Transmembrane signaling in the sensor kinase DcuS of Escherichia coli: A long-range piston-type displacement of transmembrane helix 2

Abstract

The C4-dicarboxylate sensor kinase DcuS is membrane integral because of the transmembrane (TM) helices TM1 and TM2. Fumarate-induced movement of the helices was probed in vivo by Cys accessibility scanning at the membrane-water interfaces after activation of DcuS by fumarate at the periplasmic binding site. TM1 was inserted with amino acid residues 21-41 in the membrane in both the fumarate-activated (ON) and inactive (OFF) states. In contrast, TM2 was inserted with residues 181-201 in the OFF state and residues 185-205 in the ON state. Replacement of Trp 185 by an Arg residue caused displacement of TM2 toward the outside of the membrane and a concomitant induction of the ON state. Results from Cys cross-linking of TM2/TM2' in the DcuS homodimer excluded rotation; thus, data from accessibility changes of TM2 upon activation, either by ligand binding or by mutation of TM2, and cross-linking of TM2 and the connected region in the periplasm suggest a piston-type shift of TM2 by four residues to the periplasm upon activation (or fumarate binding). This mode of function is supported by the suggestion from energetic calculations of two preferred positions for TM2 insertion in the membrane. The shift of TM2 by four residues (or 4-6 Å) toward the periplasm upon activation is complementary to the periplasmic displacement of 3-4 Å of the C-terminal part of the periplasmic ligand-binding domain upon ligand occupancy in the citrate-binding domain in the homologous CitA sensor kinase.

Keywords: DcuS sensor kinase; SCAM; bacteria; piston-type; transmembrane signaling.

Fig. 1.

Proof of principle for differentiation between water-accessible and membrane-hidden Cys residues by Scan-SCAM. Covalently bound PEG-mal (A) induces a mass shift to DcuS during SDS/PAGE that is visualized by Western blotting using antiserum against DcuS-PAS_P (B). NSP (sequential treatment by NEM, SDS; and PEG-mal): Blocking of water-accessible Cys residues by NEM is followed by cell solubilization (SDS) and labeling of unblocked Cys residues by PEG-mal. SP (sequential treatment by SDS and PEG-mal): There is no masking with NEM to PEGylate all Cys residues of the protein. SNP (sequential treatment by SDS, NEM, and PEG-mal): Cells are permeabilized before NEM masking, and no Cys residue is PEGylated.

Fig. 2.

Accessibility of Cys residues around the first (TM1) and second (TM2) transmembrane helix with and without fumarate (A and B) and in the genetic ON-variants DcuS-W181R and W185R (C and D). (A) DcuS-dependent stimulation of dcuB-lacZ by fumarate. Expression was measured in strain IMW260 complemented with plasmid-encoded DcuS^WT (pMW181) after anaerobic growth in eM9 medium with glycerol plus DMSO, without (light gray) or with (dark gray) 50 mM sodium fumarate. (B) Schematic representation of TM1 and TM2, their accessibility pattern, and the accessibility shift induced by fumarate. Accessible (unPEGylated) Cys residues are shown in green, and inaccessible (PEGylated) Cys residues are shown in red. The plus and minus signs indicate the periplasmic and cytoplasmic sides of the membrane, respectively. (C) Fumarate-independent stimulation of dcuB-lacZ by the weak ON-variant DcuS-W181R and the strong ON-variant DcuS-W185R. Expression was measured with plasmid-encoded DcuS-W181R (pMW1545) and DcuS-W185R (pMW1546), respectively. (D) Schematic representation of TM2, its accessibility pattern in the variants DcuS-W181R and DcuS-W185R, and the accessibility shift induced by fumarate in both variants. The pattern is shown for only a selection of residues. The plus and minus signs indicate the periplasmic and cytoplasmic sides of the membrane, respectively. Scan-SCAM Western blots of all Cys variants of DcuS or selected Cys variants of DcuS-W181R and DcuS-W185R, with or without fumarate, are shown in Figs. S1 and S2, respectively.

Fig. S1.

Accessibility of Cys residues around TM1 and TM2 in the absence (−F) and presence (+F) of fumarate. SCAM Western blots of 60 single Cys variants of DcuS with substituted Cys residues around TM1 and TM2, once in the absence (−F) and once in the presence (+F) of 50 mM sodium fumarate during labeling. Accessible (unPEGylated) Cys residues are shown in green, and inaccessible (PEGylated) Cys residues are shown in red. Arrows indicate Cys residues with a shifted accessibility between both activation states.

Fig. S2.

Accessibility of selected Cys residues around TM2 in the ON variants DcuS-W181R and W185R, in the absence (−F) and presence (+F) of fumarate. SCAM Western blots of single Cys variants of the DcuS ON-variants DcuS-W181R and DcuS-W185R with substituted Cys residues TM2, once in the absence and once in the presence of 50 mM sodium fumarate during labeling. Accessible (unPEGylated) Cys residues are shown in green, and inaccessible (PEGylated) Cys residues are shown in red. Arrows indicate Cys residues with a shifted accessibility between both activation states.

Fig. 3.

Cys accessibility of TM2 in the presence of various effectors and correlation to the level of reporter gene expression: Correlation between the total number of positions with a changed accessibility between both signaling states and the level of dcuB-lacZ expression. Expression was measured in strain IMW237 after anaerobic growth in eM9 medium with glycerol plus DMSO with 50 mM of the respective effector (3-Nitropropionate: 5 mM). The dotted line represents a polynomial regression of measured values. SCAM Western blots of 22 single-Cys variants of DcuS in the presence of l-malate, maleate, l-tartrate, citrate, or 3-nitropropionate are shown in Fig. S3.

Fig. S3.

Accessibility of Cys residues around TM2 in the presence of different effectors. SCAM Western blots of 22 single-Cys variants of DcuS with substituted Cys residues around TM2 in the presence of 50 mM l-malate, 50 mM maleate, 50 mM l-tartrate, 50 mM citrate, or 5 mM 3-nitropropionate during labeling. Accessible (unPEGylated) Cys residues are shown in green, and inaccessible (PEGylated) Cys residues are shown in red.

Fig. 4.

Intermolecular oxidative disulfide cross-linking in DcuS. Single-Cys variants of DcuS around TM2 were cross-linked oxidatively in vivo using copper phenanthroline as the catalyst, once in the absence (gray) and once in the presence (blue) of fumarate. The amount of cross-linking product among total DcuS was calculated after SDS/PAGE in the Western blot by measuring the band intensities with the software GelPro Analyzer. For each Cys residue the cross-linking efficiency was determined in three independent experiments, and the arithmetic average is plotted as percentage against the respective position. Arrows indicate local maxima of the cross-linking efficiency. Western blots of the cross-linking experiments are shown in Fig. S4.

Fig. S4.

Intermolecular oxidative disulfide cross-linking in DcuS. Single-Cys variants of DcuS around TM2 were cross-linked oxidatively in vivo using copper phenanthroline as the catalyst, once in the absence (−F) and once in the presence (+F) of fumarate. Each cross-linking experiment was done in triplicate.

Fig. 5.

Estimation of the amplitude of piston-type sliding by structure comparison, Cys accessibility, and energetic considerations. (A) Change in the apparent free energy (∆∆G^app) for the insertion of TM2 at various positions compared with the predicted default state (182–202; red asterisk). The ∆G^app for the different positions was calculated by the ∆G prediction server v 1.0 (27). Calculations are based on the probability that TM helices will be inserted into the membrane and use parameters such as the identity and position of a particular amino acid and the overall length of the sequence. The black arrows indicate the C-terminal ends of TM2 in the ON and OFF signaling states as derived from Scan-SCAM. The red arrow reflects a potential longitudinal displacement of TM2 between the two signaling states. (B) Comparison of the structure of the periplasmic PAS_P domains of DcuS (pink; PDB ID #3BY8) (10) and CitA_Kp with citrate (green; PDB ID #2J80) and without citrate (gray, PDB ID #2V9A) (3). Structures were superimposed using Chimera software (42). The C terminus of CitA_Kp is shown in an enlarged view; its uplift in parallel with the helix axis (derived from DcuS-PAS_P helix α6; dotted line) is shown by a red arrow.

Fig. S5.

Change in the apparent free energy (∆∆G^app) for shifting TM1 from the preferred position to other positions. Change in the apparent free energy (∆∆G^app) for the insertion of TM1 in various positions compared with the predicted default state (21–41; red asterisk). The ∆G^app for the different positions was calculated by the ∆G prediction server v. 1.0 (27), based on the respective amino acid sequences.

Fig. S6.

Scheme for the conversion of DcuS from the OFF to the ON state. Binding of fumarate or l-malate at the DcuS dimer causes closing of the binding site and compaction of PAS_P with an uplift of α6 from PAS_P (red arrow) (3, 4, 7) and of TM2 (red arrow) by one helical turn in TM2 [shifting the periplasmic membrane–water interface from Trp181 to Trp185 (upper and lower balls)]. Pulling of TM2 at the N-terminal region of PAS_C results in the relief of PAS_C dimerization (20, 31) and kinase activation (31).