The sensor of the bacterial histidine kinase CpxA is a novel dimer of extracytoplasmic Per-ARNT-Sim domains

Histidine kinases are key bacterial sensors that recognize diverse environmental stimuli. While mechanisms of phosphorylation and phosphotransfer by cytoplasmic kinase domains are relatively well-characterized, the ways in which extracytoplasmic sensor domains regulate activation remain mysterious. The Cpx envelope stress response is a conserved Gram-negative two-component system which is controlled by the sensor kinase CpxA. We report the structure of the Escherichia coli CpxA sensor domain (CpxA-SD) as a globular Per-ARNT-Sim (PAS)-like fold highly similar to that of Vibrio parahaemolyticus CpxA as determined by X-ray crystallography. Because sensor kinase dimerization is important for signaling, we used AlphaFold2 to model CpxA-SD in the context of its connected transmembrane domains, which yielded a novel dimer of PAS domains possessing a distinct dimer organization compared to previously characterized sensor domains. Gain of function cpxA∗ alleles map to the dimer interface, and mutation of other residues in this region also leads to constitutive activation. CpxA activation can be suppressed by mutations that restore inter-monomer interactions, suggesting that inhibitory interactions between CpxA-SD monomers are the major point of control for CpxA activation and signaling. Searching through hundreds of structural homologs revealed the sensor domain of Pseudomonas aeruginosa sensor kinase PfeS as the only PAS structure in the same novel dimer orientation as CpxA, suggesting that our dimer orientation may be utilized by other extracytoplasmic PAS domains. Overall, our findings provide insight into the diversity of the organization of PAS sensory domains and how they regulate sensor kinase activation.

Table S2.Strains used in this study.

Figure S1 .
Figure S1.Expression levels of (A) cpxA chromosomal mutants and (B) plasmid-based mutations of CpxA in a Western blot using an anti-CpxA-MBP antibody from whole-cell lysate.

Figure S2 .
Figure S2.Crystal dimer structure of CpxA.(A) Ribbon cartoon diagram of the dimer with each monomer shown in a different color.The main dimer interface residue M48 is highlighted.The ability of the M48K mutation to sense (B) alkaline pH and (C) NlpE overexpression, as seen in the activity of a cpxP-lacZ transcriptional reporter.Shown are mean with standard deviation of three replicates from three independent experiments.(D) shows the expression levels of CpxA in relevant strains as determined by Western blotting with anti-CpxA-MBP antibody.

Figure S5 .
Figure S5.Alignment of the crystal structure and AlphaFold2 model monomer of E. coli CpxASD.

Figure S6 .
Figure S6.Ability of plasmid-borne CpxA D113K variant to sense (A) alkaline pH and (B) NlpE overexpression.Shown are mean with standard deviation of three replicates from three independent experiments.(C) shows the expression level of D113K compared to WT CpxA by Western blotting.

Figure S7 .
Figure S7.Western blots showing expression levels of His-tagged NlpE in strains expressing CpxA mutants.Strains cultured in identical conditions to reporter assay experiments were harvested, lysed and prepared for SDS-PAGE.After transfer, membranes were probed with antibody raised against RNAP α subunit (loading control) and His×6 (NlpE).The lane labelled "M" contains the molecular weight markers.

Figure S8 .
Figure S8.Ability of plasmid-borne CpxA E91K and E91A variants to sense (A) alkaline pH and (B) NlpE overexpression.(C) shows the expression level of D113K compared to WT CpxA by Western blotting.Shown are mean with standard deviation of three replicates from three independent experiments.

Figure S9 .
Figure S9.More PAS domain dimer hits of CpxASD.Each monomer is represented as a different colored chain (grey vs green).Protein Database (PDB) codes for each structure are listed below.

Figure S10 .
Figure S10.The ability of hyperactivated CpxA variants to sense CpxP overexpression.CpxP was induced from plasmid pTrc-cpxP with 0.1 mM IPTG for 2 hours after cells reached mid-log phase.The activity of a cpxP-lacZ reporter was used to measure activation of CpxA.Indicated CpxA variants were expressed from plasmid pK184.Shown are mean with standard deviation of three replicates from three independent experiments.

Figure S11 .
Figure S11.Conservation of N-capping motifs in cpxA sequences.(A) shows sequence motifs that are present at the N-cap site.(B) shows the lack of the presence of N-capping motifs in sequences immediately following the N-cap position in CpxA-SDEC and CpxA-SDVib.

Table S3 .
Primers used in this study.

Table S4 .
Plasmids used in this study.

Table S5 .
Modeling parameters and outputs of ColabFold.