The Mycobacterium tuberculosis (Mtb) divisome protein ChiZ (gene Rv2719c) has a 64-residue intrinsically disordered N-terminal region (NT) that forms sequence-encoded "correlated segments" — contact-rich stretches with slow backbone dynamics — and, as established by follow-up work, engages in extreme fuzzy association with anionic membranes through conserved Arg residues. What NT actually does in cell division remains unknown: whether it acts through interactions with the disordered cytoplasmic regions of the divisome partners FtsQ and FtsI, through membrane association, through multivalent fuzzy complexes combining several divisome disordered regions, or a combination, and how the sequence-dependent N-half/C-half asymmetry partitions these roles.

Background

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, divides through a divisome — the multiprotein machine that assembles at midcell and builds the septum. Division begins when the tubulin homolog FtsZ polymerizes into a ring (the Z-ring) at the inner face of the cytoplasmic membrane and anchors itself there; late-stage divisome proteins such as FtsQ and FtsI (a penicillin-binding protein involved in septal peptidoglycan synthesis) are then recruited. Because most anti-tuberculosis drugs target cell-wall synthesis and growth, the mycobacterial divisome — which differs in several respects from the classical Escherichia coli paradigm — is an active target-discovery area.

ChiZ (gene Rv2719c) is an Mtb divisome membrane protein with a cytoplasmic N-terminal region (NT, residues 1–64), a single transmembrane helix, and a periplasmic LysM domain (residues 113–165; LysM is a peptidoglycan-binding module) joined to the helix by a linker. Its name ("cell wall hydrolase interfering with FtsZ ring assembly") is partly a misnomer: the reported hydrolase activity was later shown to be a zymogram false positive produced by its high positive charge, while interference with Z-ring assembly on ChiZ overexpression — which occurs physiologically under the SOS response (the bacterial DNA-damage-induced stress response that turns on ChiZ expression) — is reproducible. ChiZ overexpression halts septation and division in mycobacteria, yet the exact role of ChiZ in cell division has remained an open question since its discovery.

NT is intrinsically disordered: it does not fold into a stable three-dimensional structure but samples an ensemble of conformations. NMR spin relaxation experiments probe the dynamics of such regions residue by residue; the transverse relaxation rate R2R_2 and the nuclear Overhauser enhancement (NOE) are elevated for residues that move slowly. Hicks, Escobar, Cross, and Zhou (Biomolecules 2020) combined NMR, small-angle X-ray scattering, and explicit-solvent molecular dynamics (MD; simulations in which water molecules are represented individually) to show that NT is disordered with a high propensity for polyproline II (PPII) backbone angles, and that blocks of residues — for example residues 11–29 — form "correlated segments": stretches stabilized by frequent intrachain salt bridges, cation–π interactions, and sidechain–backbone hydrogen bonds, which display elevated slow collective motions on the 5–10 ns timescale while local sub-nanosecond motions are suppressed. Those simulations used the AMBER14SB protein force field (the set of interaction parameters governing MD) with the TIP4PD water model, a modified four-point water model developed to keep disordered proteins appropriately expanded.

Two direct follow-ups constrain any answer. First, Hicks, Escobar, Cross, and Zhou (JACS Au 2021) characterized NT of both the isolated construct and full-length ChiZ reconstituted into anionic liposomes by solution and solid-state NMR plus extensive MD: NT associates with acidic membranes in an "extremely fuzzy" way — it stays disordered and dynamic while its conserved Arg residues, predominantly in the C-terminal half (C-half), hydrogen-bond to acidic lipid headgroups; acidic residues in the N-terminal half (N-half) compete for those Args and partly keep the N-half off the membrane; and tethering via the transmembrane helix (plus an allosteric effect of the periplasmic linker and LysM domain on helix positioning) accentuates this N/C-half asymmetry. A fuzzy complex, generally, is one where a binding partner remains dynamic and does not adopt a single bound structure. Second, Smrt, Escobar, Dey, Cross, and Zhou (Communications Biology 2023) showed that the disordered cytoplasmic N-terminal region of the divisome partner FtsQ forms an Arg/Ala-rich helix that binds both acidic lipids and the FtsZ GTPase domain, making FtsQ a potential membrane anchor of the Z-ring — evidence that disordered-region-mediated interactions really do operate in this divisome, though ChiZ–FtsQ and ChiZ–FtsI interactions themselves remain uncharacterized (an earlier bacterial adenylate cyclase-based two-hybrid study indicated that ChiZ interacts with FtsQ and FtsI but not FtsZ, implying an indirect effect on Z-ring assembly, but neither interaction has been characterized at the molecular level).

Meanwhile, the broader threads raised by the 2020 paper have largely been settled by the same and adjacent groups: the Langevin-thermostat contribution to the dynamical bias was identified and a validated correction scheme provided (Hicks, MacAinsh, and Zhou, JCTC 2021); the correlated-segment concept was generalized across eight intrinsically disordered proteins with sequence-based rules (Dey, MacAinsh, Zhou, JCTC 2022) and then encoded in the sequence-based predictor SeqDYN trained on 45 such proteins — with ChiZ NT and Mtb FtsQ themselves in the test set — with explicit-solvent MD now reported to reproduce residue-level R2R_2 in quantitative agreement with NMR (Qin and Zhou, eLife 2024); and correlated segments were recast as drivers of liquid–liquid phase separation (Zhou, JACS Au 2025). What no study has done, in vitro or in cells, is establish which interaction the ChiZ disordered region actually uses to exert its documented effect on cell division.

Problem Statement

Determine the functional mechanism by which the intrinsically disordered N-terminal region (NT, residues 1–64) of Mycobacterium tuberculosis ChiZ contributes to cell division. All three parts below are jointly required to resolve the task; part (2) characterizes whichever interaction(s) part (1) identifies as functional, and part (3) demonstrates that interaction(s) in cells. Specifically:

  1. Identify which interaction NT functionally uses — binding to the disordered cytoplasmic regions of the divisome partners FtsQ and/or FtsI, the extreme fuzzy association with the anionic inner membrane that has been characterized in vitro, multivalent fuzzy complexes combining several divisome disordered regions at the membrane surface, or a combination of these.

  2. Characterize the interaction(s) identified in (1) at residue level: the binding mode and strength — a binding affinity or free-energy estimate, or a lifetime where the interaction is transient — the sequence blocks of NT involved, and how the sequence-dependent asymmetry between the N-half (contact-rich, PPII-dominated, kept partly off the membrane by its acidic residues) and the Arg-rich conserved C-half partitions whichever functional role(s) the interaction subserves (membrane anchoring and/or partner recognition) in the full-length, transmembrane-helix-tethered protein.

  3. Demonstrate in mycobacteria that the identified interaction(s) mediate ChiZ's documented effect on cell division — interference with Z-ring assembly and septation when ChiZ is overexpressed under DNA-damage/SOS conditions — for example through mutants that selectively disrupt NT membrane association or NT partner binding while preserving ChiZ expression, membrane topology, and the transmembrane and LysM segments, with quantified cell-division phenotypes relative to wild-type complementation.

References

  1. Hicks A, Escobar C, Cross TA, Zhou H-X. Sequence-Dependent Correlated Segments in the Intrinsically Disordered Region of ChiZ. Biomolecules 2020;10(6):946. DOI 10.3390/biom10060946. https://doi.org/10.3390/biom10060946
  2. Hicks A, Escobar CA, Cross TA, Zhou H-X. Fuzzy Association of an Intrinsically Disordered Protein with Acidic Membranes. JACS Au 2021;1(1):66–78. DOI 10.1021/jacsau.0c00039. https://doi.org/10.1021/jacsau.0c00039
  3. Hicks A, MacAinsh M, Zhou H-X. Removing Thermostat Distortions of Protein Dynamics in Constant-Temperature Molecular Dynamics Simulations. Journal of Chemical Theory and Computation 2021;17(9):5920–5932. DOI 10.1021/acs.jctc.1c00448. https://doi.org/10.1021/acs.jctc.1c00448
  4. Dey S, MacAinsh M, Zhou H-X. Sequence-Dependent Backbone Dynamics of Intrinsically Disordered Proteins. Journal of Chemical Theory and Computation 2022;18(10):6310–6323. DOI 10.1021/acs.jctc.2c00328. https://doi.org/10.1021/acs.jctc.2c00328
  5. Qin S, Zhou H-X. Predicting the sequence-dependent backbone dynamics of intrinsically disordered proteins. eLife 2024;12:RP88958. DOI 10.7554/eLife.88958. https://doi.org/10.7554/eLife.88958
  6. Zhou H-X. Correlated Segments of Intrinsically Disordered Proteins as Drivers of Homotypic Phase Separation. JACS Au 2025;5(9):4361–4369. DOI 10.1021/jacsau.5c00733. https://doi.org/10.1021/jacsau.5c00733
  7. Smrt ST, Escobar CA, Dey S, Cross TA, Zhou H-X. An Arg/Ala-rich helix in the N-terminal region of M. tuberculosis FtsQ is a potential membrane anchor of the Z-ring. Communications Biology 2023;6:311. DOI 10.1038/s42003-023-04686-5. https://doi.org/10.1038/s42003-023-04686-5
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  11. Vadrevu IS, Lofton H, Sarva K, Blasczyk E, Plocinska R, Chinnaswamy J, Madiraju M, Rajagopalan M. ChiZ levels modulate cell division process in mycobacteria. Tuberculosis 2011;91:S128–S135. DOI 10.1016/j.tube.2011.10.022. https://doi.org/10.1016/j.tube.2011.10.022
  12. Escobar CA, Cross TA. False positives in using the zymogram assay for identification of peptidoglycan hydrolases. Analytical Biochemistry 2018;543:162–166. DOI 10.1016/j.ab.2017.12.016. https://doi.org/10.1016/j.ab.2017.12.016