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Cited by 2Bacterial Genetics and BiotechnologyProtein Structure and DynamicsEnzyme Structure and Function

The DOI matches its Crossref record; 68 of the 68 references it cites were found in a registry or an index.

Insights Into the Conformational Dynamics of the Cytoplasmic Domain of Metal‐Sensing Sensor Histidine Kinase ZraS

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Mahapatra, Nilima, Mahanta, Pranjal, Pandey, Shubhant, Acharya, Rudresh·2025·Proteins: Structure, Function, and Bioinformatics
DOI 10.1002/prot.26819PMC PMC12314577

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Abstract

ZraS is a metal sensor integral to ZraPSR, a two‐component signaling system found in enterobacters. It belongs to a family of bifunctional sensor histidine kinases (SHKs) and is speculated to sense zinc‐induced stress on the bacterial envelope. Information on the structure–function relationship of sensor kinases is elusive due to the lack of full‐length structures, intrinsically dynamic behavior, and difficulty trapping them in active state conformations. While the kinase domains (KDs) of a few SHKs are well characterized, they exhibit significant functional diversity attributed to their modular multi‐domain arrangement in the cytoplasmic region, combined with other signal transducing elements such as simple helices, HAMP, and PAS domains. We report the crystal structure of the entire cytoplasmic region of Escherichia coli ZraS (EcZraS‐CD) resolved at a resolution of 2.49 Å, comprising a unique helical linker and the KD. In the asymmetric unit, four molecules of ZraS assemble as homodimers trapped as two ligand‐bound occluded conformers. Our analysis using these conformers shows that modulation of the dimer bundle through segmental helical bending, sliding, and rotation leads to the reorganization of the dimerization interface during kinase activation. Further, our analysis reveals the significance of aromatic amino acid interactions and loop residues at the dimer base in regulating the directionality of rotation during autophosphorylation. We also performed an in vitro coupled assay to determine ATPase activity. Overall, our findings provide structure‐based mechanistic insights into the process of autophosphorylation in trans‐acting SHKs.

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Figures

FIGURE 1 Full‐length model and truncated crystal structure of cytoplasmic domain of EcZraS. (A) Schematic representation of different types of stimuli detected by SHKs. (B) Representation of a prototypical SHK showing direct phosphate transfer and a hybrid kinase transferring phosphate through a relay mechanism involving other intermediate domains such as HPt and receiver domains. (C) AlphaFold model of the apo form of full‐length ZraS (EcZraS‐F) displaying domain distributions across periplasmic (light magenta), transmembrane (deep teal), and cytoplasmic regions (forest, limon, lime green, and deep purple) (34). (D) Three‐dimensional crystal structure of ligand‐bound ZraS (EcZraS‐CD) trapped in an intermediate state. The overall structure of the cytoplasmic region has a simple linker domain (forest) and a kinase domain made of the DHp and CA domains. The DHp region (limon and lime green) is connected to the CA domains (deep purple) using hinges (hot pink).
FIGURE 1 Full‐length model and truncated crystal structure of cytoplasmic domain of EcZraS. (A) Schematic representation of different types of stimuli detected by SHKs. (B) Representation of a prototypical SHK showing direct phosphate transfer and a hybrid kinase transferring phosphate through a relay mechanism involving other intermediate domains such as HPt and receiver domains. (C) AlphaFold model of the apo form of full‐length ZraS (EcZraS‐F) displaying domain distributions across periplasmic (light magenta), transmembrane (deep teal), and cytoplasmic regions (forest, limon, lime green, and deep purple) (34). (D) Three‐dimensional crystal structure of ligand‐bound ZraS (EcZraS‐CD) trapped in an intermediate state. The overall structure of the cytoplasmic region has a simple linker domain (forest) and a kinase domain made of the DHp and CA domains. The DHp region (limon and lime green) is connected to the CA domains (deep purple) using hinges (hot pink).CC BY-NC-ND · PubMed Central
FIGURE 2 Structural comparison between chains cA of dimer AB and cC of dimer CD. (A) Cartoon representation of the superposed dimers of EcZraS‐CD. Chain cC (cyan) of dimer CD displays a prominent helical bend at the lower region of the DHp bundle compared to chain cA (green) of dimer AB. (B) 2Fo‐Fc map contoured at 1.0 σ for the linker and DHp helices of cA and cC. (C) Structural overlap of cA and cC displaying rotation of CA domain through an angle of nearly 20°.
FIGURE 2 Structural comparison between chains cA of dimer AB and cC of dimer CD. (A) Cartoon representation of the superposed dimers of EcZraS‐CD. Chain cC (cyan) of dimer CD displays a prominent helical bend at the lower region of the DHp bundle compared to chain cA (green) of dimer AB. (B) 2Fo‐Fc map contoured at 1.0 σ for the linker and DHp helices of cA and cC. (C) Structural overlap of cA and cC displaying rotation of CA domain through an angle of nearly 20°.CC BY-NC-ND · PubMed Central
FIGURE 3 Measured distances and angles of the cytoplasmic domain of ZraS. (A) Inter‐domain distances and angles between the CA domains of apo EcZraS‐F (dimer XY) and ligand‐bound EcZraS‐CD (dimer AB and dimer CD) structures. (B) Helix bending angles at Pro259 of helix hα1 (orange) and hα1’ (deep salmon) of dimer XY, helix hα1 (green) and hα1’ (yellow) of dimer AB, and helix hα1 (cyan) and hα1’ (light magenta) of dimer CD. (C) Structural superposition of dimer XY (orange), dimer AB (green), and dimer CD (cyan) displaying the angular rotation of CA domain upon ligand binding. (D) Distance between catalytic His254 in the DHp and ATP‐binding Asn367 in the opposite CA of apo (dimer XY) and ligand‐bound (dimer AB and dimer CD) structures.
FIGURE 3 Measured distances and angles of the cytoplasmic domain of ZraS. (A) Inter‐domain distances and angles between the CA domains of apo EcZraS‐F (dimer XY) and ligand‐bound EcZraS‐CD (dimer AB and dimer CD) structures. (B) Helix bending angles at Pro259 of helix hα1 (orange) and hα1’ (deep salmon) of dimer XY, helix hα1 (green) and hα1’ (yellow) of dimer AB, and helix hα1 (cyan) and hα1’ (light magenta) of dimer CD. (C) Structural superposition of dimer XY (orange), dimer AB (green), and dimer CD (cyan) displaying the angular rotation of CA domain upon ligand binding. (D) Distance between catalytic His254 in the DHp and ATP‐binding Asn367 in the opposite CA of apo (dimer XY) and ligand‐bound (dimer AB and dimer CD) structures.CC BY-NC-ND · PubMed Central
FIGURE 4 Nucleotide‐binding pocket and lid loop of EcZraS‐CD. (A) Surface representation of the nucleotide‐binding pocket lined by residues from N (deep teal), G1 (green cyan), F (cyan), G2 (forest), and G3 (gray50) boxes in EcZras‐CD. (B) ADP and amino acid residues flanking the nucleotide‐binding pocket of cA are shown in stick representation. Mg2+ is shown as a sphere (green). (C) Structural superposition of CA domains of chain A (green), chain B (yellow), chain C (cyan), and chain D (light magenta) of ZraS showing local uncoiling of α3 and α4. (D) Structural superposition of ATP lid loop region of PhoQ (marine) in a closed state and EnvZ (hotpink) in an open state with EcZraS‐CD (gray90). (E) Comparison of ATP lid loop region of CheA (purple) and KinB (orange) with EcZraS‐CD showing secondary structures in the loop. (F) and (G) Stick and surface representation showing change in Lys416 interaction in cA and cD. The dynamic lid loop encloses the pocket in cA interacting with the adenine ring of ADP, absent in cD. The 2Fo‐Fc map is contoured at 1.0 σ.
FIGURE 4 Nucleotide‐binding pocket and lid loop of EcZraS‐CD. (A) Surface representation of the nucleotide‐binding pocket lined by residues from N (deep teal), G1 (green cyan), F (cyan), G2 (forest), and G3 (gray50) boxes in EcZras‐CD. (B) ADP and amino acid residues flanking the nucleotide‐binding pocket of cA are shown in stick representation. Mg2+ is shown as a sphere (green). (C) Structural superposition of CA domains of chain A (green), chain B (yellow), chain C (cyan), and chain D (light magenta) of ZraS showing local uncoiling of α3 and α4. (D) Structural superposition of ATP lid loop region of PhoQ (marine) in a closed state and EnvZ (hotpink) in an open state with EcZraS‐CD (gray90). (E) Comparison of ATP lid loop region of CheA (purple) and KinB (orange) with EcZraS‐CD showing secondary structures in the loop. (F) and (G) Stick and surface representation showing change in Lys416 interaction in cA and cD. The dynamic lid loop encloses the pocket in cA interacting with the adenine ring of ADP, absent in cD. The 2Fo‐Fc map is contoured at 1.0 σ.CC BY-NC-ND · PubMed Central
FIGURE 5 Organization and core packing of DHp bundle in EcZraS‐CD. (A) Representation of DHp bundle forming modified knobs‐into‐holes packing. Knobs formed by side chains of residues at positions “a” (gray50) and “d” (purple and violet) are packed into grooves formed between ridges of neighboring helices along the dimer interface. (B) Alternating residue layers (adL1–adL6) at position “d” from helix‐1 (purple) and helix‐2 (violet) forming the inner core of DHp bundle. (C) The top view of the bundle showing the distribution of “a” and “d” residues forming the a and d layers. (D) Surface representation of “d” residues (violet and purple) in the inner core and “a” residues (gray50) lining its periphery. (E) Lateral and top views of variable interactions between Met287 in adL5 at the dimerization core interface of dimer AB and dimer CD. (F) 2Fo‐Fc electron density map contoured at 1.0 σ showing Met287 in the DHp bundle of dimers AB and CD.
FIGURE 5 Organization and core packing of DHp bundle in EcZraS‐CD. (A) Representation of DHp bundle forming modified knobs‐into‐holes packing. Knobs formed by side chains of residues at positions “a” (gray50) and “d” (purple and violet) are packed into grooves formed between ridges of neighboring helices along the dimer interface. (B) Alternating residue layers (adL1–adL6) at position “d” from helix‐1 (purple) and helix‐2 (violet) forming the inner core of DHp bundle. (C) The top view of the bundle showing the distribution of “a” and “d” residues forming the a and d layers. (D) Surface representation of “d” residues (violet and purple) in the inner core and “a” residues (gray50) lining its periphery. (E) Lateral and top views of variable interactions between Met287 in adL5 at the dimerization core interface of dimer AB and dimer CD. (F) 2Fo‐Fc electron density map contoured at 1.0 σ showing Met287 in the DHp bundle of dimers AB and CD.CC BY-NC-ND · PubMed Central
FIGURE 6 Variable helical interface interactions of DHp and linker domain in EcZraS‐CD. (A) Distance between aromatic amino acid residues Tyr269 and Phe270 of chains cA and cB in dimer AB. (B) Bundle base interactions in dimer AB regulating bundle rotation. Interacting residues are shown in stick representation, and non‐interacting residues are shown in line representation. (C) Residue interactions at the linker interface of dimers AB and CD. Distortion in the alternate layering arrangement of the core residues is caused due to intervention by residues in polar clusters. The core “d” residues interacting at the interface in each dimer are labeled in violet, with the 2Fo‐Fc maps contoured at 1.0 σ. The dotted circles highlight the position of linker helix bending initiating at Leu242 in cA and cC.
FIGURE 6 Variable helical interface interactions of DHp and linker domain in EcZraS‐CD. (A) Distance between aromatic amino acid residues Tyr269 and Phe270 of chains cA and cB in dimer AB. (B) Bundle base interactions in dimer AB regulating bundle rotation. Interacting residues are shown in stick representation, and non‐interacting residues are shown in line representation. (C) Residue interactions at the linker interface of dimers AB and CD. Distortion in the alternate layering arrangement of the core residues is caused due to intervention by residues in polar clusters. The core “d” residues interacting at the interface in each dimer are labeled in violet, with the 2Fo‐Fc maps contoured at 1.0 σ. The dotted circles highlight the position of linker helix bending initiating at Leu242 in cA and cC.CC BY-NC-ND · PubMed Central
FIGURE 7 Proximal CA–DHp domain interfaces and enzyme kinetics plot of EcZraS‐CD (A) Cartoon representation of the CA–DHp interface of dimer AB and dimer CD. Sticks show the interacting residues at the interface in either dimer (2Fo‐Fc maps contoured at 1.0 σ). (B) Electrostatic surface potential representation of interacting interfaces of CA and DHp domain. ADP bound to the nucleotide‐binding pocket, with a highly positive electrostatic potential is shown in surface and stick representation. (C) Kinetic characterization of EcZraS‐CD autophosphorylation using pyruvate kinase–lactate dehydrogenase coupled assay. The curve shows ATP dependence on initial velocities of autophosphorylation.
FIGURE 7 Proximal CA–DHp domain interfaces and enzyme kinetics plot of EcZraS‐CD (A) Cartoon representation of the CA–DHp interface of dimer AB and dimer CD. Sticks show the interacting residues at the interface in either dimer (2Fo‐Fc maps contoured at 1.0 σ). (B) Electrostatic surface potential representation of interacting interfaces of CA and DHp domain. ADP bound to the nucleotide‐binding pocket, with a highly positive electrostatic potential is shown in surface and stick representation. (C) Kinetic characterization of EcZraS‐CD autophosphorylation using pyruvate kinase–lactate dehydrogenase coupled assay. The curve shows ATP dependence on initial velocities of autophosphorylation.CC BY-NC-ND · PubMed Central
FIGURE 8 Model of autokinase activation in EcZraS‐CD. Schematic representation of the autokinase activity in the cytoplasmic domain of ZraS illustrates transition from an inactive apo form to an active ligand‐bound form mediated through intermediate conformers, state‐1 (dimer AB) and state‐2 (dimer CD). Conformational transition in the cytoplasmic region is facilitated through dimer interface reorganization driven by helical bending, scissoring, registry shift, and domain rotation.
FIGURE 8 Model of autokinase activation in EcZraS‐CD. Schematic representation of the autokinase activity in the cytoplasmic domain of ZraS illustrates transition from an inactive apo form to an active ligand‐bound form mediated through intermediate conformers, state‐1 (dimer AB) and state‐2 (dimer CD). Conformational transition in the cytoplasmic region is facilitated through dimer interface reorganization driven by helical bending, scissoring, registry shift, and domain rotation.CC BY-NC-ND · PubMed Central

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Research works printed but not linked: 3

  1. 10.1128/mmbr.53.4.450‐490.1989 · DOI
  2. L DeLano W., 2002, Pymol: An Open‐Source Molecular Graphics Tool, CCP4 Newsl. Protein Crystallogr, 40, 1
  3. 10.2142/biophysico.16.0 · DOI

Metadata

Authors
Mahapatra, N., Mahanta, P., Pandey, S., & Acharya, R.
Affiliations
National Institute of Science Education and Research (IN)
Corresponding author
Acharya, Rudresh
ORCID
Venue
Proteins: Structure, Function, and Bioinformatics
Publisher
Wiley
Published
2025-03-10
Volume
93
Issue
9
Pages
1465-1480
ISSN
  • 0887-3585 (ISSN-L)
  • 1097-0134
DOI
10.1002/prot.26819
PMC
PMC12314577
OpenAlex
W4408301829
Cited by
2
References
68
Citations by year
2025: 2
FWCI
1.17
Type
journal article
Language
en
Open access
Licence
Locations
  • Proteins Structure Function and Bioinformatics (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
  • National Institute of Science Education and Research (RIN4002)
  • Council of Scientific and Industrial Research, India (09/1002(0045)/2019‐EMR‐I)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
Keywords
sensor histidine kinase, two-component systems, zinc ion detection, Escherichia coli, kinase domain, homodimer, ATPase activity, conformational dynamics
MeSH terms
Histidine Kinase, Protein Domains, Binding Sites, Cytoplasm, Escherichia coli, Models, Molecular, Protein Binding, Protein Conformation, Zinc, Signal Transduction, Crystallography, X-Ray, Escherichia coli Proteins, Protein Multimerization

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CoChat. (2026). Citation check: Insights Into the Conformational Dynamics of the Cytoplasmic Domain of Metal‐Sensing Sensor Histidine Kinase ZraS. https://app.cochat.ai/papers/10.1002/prot.26819
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Mahapatra, N., Mahanta, P., Pandey, S., & Acharya, R. (2025). Insights Into the Conformational Dynamics of the Cytoplasmic Domain of Metal‐Sensing Sensor Histidine Kinase ZraS. Proteins: Structure, Function, and Bioinformatics. https://doi.org/10.1002/prot.26819

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