PMC

Stereoview of the ChpT recognition surface for RRs. A. Residues (shown in yellow/blue/red ball-and-sticks) in the ChpT DHp region (green) hypothesized to be involved in binding RRs or phosphotransfer. The residues mutated in this study are labeled in red colored texts. B. Structural alignments of the DHp regions of ChpT (green), HK853 (orange) and Spo0B (blue). ChpT has a shorter α1 helix like Spo0B compared to HK853. Histidine residues involved in phosphotransfer are shown as balls-and-sticks (see also ).

Computational models of ChpT bound to its cognate RR domains. A. Schematic of the CckA-ChpT-CtrA-CpdR signaling pathway. B. Interaction site sequence conservation logos throughout alphaproteobacteria for ChpT, CckA-RD, CtrA, and CpdR. C. Stereoview of homology models of CckA (blue), CtrA (yellow), or CpdR (magenta) docked onto ChpT (green). D. Close-up views of the ChpT DHp-RR binding interface. Potential residues likely involved in binding are drawn as sticks and labeled.

The CckA-ChpT-CtrA-CpdR signaling pathway controls cell cycle progression in Caulobacter. A. Schematic of the CckA-ChpT-CtrA-CpdR signaling pathway. B. Domain organization of the TCS proteins in the CckA-ChpT-CtrA-CpdR pathway (transmembrane helix, TM; Per-Arnt-Sim sensor domain, PAS; dimerization and histidine phosphotransfer domain, DHp; catalytic and ATP-binding domain, CA; receiver domain, RD; helix-turn-helix DNA binding domain, HTH; domain of unknown function, DUF). Vertical arrows point to residues involved in phosphotransfer.

An essential surface on ChpT governs its RR interactions. A. For each ChpT mutant, ChpT~P was generated by incubation with FLAG-CckA, then subsequently purified to remove FLAG-CckA and ATP. Purified ChpT~P mutants were incubated for 10 s with either (1) ChpT~P only, (2) ChpT~P + SUMO-CtrA, (3) ChpT~P + CpdR, (4) ChpT~P + FLAG-CckA. B. A phosphotransfer assay between CckA and ChpT variants with point mutations in the putative RR binding region. CckA~P autophosphorylated in [γ-³²P] ATP was mixed with each ChpT variant and allowed to react for 10s before quenching. The phosphoproteins were separated by SDS-PAGE and imaged by phosphor storage. Negative control lane was a reaction mixture lacking ChpT. C. Quantitation of three replicate phosphotransfer assays between CckA~P and ChpT variants with mean intensity and standard deviation of each ChpT~P band shown. D. Classification of ChpT mutants as deficient in CckA-ChpT phosphotransfers, deficient in all phosphotransfers, or deficient in phosphotransfers between CckA-ChpT and ChpT-CtrA. Large black arrows indicate efficient phosphotransfer between signaling partners, whereas small, dashed, red arrows indicate diminished phosphotransfer (see also ).

The ChpT crystal structure shows a pseudo-HK fold. A. The annotated ChpT primary sequence with the secondary structure elements shown above (the 3₁₀ helix in red and indicated by an arrow). Conserved residues among ChpT orthologs are highlighted in colors (red, acidic; blue, basic; yellow, polar uncharged; and green, hydrophobic nonpolar). Magenta dots denote the residues at the DHp-CA interfaces, and the black triangle denotes the site of phosphorylation (His33). Residues that are subjected to point mutations are denoted by down arrows. The expected locations for degenerate HK sequence motifs are shown at the bottom (in quotes and up arrows). The domain boundary between the DHp and CA domains is located at residue 87. B. The domain organization of a ChpT monomer colored by domain (CA - red, DHp - blue). His33 and conserved arginines on the surface of CA domain are shown as sticks. C. A ChpT homodimer contains a four-helix bundle formed by two DHp domains. One ChpT molecule is colored magenta; the other is colored sea-green. His33, the site of phosphorylation, is shown in sticks. A circle highlights the site for interaction with RRs. D. A coupled-enzyme assay confirms ChpT cannot catalyze ATP hydrolysis. E. An ATP filter binding assay confirms ChpT cannot bind ATP (see also ).

A. Surface plasmon resonance demonstrates that His₆-ChpT and His₆-ChpT-A41R interact with CckA_70–691, but His₆-ChpT-G45R does not. Each SPR trace represents an increasing concentration of CckA-RD (1.6, 3.2, 6.3, 12.5, 25, 50, 100 and 200 µM). The mean response units (RU) from the CckA-ChpT interaction are plotted as a function of the CckA-RD concentration and fit to a one site specific binding model. Error bars represent the maximum deviation from the mean. B. ChpT point mutations along the putative RR binding interface affect Caulobacter crescentus morphology. Phase contrast micrographs of Caulobacter NA1000 strains whose sole chpT copy is either wild-type chpT-mcherry or a chpT-mcherry variant harboring a chpT RR binding mutation. C. Overexpression of chpT-mcherry-L64D overcomes cell morphology defects. Western blot analysis of ChpT-mCherry levels using an anti-RFP antibody confirms ChpT-mCherry L64D overexpression at 0.5mM vanillate relative to ChpT-mCherry expression levels at 5 µM vanillate. Probing the same samples with anti-PopZ sera provides a loading control. Phase contrast micrographs of chpT-mcherry-L64D cells reveal normal morphologies at 0.5 mM vanillate (see also ).