remains elusive. It is currently unclear whether activation is driven by substrate binding and/or transport through the SLC5 domain, ligand binding to the intracellular PAS domain, coordinated conformational coupling between all domains of the protein, or some combination of all these factors (Monteagudo‐Cascales et al. ²⁰²²). Progress toward mechanistic clarity on this process has largely been hampered by a lack of structural information. Beyond a crystal structure of an isolated STAC domain from an unrelated archaeal protein (Korycinski et al. 2015), no high‐resolution structures of CbrA or its isolated domains have been reported. As a result, the molecular basis of substrate recognition, conformational communication between membrane and cytosolic domains, and the integration of these events into global regulatory outputs remain poorly understood.

Here, we address this knowledge gap by determining a cryo‐electron microscopy (cryo‐EM) structure of CbrA, which reveals the overall architecture of the SLC5‐STAC domains and delineates a clearly defined and conserved histidine binding pocket within the SLC5 transporter domain. Unexpectedly, a small peptide (CbrX) that is encoded immediately upstream of the transceptor remained stably bound throughout purification and cryo‐EM imaging. Together with extensive molecular dynamics simulations, our studies reveal the molecular determinants of histidine binding to the SLC5 domain, identify a protonation dependent conformational switch that likely drives membrane transport, and provide a structural framework to begin understanding how membrane transport and histidine kinase signaling activities are coupled in this central regulator of Pseudomonas metabolism and biology.

2. RESULTS

2.1. Expression, purification, and cryo‐EM reconstruction of CbrA

Based on previously available biochemical and microbiological data, we began our studies using the CbrA system from P. putida KT2440 (Monteagudo‐Cascales et al. ²⁰¹⁹; Wirtz et al. ²⁰²⁰). CbrA is highly conserved among common Pseudomonas species, with the protein from P. putida displaying 82% sequence identity to P. aeruginosa PAO1, and ~92% sequence identity to P. fluorescens (Figure S1, Supporting Information). CbrA has an overall molecular weight of 109 kDa, and in all genomes analyzed is overlapped in an operon with a small (~58 amino acid) upstream peptide called CbrX (Figure 1b). Previous analysis in P. putida has demonstrated that CbrA is translationally coupled to CbrX, with transcription of both genes being driven from a single upstream P_cbrA promoter (Monteagudo‐Cascales et al. ²⁰¹⁹). AlphaFold predictions suggest that CbrX adopts an alpha‐helical hairpin configuration with two alpha‐helices separated by a short loop (Figure 1c). The hydrophobic amino acid composition of the two CbrX alpha‐helices suggests that the peptide may be anchored within the lipid membrane. However, the cellular localization and overall functional role of CbrX beyond translational coupling to CbrA has yet to be determined.

To facilitate expression and purification of CbrA for structural studies, we constructed a pETDuet‐1 expression vector containing full‐length CbrX and CbrA genes from P. putida each driven by individual T7 promoters (Figure S2a). CbrA was tagged with an 8x‐histidine tag at the C‐terminus for purification purposes, and CbrX was left untagged. Following heterologous expression of CbrX and CbrA together from this single plasmid in C41(DE3) E. coli, isolated membrane fractions were solubilized in lauryl maltose neopentyl glycol (LMNG) detergent, and CbrA was purified by two‐step Co²⁺‐Talon affinity and size‐exclusion chromatography. The resultant size‐exclusion chromatogram and SDS‐PAGE of peak fractions revealed a relatively monodisperse preparation of full‐length CbrA at the expected molecular weight of ~109 kDa (Figure S2b–d). Based on calibrated molecular weight standards, the elution volume of CbrA on a Superdex 200 Increase 10/300 GL size‐exclusion column (~11.8 mL) was most consistent with a monomer of CbrA plus the associated mass of an LMNG micelle.

As an initial screening step, the detergent solubilized preparations of purified full‐length CbrA were plunge‐frozen on cryo‐EM grids and imaged on a Talos Arctica 200 keV electron microscope. The resultant micrographs displayed an even distribution of seemingly small particles in thin ice (Figure S2e). 2D averages calculated from particles extracted from these micrographs show a small membrane protein embedded within a detergent micelle, with a small protrusion from the detergent micelle corresponding to the intracellular STAC domain (Figure S2f). From the 2D averages alone it is readily apparent that detergent solubilized full‐length CbrA is purified in monomeric form consistent with the elution volume observed with size‐exclusion chromatography, and also that the cytosolic PAS‐DHp‐CA domains are completely disordered and not visible in the averages (Figure S2f). Based on this preliminary screening dataset we constructed a truncated version of CbrA in which the flexible and disordered PAS‐DHp‐CA domains were removed, leaving an expression plasmid with only CbrX and the SLC5‐STAC domains of CbrA which encompass a calculated protein mass of ~66 kDa. Purification of this truncated construct resulted in significantly improved overall protein yield and monodispersity as assessed by size‐exclusion chromatography (Figure S3a–c).

For high‐resolution cryo‐EM studies we prepared plunge frozen grids of the truncated SLC5‐STAC CbrXA construct which were imaged at 300 keV on a Titan Krios G4i. The resultant dataset revealed particle distribution and calculated 2D averages (Figure S3d,e) that were highly similar to those obtained with the full‐length CbrA construct, further indicating that the PAS‐DHp‐CA domains are disordered in the full‐length construct. Subsequent 3D classification and reconstruction (Figure S4) revealed a predominant single conformation of the CbrA SLC5‐STAC domains which was reconstructed to a final overall resolution of ~2 Å (Figures 2a and S5a–c and Table S1), with most internal regions of the membrane embedded SLC5 domain resolved at resolutions higher than 2 Å (Figure S5d). At such resolution the individual rotamer states of most residues are easily discerned (Figure S5e), and holes are observed in the coulomb potential map for most aromatic sidechains (Figure S5f). The high resolution of the final map greatly facilitated refinement of an atomic model, and also revealed several well‐ordered water molecules throughout different regions of the SLC5‐STAC domain (Figure S5g).

FIGURE 2.

Structural analysis of CbrA SLC5‐STAC construct. (a) Rotated views of the high resolution cryo‐EM map of the CbrA SLC5‐STAC domains. The SLC5 domain is colored blue, STAC domain is colored orange, and CbrX is shown in cyan. Copurified lipid/detergent moieties are shown in magenta. The boundary of the detergent micelle is shown as a transparent white surface. (b) Zoomed in view of co‐purified CbrX demonstrating clear sidechain density throughout the peptide. A lipid like density (magenta) inserts between the two helices of CbrX. (c) Diagram showing the interaction between SLC5 and STAC domains in CbrA. Coloring scheme is the same as in (a). Dashed box inset provides a zoomed in view of the specific interactions between SLC5 and STAC domains. Hydrogen bonds are shown as dashed black lines. (d) Growth curves of WT, ∆cbrXAB, and complemented strains of P. putida in M9 minimal media with histidine as a sole carbon and nitrogen source. CbrX is not required for growth on histidine. The SLC5 or SLC5‐STAC constructs which lack intracellular histidine kinase domains of CbrA are not sufficient to rescue growth on histidine. Data points represent the average of three (N = 3) biological replicates that were each collected in technical triplicate. Error bars represent standard error of the mean (SEM) among biological replicates.

2.2. Structure of the CbrXA SLC5‐STAC domains

The high‐resolution cryo‐EM map of CbrA SLC5‐STAC domains revealed all 13 TM helices of CbrA, with several copurified lipid/detergent‐like densities also visible around the periphery of the transmembrane region (Figure 2a). Most surprisingly, clear density for the short CbrX peptide was also present in the final reconstructed cryo‐EM map (Figure 2a,b), indicating that CbrX forms a stable interaction with CbrA even after solubilization from membranes with detergent. In the reconstructed map, CbrX adopts an alpha‐helical hairpin configuration with both the amino and carboxy termini facing the intracellular region, and a short loop that connects both helices facing the periplasmic space. CbrX was positioned with both helices packed against TM₃ and TM₈ of the CbrA SLC5 domain, and underneath a structured alpha‐helical loop in the periplasmic space that connects CbrA TM_6/7 (Figure 2a,b). Within the cryo‐EM map, a thin acyl chain‐like density likely corresponding to a co‐purified lipid or detergent molecule is observed inserted between the two CbrX TM helices (Figure 2b). As elaborated upon in the discussion, the position of this lipid‐like density suggests a possible explanation for purification of CbrA in a monomeric form, rather than as a dimer, which is typical of most histidine kinase family proteins.

The STAC domain was also directly visible in the final cryo‐EM map and adopted a four‐helix bundle similar to the previously determined crystal structure of an isolated STAC domain from Archaeoglobus fulgidus (Korycinski et al. 2015). The STAC domain is connected to the SLC5 domain through a 30‐residue linker extending from TM₁₃ in the SLC5 domain. The N‐terminal region of this linker (residues 497–507) is ordered as an alpha‐helix that runs parallel to the membrane plane, with the remainder (residues 509–517) disordered and not visible in the map. The STAC domain is positioned beneath the membrane plane with the tips of STAC domain helices 3 and 4 inserted into a pocket formed at the base of the SLC5 transporter domain (Figure 2c). Most of the interactions between the SLC5 and STAC domains are hydrophobic in nature, with hydrogen bonds also observed between N531 and the backbone carbonyl of G579, and the sidechain hydroxyl of T115 and S582 (Figure 2c). Together, these interactions anchor the STAC domain against the cytoplasmic face of the SLC5 transporter domain in a defined orientation, suggesting that STAC serves as a structurally integrated appendage rather than a flexibly tethered accessory. This positioning suggests that the STAC domain may sense conformational changes within the SLC5 transporter core and potentially relay them to the downstream histidine kinase domains.

To assess the functional significance of CbrX and different domains of CbrA, we constructed a markerless knockout of the entire CbrXAB operon in P. putida KT2440 using allelic exchange (Hmelo et al. 2015). Consistent with previous studies, growth of wild‐type and ∆cbrXAB strains in defined minimal media revealed that cbrXAB is essential for growth when L‐histidine is supplied as the sole carbon and nitrogen source (Monteagudo‐Cascales et al. ²⁰¹⁹; Zhang et al. ²⁰¹⁵) (Figure 2d). Complementation of the knockout strain was achieved by mini‐Tn7 based insertion of a single copy of the wild‐type cbrXAB operon with native promoters at the attTn7 site downstream of the glmS gene (Choi and Schweizer 2006), which complemented growth on histidine as a sole carbon and nitrogen source back to levels seen with wild‐type P. putida (Figure 2d). When the ∆cbrXAB knockout strain was complemented with a cbrAB operon lacking the gene encoding the CbrX peptide, growth was also restored to levels comparable to the wild‐type strain (Figure 2d). This result demonstrates that while CbrX is translationally coupled to CbrA (Monteagudo‐Cascales et al. ²⁰¹⁹), production of the CbrX peptide is not required to produce a functional copy of CbrA and complement growth on L‐histidine. Similarly, while previous studies have demonstrated that the SLC5 domain of CbrA alone can mediate histidine uptake in P. putida (Wirtz et al. 2020), the functional significance of such uptake remained unclear. When we complemented the ∆cbrXAB strain with a cbrXAB operon that contained only the SLC5 or SLC5‐STAC domains of CbrA, no growth on histidine as a sole carbon/nitrogen source was observed (Figure 2d). These results indicate that any histidine transport capacity of the SLC5 or SLC5‐STAC domains is insufficient for growth of P. putida on histidine, and that a full‐length CbrA protein complete with intracellular histidine kinase domains is required for complementation, which corroborates findings in Pseudomonas fluorescens SBW25 (Zhang et al. 2015).

2.3. Histidine binding in the SLC5 domain

Previous studies have demonstrated that the CbrA SLC5 domain can bind histidine and mediate proton gradient driven uptake of the amino acid from the extracellular environment (Wirtz et al. 2020). In our cryo‐EM map the TM helices of the SLC5 domain are arranged in a classic LeuT‐type membrane transporter fold in an outward occluded state (Figure 3a,b) (Licht et al. 2024). TM_2,3,7,8 and TM_4,5,9,10 form “bundle” and “hash” subdomains, with TM_6,11 forming gating helices on the periphery of the transporter core (Del Alamo et al. 2022). The TM helices are arranged with TM_3‐6 and TM_8‐11 in an inverted repeat architecture, with helical breaks in TM₂ and TM₇ forming a central transport substrate pocket analogous to that seen in other LeuT type transporters (Del Alamo et al. 2022; Edwards et al. ²⁰¹⁸; Licht et al. ²⁰²⁴). The cryo‐EM map revealed a distinct density within these helical break regions that is not attributable to CbrXA, and is consistent with the size and shape of a free histidine molecule (Figures 3c,d and S6a,b). Analysis of the cryo‐EM map at different threshold levels reveals that the ligand density is significantly weaker than the surrounding protein density, suggesting that the copurified ligand is present at reduced occupancy relative to the protein and/or that its binding within the pocket is weakly constrained by surrounding residues (Figure S6c). Despite this potential reduced occupancy or weak binding, based on the shape of the observed density, its location in the canonical LeuT‐type transport pocket, and previous reports of histidine transport by CbrA (Wirtz et al. 2020; Zhang et al. ²⁰¹⁵), we have modeled this extra density as a co‐purified free histidine molecule (Figures 3c and S6).

FIGURE 3.

Histidine binding pocket within the SLC5 domain. (a) Overall topology of the CbrA SLC5 domain colored according to conventional LeuT type transporter folds. TM helices corresponding to the hash subdomain are colored green, the bundle subdomain colored blue, and the gating helices colored magenta. Extra TM helices outside the conventional LeuT topology are colored gray. The STAC domain is shown in orange beneath the SLC5 domain. (b) Topology diagram of the SLC5 TM helices colored the same as in (a). TM_3‐6 and TM_8‐11 form two inverted repeats highlighted with transparent yellow triangles. Helical break regions are identified in TM₂ and TM₇, which form the central ligand binding pocket outlined in a dashed red circle. (c) View of the central transport pocket in the SLC5 domain showing density for a copurified molecule of the approximate size and shape of histidine. Individual TM helices are colored as in pane (b). The Na2 site characteristic of LeuT type transporters is occupied by the sidechain of K196. (d) Rotated view of panel (c) showing the histidine binding pocket as viewed from the periplasmic space.

The copurified histidine captured within the center of the CbrA SLC5 domain is oriented with the imidazole ring moiety pointing toward the periplasmic space (Figures 3c,d and S6). The substrate binding pocket is lined by several hydrophobic sidechains, including A56, W55, Y51, Y75, F248, and F406 of CbrA (Figures 3c,d and S6a,b). Just above the bound histidine we observed two alternate rotamer states of the F71 sidechain, one of which partially seals the substrate binding pocket from the periplasmic space (Figure S6d). The carboxylate moiety of the bound histidine interacts closely with S53 in the helical break region of CbrA TM₂ (Figures 3c and S6a). On the opposite side of this helical break is the canonical Na2 site found in various sodium coupled LeuT type transporters (Del Alamo et al. 2022). Consistent with previous reports of histidine transport by CbrA being independent of a sodium gradient and dependent on the proton motive force (Wirtz et al. 2020), the Na2 site in CbrA is occupied by the sidechain of K196 (Figure 3c,d). The pKa of the K196 sidechain amino group as calculated with PROPKA (Olsson et al. 2011) is 7.36, consistent with the lower pKa seen with similarly placed lysine sidechains in other proton coupled LeuT type transporters (Jungnickel et al. 2018; Shaffer et al. ²⁰⁰⁹). The low pKa of the K196 sidechain suggests that this residue may act as a titratable residue to bias the conformation of the helical break region in TM₂ similar to Na⁺ binding in sodium dependent transporters (Del Alamo et al. 2022), thus modulating SLC5 domain dynamics for histidine transport.

2.4. Water permeation in the CbrA SLC5 domain

In addition to greatly facilitating refinement of an atomic model, the high resolution obtained from 3D reconstruction of the CbrA SLC5‐STAC domains revealed many water molecules directly visible in the cryo‐EM map (Figures 4a and S5g). Several well‐ordered water molecules are observed within the vicinity of the bound histidine, and also within the Na2 site near the helical break in TM₂ that is occupied by the K196 sidechain (Figure 4b). Many water molecules are also observed in the cytoplasmic vestibule where the STAC domain interacts with the hash subdomain of the SLC5 transporter (Figure 4c). The presence of these well‐ordered water molecules in the cryo‐EM map demonstrates that a significant proportion of the transport pathway through the SLC5 transporter core remains solvated following solubilization in detergent.

FIGURE 4.

Water permeation analysis. (a) Waters observed in the cryo‐EM map of the CbrXA SLC5‐STAC domains. Waters are shown as red spheres. CbrX is colored cyan, the CbrA SLC5 domain is shown in transparent light blue, and the STAC domain is shown in orange. (b) Close‐up view of the cryo‐EM resolved waters positioned near K196 in TM₆, and the periphery of the histidine binding pocket. Waters are shown as red spheres with the corresponding coulomb potential map shown in gray mesh. (c) View of waters (red spheres) resolved in the cryo‐EM map (gray mesh) in the cleft beneath the SLC5 domain (blue) that is occupied by the STAC domain (orange). (d) Single snapshot from one of the MD simulation trajectories with the CbrXA SLC5‐STAC domains and waters within 3 Å of the protein. Coloring scheme is the same as in panel (a). (e) Close‐up view of HIS binding pocket, TM₆ (K196) and TM₂ (break region) with the volume map of water occupancy from MD simulations shown in black transparent solid. The volume map is overlayed on the cryo‐EM structure with waters shown in red. The occupancy map of dynamic waters (MD) completely confine all the static waters observed in the cryo‐EM map. (f) Time evolution of average water occupancy within 1 nm of the CA atom of histidine (top panel) and K196 residue across protonated (in red) and deprotonated (in blue) MD simulations. The shaded region shows standard error of mean across all five replicates.

While the cryo‐EM structure reveals the locations of ordered water molecules, it cannot capture their dynamic exchange or the transient formation of hydrated pathways that may facilitate substrate translocation and/or proton transfer through the transporter core. To investigate the dynamics of these hydration networks, as well as the conformational dynamics of CbrA, we performed all‐atom molecular dynamics (MD) simulations of the CbrA SLC5–STAC domains embedded in a lipid bilayer. A visual representation of the simulation box with all the components is shown in supplemental data (Figure S7a). Due to the near‐neutral pKa of the conserved K196 that is central to the Na2 site, we decided to carry out two separate sets of MD simulations with the K196 side‐chain held fixed in either protonated or deprotonated state, treating protonation as a controlled perturbation rather than a dynamically sampled variable. For each of these two systems, henceforth denoted as “K196 protonated” and “K196 deprotonated,” we simulated five independent replicates totaling approximately 5 μs of aggregate simulation time for each. Importantly, the water molecules observed in the cryo‐EM map were not included in the initial setup of the simulation systems, and water was instead restricted to the bulk aqueous regions outside the lipid membranes while setting up the molecular dynamics simulations. Hence, the water molecules that permeated the CbrA transporter core in the MD simulations originated from the external solvent.

Analysis of the MD trajectories revealed that water molecules consistently permeated into the core of the CbrA SLC5 domain and occupied positions that closely correspond to those observed in the cryo‐EM structure. The permeated water molecules formed hydrogen‐bonded networks extending from the bulk solvent toward the helical break in TM₂ around the histidine binding pocket and K196 residue (Figure S7b–d). A representative MD snapshot (Figure 4d) of water molecules inside CbrA was extracted once a steady state of water influx had been attained. In this static picture, we observed a strong correlation between the positions of permeated waters in simulations with that of the stably bound waters in the experimental cryo‐EM structure. Subsequently, utilizing the MD trajectories we constructed a density map of permeated waters in the SLC5 domain (Figure 4e). This dynamic representation revealed a strong spatial overlap between the probability density of waters seen in MD with that of cryo‐EM resolved waters. Furthermore, water molecules consistently infiltrated the Na2 site region surrounding the conserved K196 residue and the histidine‐binding pocket in MD simulations, albeit with different frequencies in protonated and deprotonated systems (Figure S7c,d). The time evolution of water permeation in CbrA (Figure 4f) showed that the average number of waters within 1 nm of the Cα atom of K196 (top panel) or histidine substrate (bottom panel) is typically 3–5 times higher in K196 deprotonated simulations compared to its protonated counterpart. This pattern is prominent across all replicates once a steady state of water influx has been achieved, typically around the 400 ns timescale. The preferential hydration in deprotonated K196 systems indicates a relationship between the protonation state of the K196 and water permeation through the transporter core, hinting at a potential impact in the histidine transport mechanism.

Overall, the strong correspondence between water molecules identified experimentally by cryo‐EM and those sampled in the dynamic MD trajectories (Figure 4e) indicates that hydration within the CbrA SLC5 transporter represents an intrinsic and dynamically accessible feature of the protein rather than an artifact of experimental preparation. Water permeation near the Na2 site, captured in both cryo‐EM and MD simulations, suggests that the local environment surrounding K196 could support plausible pathways for solvent‐mediated proton access. Such proton access could plausibly lead to biasing of the K196 protonation state, leading to altered conformational states of the transporter to support histidine transport.

2.5. K196 (de)protonation and its effect on CbrA conformation space

Given the conserved placement of K196 within the Na2 site of the CbrA SLC5 domain and the intriguing observation of water permeation near this residue in CbrA, we decided to examine whether the protonation state of K196 would also influence the conformational landscape of the SLC5‐STAC domains and have an effect on histidine transport, as has been observed for similarly placed lysine residues in other proton‐coupled SLC transporters (Jungnickel et al. 2018; Shaffer et al. ²⁰⁰⁹; Wirtz et al. ²⁰²¹). The previously mentioned MD simulation trajectories of the CbrXA SLC5–STAC domains in either protonated or deprotonated K196 state were analyzed to understand the histidine motion and relevant conformational changes. First, to assess the stability of binding and probable transport of histidine in CbrA, we estimated the root mean square deviation (RMSD) of histidine coordinates in both protonated and deprotonated MD trajectories (Figure 5a). The average RMSD across five replicate simulations revealed noticeable differences in histidine motion between the K196 protonated and deprotonated systems, with the deprotonated systems showing a steady increase in the RMSD post 600 ns compared to the protonated systems. Next, we computed the average RMSD of the TM₂ break region heavy atoms (Figure 5b) to understand the motion of CbrA in each of the two systems. Consistent with histidine deviation, we noticed that the deprotonated ensembles have a 2‐fold higher RMSD in the break region compared to the protonated ensembles. Interestingly, both histidine and break region RMSDs in deprotonated simulations typically start to show this upward trend around 500–600 ns, after the steady state of water permeation is achieved (~400 ns), which may point to a subtle correlation between the K196 protonation dependent hydration with that of the conformational dynamics of the transporter.

FIGURE 5.

RMSD and residue contact analysis from MD simulations. Average root mean square deviations (RMSD) of histidine (a) and TM₂ break region (b) across five replicates of protonated (blue) and deprotonated (red) simulations with shaded areas showing the standard error across five replicate simulations. (c) Heatmap of intermolecular interactions between histidine and CbrA residues across five replicates of protonated and deprotonated simulations. The color scheme represents the percentage of snapshots in a replicate that have contact between histidine and the specific amino acid. Amino acids labeled in black primarily form contacts in deprotonated CbrA simulations. (d) Representative snapshots from MD trajectories highlighting different histidine‐CbrA interactions near the binding pocket (top panel) and after the downward transport (bottom panel).

Examining the intermolecular interactions between the bound histidine molecule and CbrA from the MD trajectories highlights significant residues that contact histidine at different timepoints of the trajectories. Histidine‐CbrA interactions were quantified across five replicates of protonated and deprotonated K196 simulations (Figure 5c). The set of significant residues is determined by a threshold that the residue and histidine contact must be present in at least 10% of the frames in either of the two systems, and contact is defined when the histidine:CbrA heavy atom distances are less than 3.5 Å (Bose et al. 2023; Bose et al. ²⁰²⁶). The contact maps of the protonated simulations show a high probability of histidine interactions with Y51, S53, W55, A56, C74, and Y75 residues (Figure 5c,d, top panel). However, this pattern is significantly shifted in deprotonated simulations, where unique interactions with F248, S251, H257, and F406 were observed in our MD trajectories that were not seen in protonated counterparts (Figure 5c,d, bottom panel). It should be noted that these new contacts in the deprotonated system are only possible after substantial displacement of histidine and considerable motion in the break region of CbrA TM₂.

Histidine transport mediated by CbrA is expected to involve conformational transitions that occur on timescales beyond those accessible by conventional unbiased MD simulations. However, the simulations performed here provide insight into slower collective motions and conformational states explored within the accessible simulation timescale. To examine these slower motions within the conformational landscape sampled by our simulations, we carried out combined time‐lagged independent component analyses (tICA) using all simulation trajectories. In this dimensionality reduction technique, the slow conformational motions are resolved into independent components (collective modes) enabling us to study progress along these slower modes (Bose et al. 2023; Bose et al. ²⁰²⁶). We trained 2‐dimensional tICA models using the combined dataset of protonated and deprotonated simulation features, one each with histidine coordinates and aligned break region heavy atom coordinates respectively. The tICA models are trained with a combined dataset (protonated and deprotonated) to ensure consistency of the definitions of independent components across both systems. We compared the free energy distributions in the tICA space between protonated and deprotonated systems with histidine positions as features toward the tICA model (Figure 6a). The protonated ensembles have two energetically stable, localized, free energy minima (a) and (b), where the backbone of S53 interacts with histidine by forming H‐bonds with the carboxyl moiety of the ligand. In each of these energetic basins the histidine molecule is oriented in different directions (Figure 6c). On the other hand, deprotonated ensembles have three relatively spread‐out shallow basins. In general, the free energy distribution of the deprotonated system showed a relatively high spread of conformational sampling. Interestingly, one of the stable basins (d) captures histidine in a slightly lower pocket along the channel enabling an H‐bond between the ligand amino group and the S53 backbone carbonyl.

FIGURE 6.

Time‐lagged independent component analysis. Free energy distributions along tIC‐1 and tIC‐2 with (a) histidine coordinates as a feature, and (b) heavy atoms of the break region as a feature for tICA training in protonated and deprotonated systems. (c) Representative conformations and important residues captured in highlighted energy basins are shown. Red lowercase letters correspond to the energy basins highlighted in panel (a) and (b). The TM2 break region is shown in purple in all snapshots with Histidine and K196 shown in licorice. Orange transparent surface corresponds to the average position of histidine (basin a–d) or the TM₂ break region (basin e–f) across five replicate MD simulations.

From the second tICA model, trained on the heavy atom coordinates of the break region residues, we observed one remarkably stable free energy minima (e) in the protonated ensembles suggesting no significant motion in the break region. In fact, we noticed the break region forming a stable helical turn in >50% of the protonated ensembles. Also, in the protonated system the Y51 sidechain forms a barrier at the base of the histidine binding pocket, preventing diffusion of the ligand to the intracellular space (Figure 6e). Interestingly, the deprotonated system demonstrated enhanced flexibility of the break region with three distinct basins and internal conversions. While we observed a broad, sufficiently stable basin in similar free energy space as the one in the protonated system, the other two shallower basins appear at the two extremes of tIC‐1 revealing unique ensembles of conformations. The break region attains a flexible loop conformation in both (f) and (g) basins. Remarkably, the ensemble (g) showed a considerable conformational shift in the break region, in particular the sidechain of Y51, which shifted away from its native gating location. This resulted in substantial downward transport of the HIS molecule toward the intracellular space, correlating to an RMSD of 10 Å in the histidine molecules in this basin compared to basin (e) or (f). These findings suggest that the protonation state of K196 can modulate the flexibility of the break region and subsequent orientation of Y51 as one component of enabling conformational transitions to mediate histidine transport through CbrA.

Together, our cryo‐EM structure and molecular dynamics simulations reveal that the CbrA SLC5 domain retains hallmark features of proton‐coupled amino acid transporters (Jungnickel et al. 2018; Shaffer et al. ²⁰⁰⁹). These features include a conserved Na2 site that is occupied by a lysine residue with a near‐neutral pKa that likely facilitates amino acid transport by biasing transporter conformations through a (de)protonation mechanism.

3. DISCUSSION

The ability of Pseudomonadaceae to thrive across diverse ecological niches relies on the capacity to sense and metabolize fluctuating nutrient sources. Central to this adaptability is CbrA, an unusual fusion protein that combines an SLC5‐family membrane transporter with a cytosolic histidine kinase, enabling coordinated regulation of carbon and nitrogen metabolism (Monteagudo‐Cascales et al. ²⁰²²). Although CbrA is essential for the utilization of substrates such as histidine, how membrane transport events are mechanistically coupled to downstream signaling and metabolic control has remained poorly understood (Wirtz et al. 2020; Zhang et al. ²⁰¹⁵). Here, we addressed this gap by determining a high‐resolution cryo‐EM structure of the CbrA SLC5–STAC domains, revealing the architecture of the membrane transporter region and a conserved histidine‐binding pocket. Integrated with all‐atom molecular dynamics simulations, these data provide a framework to begin understanding how ligand recognition and transporter conformational dynamics may be communicated across domains to allow CbrA to function as a global metabolic regulator in Pseudomonadaceae.

CbrA is translationally coupled to the short peptide CbrX (Monteagudo‐Cascales et al. ²⁰¹⁹), and our cryo‐EM structure reveals that CbrX is a membrane‐integrated peptide that forms a stable interaction with the CbrA SLC5 transporter domain (Figure 2a,b). Although histidine kinases classically function as dimers (Bhate et al. 2015), heterologously expressed CbrXA purified in detergent was predominantly monomeric (Figures S2 and S3). In contrast, AlphaFold models predict a dimeric configuration of CbrA that is consistent with canonical histidine kinase architectures (Figure S8a,b), in which two CbrX peptides appear to mediate contacts between opposing CbrA SLC5 domains (Figure S8c). We suspect that detergent solubilization weakens interactions between CbrX peptides at the dimer interface between opposing CbrA monomers, leading to the monomeric preparations of CbrXA obtained here. This idea is supported by the observation of acyl‐chain‐like density interdigitating between the TM helices of CbrX (Figure 2b). Together, these observations suggest that a native membrane environment and specific lipid interactions may be required to stabilize the functional oligomeric state of CbrXA. However, it is important to note that P. putida growth assays demonstrate that CbrX is not essential for CbrA‐dependent growth on histidine as a sole carbon and nitrogen source (Figure 2d). Thus, while CbrX copurifies with CbrA, and AlphaFold models suggest that it may contribute to CbrA dimer stabilization, the precise mechanistic role of CbrX in modulating CbrA signaling remains unresolved. An alternative model that cannot yet be excluded postulates that binding of CbrX to CbrA may prevent dimerization and activation of CbrA, and that CbrX dissociation may be facilitated by histidine binding and/or transport. Future biophysical assays with elaborate membrane reconstitution methods and/or in vivo assays will be required to investigate the mechanistic role of CbrX, and the oligomeric state of CbrA in a (near) native membrane context.

The high‐resolution structure of the CbrA SLC5–STAC domains presented here provides a framework for understanding how histidine sensing and transport are mechanistically encoded within the transmembrane region of CbrA. Our cryo‐EM structure revealed that the SLC5 domain of CbrA forms a classical LeuT type transporter topology with a histidine binding pocket that is analogous to the transport pocket identified in other SLC family amino acid transporters (Figure 3a–c) (Jungnickel et al. 2018; Shaffer et al. ²⁰⁰⁹). The CbrA K196 sidechain is pointed into the Na2 site characteristic of other proton coupled SLC transporters (Figure 3c,d), with a calculated pKa suggesting that this residue may undergo protonation‐dependent conformational changes during the transport cycle (Jungnickel et al. 2018; Shaffer et al. ²⁰⁰⁹). Molecular dynamics simulations with protonated and deprotonated K196 supported this hypothesis, as significant deviations in the helical break of TM₂ were observed only when K196 was deprotonated (Figure 5b). This deviation in the TM₂ helical break aided in a relatively higher movement and a downward motion of the histidine substrate toward the cytosol. The free energy landscapes along two of the slowest conformational modes in tICA space (Figure 6a,b) show that both histidine and the TM₂ break region sample a diverse ensemble of conformations when K196 is deprotonated compared to protonated. The break region, in its fully flexible loop structure, facilitated the gating residue (Y51) to adopt an alternate conformation that allows the histidine to transition significantly toward the cytoplasm. These free energy landscapes along slower modes provide a preliminary mechanistic understanding of a long time‐scale histidine transport process. However, our simulations were performed with fixed protonation states of K196 and therefore do not capture potential dynamic changes in residue protonation that may occur during the transport cycle. Given that K196 exhibits a near‐neutral calculated pKa, future studies incorporating constant‐pH molecular dynamics will be valuable for investigating how dynamic protonation state changes of K196 and other residues influence conformational transitions and histidine transport. Additionally, while our simulations reveal slower collective motions within the accessible timeframe sampled, they do not capture the complete conformational landscape associated with a full histidine transport cycle. Future simulations using enhanced sampling methods will be required to access conformational states that simply cannot be obtained in the ~1 μs regime sampled here. An exciting avenue for future work will be to combine constant pH with enhanced sampling methods to understand how dynamic protonation state changes couple to long timescale conformational transitions associated with a complete membrane transport cycle.

Our MD simulations indicate an increased structural plasticity of the break region and substrate histidine with substantially more waters permeating in K196 deprotonated simulations. Previous studies have demonstrated that in other proton‐coupled amino acid transporters such as MjApcT (Shaffer et al. 2009), HutT (Wirtz et al. 2021), and GkApcT (Jungnickel et al. 2018), a lysine pointing into the conserved Na2 site is essential for substrate transport. Analysis of mutational effects on CbrA in P. putida are complicated by the fact that CbrA signaling induces expression of the high‐affinity histidine transporter HutT, which serves as the primary histidine importer (Figure 1a) (Wirtz et al. 2021). Thus, even partial signaling by CbrA variants may therefore be sufficient to activate HutT expression, masking potential defects in CbrA‐mediated histidine transport. Furthermore, both CbrA (Figure 2d) and HutT (Wirtz et al. 2021) are essential for growth of P. putida on histidine as a sole carbon source, which significantly complicates development of growth experiments to assay CbrA histidine transport capacity in a native context. These observations highlight the need for development of in vitro assays that decouple CbrA transport activity from downstream transcriptional responses. Although extensive efforts by us and other groups to functionally reconstitute CbrA into proteoliposomes have thus far been unsuccessful (Wirtz et al. 2020; Zhang et al. ²⁰¹⁵), future advances in membrane reconstitution strategies and/or modulation of lipid composition may enable direct interrogation of CbrA transport and signaling mechanisms in a controlled in vitro system.

Mechanistic interpretation of CbrA function is strengthened by the resolution of our cryo‐EM reconstruction. Achieving sub–2 Å resolution cryo‐EM reconstructions (Figure S5) for a ~66 kDa membrane protein assembly is rare and enabled direct visualization of features typically inaccessible in cryo‐EM studies of small membrane transporters. This level of detail allowed visualization of ordered water molecules throughout the CbrA SLC5–STAC domains, including within the histidine‐binding pocket, surrounding the titratable K196 residue, and extending into a solvent‐accessible vestibule beneath the SLC5 domain where the STAC domain docks (Figures 4a–c and S5g). The presence of structured waters in these regions suggests potential pathways for proton and solvent coordination that may facilitate conformational transitions during transport and signaling. Consistent with this interpretation, all‐atom molecular dynamics simulations revealed dynamic water permeation into these same cavities (Figure 4d–f), supporting the notion that hydration plays a role in stabilizing intermediate conformational states of the transporter. Together, the convergence of high‐resolution structural data and molecular dynamics affirms the importance of solvent‐mediated interactions in shaping the functional landscape of the CbrA transmembrane region.

In our cryo‐EM reconstruction the STAC domain forms an extensive and well‐ordered interface beneath the SLC5 transporter core. This positioning places the STAC domain in an ideal location to potentially sense and respond to conformational changes within the SLC5 transporter domain. Rather than serving as a passive linker, the tight packing and geometry of the STAC–SLC5 interface suggest that the STAC domain may act as a mechanical or allosteric coupling element, relaying transporter motions toward the downstream histidine kinase domains. Subtle rearrangements within the SLC5 domain that are driven by ligand binding, protonation state, or hydration changes could plausibly be transmitted through the STAC domain to modulate kinase activity. This architecture supports a model in which CbrA may operate as a true transceptor, integrating transport‐derived conformational signals directly into regulatory outputs, and provides a structural basis for how membrane‐localized events may control cytosolic signaling in this system.

In summary, our work provides a high‐resolution structural and dynamic framework for beginning to understand how CbrA integrates membrane transport with global metabolic regulation in Pseudomonadaceae. By revealing the architecture of the SLC5–STAC domains, identifying a conserved histidine‐binding pocket, and uncovering hydration and protonation‐dependent conformational dynamics within the transporter core, this study establishes key molecular features that underlie the overall function of CbrA. The close association of the STAC domain with the SLC5 transporter further suggests a direct route for coupling transport‐derived motions to histidine kinase activation, offering new insight into how transceptors may have evolved to coordinate environmental sensing with transcriptional control. Future efforts aimed at analyzing and visualizing CbrXA oligomeric states, reconstituting functional CbrA in defined membrane environments, and probing conformational and ligand binding‐dependent coupling between CbrA domains will be essential to fully elucidate how transport, signaling, and metabolism are integrated within this unusually sophisticated regulatory protein.

4. MATERIALS AND METHODS

4.1. Protein expression and purification

The overlapped genes encoding cbrX and cbrA from P. putida KT2440 were codon optimized for E. coli expression and cloned into a pET28A vector with a C‐terminal 8xHIS tag by TWIST Bioscience (San Francisco, CA). The individual cbrX and cbrA‐8xHIS genes were PCR amplified from this vector individually and assembled into a pETDUET‐1 vector under the control of individual T7 promoters using NEB HiFi DNA Assembly according to manufacturer's protocols. The truncated CbrXA construct containing only CbrX and the CbrA SLC5‐STAC domains was generated using the NEB Q5 Mutagenesis kit according to manufacturer's protocols. The resulting expression plasmids were sequence verified by whole plasmid sequencing at Plasmidsaurus (San Francisco, CA) before being transformed into chemically competent C41(DE3) E. coli. A single colony was grown overnight in 100 mL of Lennox Broth (LB) media with 100 μg/mL carbenicillin at 37°C with shaking at 250 rpm. The starter culture was used to inoculate 4 L of Terrific Broth (TB) media with 100 μg/mL carbenicillin and several drops of antifoam‐204 in 3 L baffled Fernbach flasks. Cultures were grown at 37°C with shaking at 180 rpm to an OD600 of 0.8 before reducing the temperature to 16°C and inducing protein expression with 1 mM Isopropyl thiogalactoside (IPTG). Protein expression was allowed to proceed overnight before harvesting cultures via centrifugation. Bacterial pellets were flash frozen in liquid nitrogen and stored at −80°C.

For purification, bacterial pellets were thawed and resuspended with a Dounce homogenizer in lysis buffer (50 mM HEPES (pH 8), 300 mM KCl, 5 mM β‐mercaptoethanol, 10% (w/v) glycerol, 2 mM MgCl₂) with protease inhibitors (1 μg/mL pepstatin A, 1 μg/mL leupeptin, 1 μg/mL aprotinin, 0.6 mM benzamidine) and ~5 μg of recombinantly produced Serratia marcescens extracellular nuclease. The resuspended cell slurry was then lysed via sonication on ice, and the lysate was centrifuged at 4000g for 20 min to remove large debris and unbroken cells followed by ultracentrifugation at 100,000g to isolate the membrane fraction. Membranes were resuspended in a Dounce homogenizer on ice in lysis buffer with protease inhibitors and solubilized by adding LMNG detergent to a final concentration of 1% (w/v) and stirring at 4°C for 1 h. Insoluble debris was removed by ultracentrifugation at 100,000g for 1 h before proceeding with two‐step affinity and size‐exclusion chromatography.

Detergent solubilized CbrXA complexes were purified by applying the solubilized membrane fraction to 1 mL Co²⁺ TALON affinity resin in a gravity flow column format. The resin was subsequently washed in buffer A (25 mM HEPES (pH 8), 150 mM KCl, 5 mM β‐mercaptoethanol, 10% (w/v) glycerol, 0.005% LMNG, and 20 mM imidazole) to remove contaminants. CbrXA was then eluted in buffer A containing 250 mM imidazole before being concentrated in a 100 kDa MWCO centrifugal concentrator. The concentrated protein was then injected on to a Superdex 200 Increase 10/300GL column in 25 mM HEPES (pH 8), 150 mM KCl, 5 mM β‐mercaptoethanol, and 0.005% LMNG. Peak fractions were pooled and concentrated to ~6 mg/mL in a 100 kDa MWCO centrifugal concentrator for subsequent cryo‐EM experiments. Purity of the final samples was assessed by SDS‐PAGE analysis and staining with Aquastain (Bulldog Bio, Portsmouth, NH).

4.2. Cryo‐EM imaging and data processing

Purified CbrXA constructs were vitrified by applying 3 μL of purified protein to Quantifoil R1.2/1.3200 mesh Cu grids that had been glow‐discharged in a Pelco EasyGlow for 45 s at 15 mA. Grids were plunge frozen by blotting for 4 s on a Vitrobot Mark IV set to 100% relative humidity (RH), 4°C, and a blot force of 1. Blotted grids were plunge frozen in liquid ethane cooled by liquid nitrogen. An initial screening dataset was collected on a Talos Arctica 200 kV microscope equipped with a Selectris energy filter and Falcon IVi detector, and the final high‐resolution dataset of the truncated CbrXA construct was collected on a Titan Krios G4i equipped with a cold‐FEG, fringe‐free illumination, and a Selectris‐X energy filter with a 10 eV slit width. Movies were collected in .EER format using beam‐image shift acquisition targeting ~59 holes per stage move. Movies were collected with a pixel size of 0.731 Å/pixel, a total dose of 45 e⁻/Ų, and a defocus range of −0.5 to −1.5 μm (Table S1).

All cryo‐EM image processing steps were performed in CryoSPARC (Punjani et al. 2017). Raw .EER movies without upsampling were corrected for beam induced motion and the contrast transfer function (CTF) using patch‐motion correction and patch‐CTF correction. Blob‐based particle picking was performed on denoised micrographs, and 2D classification was used to generate 2D averages for template‐based picking. Extracted particles were subjected to multiple rounds of 2D classification followed by ab initio reconstruction, heterogeneous refinement, and non‐uniform refinement in CryoSPARC. The final resolution of cryo‐EM maps was determined from Fourier Shell Correlation (FSC) of independently refined half‐maps using a Gold‐standard cutoff of 0.143 (Scheres and Chen 2012). Data collection and refinement statistics are reported in Table S1.

4.3. Atomic model building and refinement

The atomic model for CbrXA SLC5‐STAC domains was built by first rigid body docking an AlphaFold (Jumper et al. 2021; Varadi et al. ²⁰²³) model of the CbrXA complex generated with localColabFold (Mirdita et al. 2022) into the cryo‐EM map using UCSF Chimera (Pettersen et al. 2004). The model was manually adjusted in COOT (Emsley et al. 2010) to properly fit the cryo‐EM density, and refined in real‐space using phenix.real_space_refine in the PHENIX software suite (Adams et al. 2010). Iterative rounds of real‐space refinement in PHENIX and manual model adjustment in COOT were performed to optimize model geometry and fit to the experimental cryo‐EM map. All models were assessed for appropriate stereochemical properties and fit to the experimental cryo‐EM map using MolProbity (Williams et al. 2018) as implemented in PHENIX (Table S1). Figures of cryo‐EM maps and atomic models were created using UCSF Chimera or UCSF ChimeraX (Pettersen et al. 2021).

4.4. Pseudomonas putida strain construction and bacterial growth assays

Knockout strains (∆cbrXAB) of P. putida KT2440 were generated by allelic exchange (Hmelo et al. 2015). DNA fragments encoding ~750 base pairs upstream and downstream of cbrXAB were PCR amplified from P. putida KT2440 genomic DNA and assembled into a pEXG2 vector (Rietsch et al. 2005). Deletion constructs were designed to maintain the start codon of cbrX and ~42 nucleotides at the 3′ end of cbrB. This cloning strategy removes the bulk of cbrXAB and instead produces a short (~14 amino acid) peptide to avoid potential ectopic effects to neighboring open reading frames. The resulting plasmids were transformed into P. putida KT2440 using electroporation, and merodiploids were selected by plating on LB‐agar plates containing 30 μg/mL gentamycin and confirmed by colony PCR with primers upstream and downstream of the cbrXAB operon. Confirmed merodiploids were then counter‐selected by plating on LB‐agar plates with 15% (wt/vol) sucrose. Sucrose insensitive colonies were screened for deletion of cbrXAB by colony PCR and further confirmed by nanopore sequencing at Plasmidsaurus.

To complement knockout strains of P. putida, the wild‐type cbrXAB sequence including the upstream P_cbrA promoter was cloned into a pUC18T‐mini‐Tn7T‐Gm vector (Choi and Schweizer 2006) using HiFi DNA Assembly. The resulting plasmid was then used as a template to make domain deletion constructs using the NEB Q5 Mutagenesis Kit according to manufacturer protocols. The resulting plasmids along with a pTNS2 helper plasmid were transformed into the ∆cbrXAB strains by electroporation and plated on LB‐agar containing 30 μg/mL gentamycin (Choi and Schweizer 2006). Transformants that had integrated cbrXAB at the mini‐TN7 site downstream of glmS were screened by colony PCR followed by nanopore sequencing at Plasmidsaurus.

Bacterial growth assays were performed by growing a single colony of each P. putida strain in 3 mL LB medium overnight at 30°C with shaking at 250 rpm. The following morning 1 mL of culture was pelleted by centrifugation and resuspended in 7 mL PBS before being diluted to a final OD600 of 0.05 in M9 minimal medium (42.2 mM Na₂HPO₄, 22 mM KH₂PO₄, 8.6 mM NaCl, 2 mM MgSO₄, trace elements) containing L‐histidine (20 mM) as a sole carbon and nitrogen source. Cells were plated in clear 96‐well plates (200 μL/well) and growth at 30°C was monitored at OD600 in a Molecular Devices iD5 plate reader with read intervals of 10 min with continuous medium intensity double orbital shaking. Growth assays were performed in biological triplicate.

4.5. Molecular dynamics simulations and analysis

The structure of CbrXA built from cryo‐EM data was embedded in a POPC bilayer using CHARMM‐GUI membrane builder (Feng et al. 2023). The obtained membrane‐protein systems were neutralized, and NaCl was added to achieve a salt concentration of 150 mM and solvated with TIP3P water molecules extending at least 20 Å on each side. The final systems containing ~205,000 atoms with the box size (132 Å × 132 Å × 126 Å) were made periodic on all three directions and periodic boundary conditions were applied. Another system with deprotonated K196 was also prepared identically to investigate the impact of protonation state on conformational space of CbrXA and corresponding histidine motion. All simulations were performed using OpenMM 8 (Eastman et al. 2024) with the CHARMM36m forcefield (Huang et al. 2017) using the Langevin integrator (Izaguirre et al. 2010) with 2 fs timestep. The systems were first energy minimized to relax the protein and lipids and remove steric clashes. The minimized systems were subjected to NVT equilibrations, followed by NPT equilibrations and MD production in NPT ensemble at 310 K and 1 atmospheric pressure using Monte Carlo barostat (Åqvist et al. 2004). The force‐based switching method was used for smoothing vdW interactions over the distance range 10–12 Å, and the particle‐mesh Ewald (PME) method (Essmann et al. 1995) was used for long‐range electrostatic interactions. Five independent replicates were simulated for each protonation state ranging between 900 and 1100 ns with different initial velocities to ensure reproducibility and adequate sampling of the conformational dynamics. The simulation trajectories were visualized using VMD (Humphrey et al. 1996) and molecular graphics in the figures were rendered in both VMD and UCSF Chimera X (Pettersen et al. 2021). Water occupancy maps were generated by combining trajectories from all simulations and analyzing them with the VolMap plugin in VMD. Trajectory frames after 400 ns were included, since we notice a steady state in water permeation, that is, a plateau in water count around the substrate and the break region, around that time‐point in our simulation. Water occupancy within 10 Å of the substrate histidine or CbrA residue K196 was quantified by computing an average occupancy grid at an isosurface threshold of 0.1, corresponding to water being present in a voxel in 10% of snapshots. The resulting occupancy maps were overlaid onto the cryo‐EM–derived atomic coordinates to compare experimentally observed water positions with those identified in the MD simulations.

Subsequent analyses such as (i) computing root mean square deviations (RMSD) of substrate histidine and break region residues, (ii) building contact maps of histidine with CbrA along the trajectories, and (iii) carrying out time‐lagged independent component analysis (tICA) to extract slow motions from the MD ensembles were performed using in‐house scripts (available on MD_Interpret), MDtraj libraries (McGibbon et al. ²⁰¹⁵), and Deeptime packages (Hoffmann et al. 2022). To remove global translational and rotational motion, the deprotonated and protonated MD trajectories were mean‐centered and aligned to their respective post‐equilibration reference frames. The backbone atoms were used as the alignment indices for all trajectory superpositions. It should be noted that there is negligible difference between the post‐equilibration structures of deprotonated and protonated system (backbone RMSD <0.2 nm). The feature sets to train tICA models were built using either the aligned histidine substrate coordinates or the aligned TM2 break region heavy atoms. The tICA method was used to identify the slow collective motions underlying the MD trajectories and to construct a reduced representation of the conformational landscape (Pérez‐Hernández et al. ²⁰¹³). In this framework, structural descriptors of the system (the aligned coordinates of histidine or break region residues) are linearly transformed into a set of orthogonal coordinates that maximize time‐lagged autocorrelation at a chosen lag time. This procedure provides an approximation to the slow eigenmodes of the underlying dynamical propagator that governs the time evolution of the molecular system. By emphasizing motions that relax slowly relative to the lag time, tICA filters out fast fluctuations and isolates collective coordinates associated with transitions between metastable conformational states. The leading time‐lagged component(s) therefore provide a kinetically meaningful low‐dimensional coordinate system for analyzing conformational transitions in the simulation trajectories (Bose et al. 2026). It should be noted, to preserve the continuity of the time‐lagged dataset, we ensured that each [t ₀, t _lag] that forms a time‐lagged pair is from a specific run of a specific (protonated/deprotonated) system. In this work, we examined a range of model parameters, including lag times from 10 to 200 ns and tICA dimensionalities between 2 and 5. Across this range, the qualitative structure of the free‐energy landscape projected onto the leading time‐lagged independent components (tIC1 and tIC2) remained largely unchanged, indicating that the dominant slow dynamical modes captured by the tICA models are robust to the choice of these parameters. We have used a sufficiently large lag time of 100 ns and two component tICA model to present our free energy analysis in section 2, with similar free energy distributions using different model parameters are provided in the Supporting Information.

4.6. Statistical analysis

Pseudomonas putida growth assays were performed in biological triplicate (N = 3) by starting growth protocols from independently picked individual bacterial colonies. Biological replicates were also measured in technical triplicate, and the reported values represent the average of values obtained across biological replicates (measured in technical triplicate). Where appropriate, error bars represent the standard error of the mean (SEM) across biological replicates.

AUTHOR CONTRIBUTIONS

Melanie A. Orlando: Writing – review and editing; formal analysis; investigation. Benjamin J. Orlando: Conceptualization; funding acquisition; writing – original draft; writing – review and editing; investigation; formal analysis; supervision. Matthew W. Faber: Investigation; formal analysis. Tejas Shah: Investigation; writing – review and editing; writing – original draft; formal analysis. Samik Bose: Investigation; writing – original draft; writing – review and editing; formal analysis; supervision.

FUNDING INFORMATION

Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM146721 to BJO. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Supporting information

Figure S1. Sequence alignment of CbrA proteins.

Figure S2. Purification and cryo‐EM analysis of full‐length CbrXA.

Figure S3. Structural analysis of truncated CbrXA.

Figure S4. Cryo‐EM data processing for truncated CbrXA.

Figure S5. Cryo‐EM data analysis for CbrXA SLC5‐STAC construct.

Figure S6. CbrA histidine binding pocket.

Figure S7. Molecular dynamics simulations.

Figure S8. tICA features and analysis.

Figure S9. AlphaFold models of CbrXA.

Table S1. Cryo‐EM data collection and refinement statistics.

Orlando MA, Shah T, Faber MW, Bose S, Orlando BJ. Structure and conformational dynamics of the Pseudomonas CbrA transceptor. Protein Science. 2026;35(9):e70775. 10.1002/pro.70775

DATA AVAILABILITY STATEMENT

Atomic coordinates and associated electron microscopy maps for the structure reported in this publication have been deposited in the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB) under the following accession number: CbrXA SLC5‐STAC (PDB 10ED, EMDB 75104). All files required to visualize and reproduce molecular dynamics simulations are freely available on Zenodo under the following DOI (https://doi.org/10.5281/zenodo.18867874). The analysis codes for RMSD, tICA, and water permeation are available on Github (https://github.com/SamikBose/MD_Interpret). All requests for plasmids, bacterial strains, or other materials will be honored upon request to BJO with an appropriate Materials Transfer Agreement issued by Michigan State University.