Molecular insights into Escherichia coli Cpx envelope stress response activation by the sensor lipoprotein NlpE

Abstract

Bacterial two-component signal transduction systems provide sensory inputs for appropriately adapting gene expression. These systems rely on a histidine kinase that phosphorylates a response regulator which alters gene expression. Several two-component systems include additional sensory components that can activate the histidine kinase. In Escherichia coli, the lipoprotein NlpE was identified as a sensor for the Cpx cell envelope stress response. It has remained unclear how NlpE signals to Cpx in the periplasm. In this study, we used a combination of genetics, biochemistry, and AlphaFold2 complex modeling to uncover the molecular details of how NlpE triggers the Cpx response through an interaction with the CpxA histidine kinase. Remarkably, only a short loop of NlpE is required to activate the Cpx response. A single substitution in this loop inactivates NlpE signaling to Cpx and abolishes an in vivo biochemical NlpE:CpxA interaction. An independent AlphaFold multimer prediction supported a role for the loop and predicted an interaction interface at CpxA. Mutations in this CpxA region specifically blind the histidine kinase to NlpE activation but preserve the ability to respond to other cell envelope stressors. Hence, our work additionally reveals a previously unrecognized complexity in signal integration by the CpxA periplasmic sensor domain.

Figure 1:. Lipoprotein trafficking and response to defects in pathway.

(A) Lipoproteins are secreted through the Sec translocon and modified through several steps at the inner membrane (IM). Mature triacylated lipoproteins are extracted by the LolCDE ATPase complex. The periplasmic chaperone LolA then transports the lipoproteins to the outer membrane (OM) and transfers to the OM acceptor, LolB. LolB then inserts them into the OM. When there are defects in the trafficking pathway, lipoproteins accumulate at the IM and sensor lipoprotein NlpE signals to the Cpx two-component stress response system. Compound 2 (Cpd2) and Globomycin (Glb) are two inhibitors specific to the Lol pathway and result in potent Cpx activation. (B) Overview of the NlpE random mutagenesis screening strategy used to identify mutants defective in signaling to the Cpx stress response system.

Figure 2:. Mutations in NlpE loop 73–75 strongly impair signaling to Cpx.

(A) NlpE residues R73, T74, and A75 highlighted on the crystal structure of E. coli NlpE (PDB 2Z4H). (B) Whole cell lysates of induced cultures were probed with anti-FLAG to detect NlpE_1–121-FLAG protein levels. The variants are produced at levels that are equivalent to wildtype. R73Q, T74I, and A75T (not shown) are all representative. (C and D) NlpE expression was induced with 0.01% Arabinose. Culture growth was measured using A_600nm. Cpx activation was measured by GFP fluorescence (FL) produced from a PcpxP–GFP transcriptional reporter plasmid. Fold-change was calculated by normalizing GFP per cell values (FL/A_600nm) values to the strain containing pBAD18 vector control. Data are average of n=4 biological replicates ± standard deviation of the mean.

Figure 3:. Loop 73–77 is the only NlpE NTD loop that is critical for signaling to Cpx.

(A) Structural representation of NlpE_1–121 β-barrel loops, from crystal structure (PDB 2Z4H). The residues of each loop were all replaced with alanine residues. (B) NlpE variant expression was induced with 0.01% arabinose and Cpx activation was measured via chromosomal transcriptional reporter PcpxP-lacZ. Fold change was calculated by normalizing lacZ activity to the strain containing pBAD18 vector control. Data are average of n=4 biological replicates ± standard deviation of the mean. Significant difference in fold change compared to WT was determined by one-way ANOVA; * represents p<0.0001.

Figure 4:. NlpE_1–121(R73Q) is unable to protect against lipoprotein trafficking defects.

(A,B) Strains were grown for 100 minutes before being treated with sub- MIC amounts of either Compound 2, Globomycin, or DMSO vehicle control. Culture growth was measured using A_600nm. Cpx activation was measured by GFP fluorescence (FL) produced from a PcpxP–GFP transcriptional reporter plasmid. Fold-change was calculated by normalizing GFP per cell values (FL/A_600nm) values to the DMSO mock treated controls. Data are average of n=3 biological replicates ± standard deviation of the mean. (C) LolB depletion strains were grown overnight with 0.2% arabinose and plated in a 10-fold serial dilution on LB and LB + 0.2% arabinose plates. Plates without arabinose represent LolB deplete conditions since the only copy of lolB is under the araBAD promoter on pBAD18. (D) Whole cell lysates of induced cultures were probed with anti-FLAG to detect NlpE_1–121-FLAG protein levels. NlpE_1–121(WT) and NlpE_1–121(R73Q) are expressed at comparable levels chromosomally.

Figure 5:. NlpE_1–121(R73Q) disrupts interaction to CpxA.

(A) AlphaFold multimer algorithm predicts NlpE NTD and CpxA periplasmic sensor domain physically interact at NlpE(R73) and a combination of CpxA(D136), CpxA(E138), and CpxA(D139). (B) Co-purification of NlpE during CpxA purification. CpxA-Strep was purified using StrepTactin resin from strains also expressing IM-targeted NlpE(DD)-FLAG. Eluates were visualized by SDS-PAGE and BlueStain protein stain. A small amount of stained protein migration front is visible at right and just below the 13 kDa marker. (C) Purification samples had equivalent levels of CpxA-Strep and respective NlpE proteins in the input fractions and assessed by immunoblotting. The R73Q variant failed to co-purify with CpxA-Strep.

Figure 6:. CpxA residues modeled by AlphaFold to interact with NlpE are important for sensing NlpE.

(A) NlpE expression was induced with 0.01% Arabinose. Culture growth was measured using A_600nm. Cpx activation was measured by GFP fluorescence (FL) produced from a PcpxP–GFP transcriptional reporter plasmid. Fold-change was calculated by normalizing GFP per cell values (FL/A_600nm) values to the strain containing pBAD18 vector control. Data are average of n=3 biological replicates ± standard deviation of the mean. (B) Whole cell lysates of cultures expressing CpxA variants were probed with anti-Strep to detect CpxA-Strep protein levels. All variants are expressed comparably to CpxA.

Figure 7:. NlpE-blind CpxA mutants can still signal in response to PapG and PapE overexpression stress.

(A and B) Plasmid-encoded PapG and PapE expression was induced with IPTG after 60 minutes of growth. Culture growth was measured using A_600nm. Cpx activation was measured by GFP fluorescence (FL) produced from a PcpxP–GFP transcriptional reporter plasmid. Fold-change was calculated by normalizing GFP per cell values (FL/A_600nm) values to the vector control strain that lacks either PapG or PapE. Data are average of n=3 biological replicates ± standard deviation of the mean.

Figure 8:. Model for Cpx Activation.

Under normal growth conditions, lipoproteins are trafficked to the outer membrane (OM) via the Lol pathway. Since NlpE is trafficked so efficiently, it does not interact with CpxA and the Cpx system remains off. CpxP maintains this closed conformation. When there are trafficking defects, NlpE accumulates at the inner membrane (IM) and outcompetes CpxP to bind to CpxA. We propose that this interaction is electrostatic, where NlpE(R73) binds to a negatively charged region of the CpxA periplasmic sensor domain (D136, E138, and/or D139).