Transmembrane signaling and cytoplasmic signal conversion by dimeric transmembrane helix 2 and a linker domain of the DcuS sensor kinase
2020; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1074/jbc.ra120.015999
ISSN1083-351X
AutoresMarius Stopp, Philipp Aloysius Steinmetz, Christopher Schubert, Christian Griesinger, Dirk Schneider, Gottfried Unden,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoTransmembrane (TM) signaling is a key process of membrane-bound sensor kinases. The C4-dicarboxylate (fumarate) responsive sensor kinase DcuS of Escherichia coli is anchored by TM helices TM1 and TM2 in the membrane. Signal transmission across the membrane relies on the piston-type movement of the periplasmic part of TM2. To define the role of TM2 in TM signaling, we use oxidative Cys cross-linking to demonstrate that TM2 extends over the full distance of the membrane and forms a stable TM homodimer in both the inactive and fumarate-activated state of DcuS. An S186xxxGxxxG194 motif is required for the stability and function of the TM2 homodimer. The TM2 helix further extends on the periplasmic side into the α6-helix of the sensory PASP domain and on the cytoplasmic side into the α1-helix of PASC. PASC has to transmit the signal to the C-terminal kinase domain. A helical linker on the cytoplasmic side connecting TM2 with PASC contains an LxxxLxxxL sequence. The dimeric state of the linker was relieved during fumarate activation of DcuS, indicating structural rearrangements in the linker. Thus, DcuS contains a long α-helical structure reaching from the sensory PASP (α6) domain across the membrane to α1(PASC). Taken together, the results suggest piston-type TM signaling by the TM2 homodimer from PASP across the full TM region, whereas the fumarate-destabilized linker dimer converts the signal on the cytoplasmic side for PASC and kinase regulation. Transmembrane (TM) signaling is a key process of membrane-bound sensor kinases. The C4-dicarboxylate (fumarate) responsive sensor kinase DcuS of Escherichia coli is anchored by TM helices TM1 and TM2 in the membrane. Signal transmission across the membrane relies on the piston-type movement of the periplasmic part of TM2. To define the role of TM2 in TM signaling, we use oxidative Cys cross-linking to demonstrate that TM2 extends over the full distance of the membrane and forms a stable TM homodimer in both the inactive and fumarate-activated state of DcuS. An S186xxxGxxxG194 motif is required for the stability and function of the TM2 homodimer. The TM2 helix further extends on the periplasmic side into the α6-helix of the sensory PASP domain and on the cytoplasmic side into the α1-helix of PASC. PASC has to transmit the signal to the C-terminal kinase domain. A helical linker on the cytoplasmic side connecting TM2 with PASC contains an LxxxLxxxL sequence. The dimeric state of the linker was relieved during fumarate activation of DcuS, indicating structural rearrangements in the linker. Thus, DcuS contains a long α-helical structure reaching from the sensory PASP (α6) domain across the membrane to α1(PASC). Taken together, the results suggest piston-type TM signaling by the TM2 homodimer from PASP across the full TM region, whereas the fumarate-destabilized linker dimer converts the signal on the cytoplasmic side for PASC and kinase regulation. Membrane-anchored bacterial histidine kinases typically perceive ambient stimuli via their extra-cytoplasmic sensor domains (1Mascher T. Helmann J.D. Unden G. Stimulus perception in bacterial signal-transducing histidine kinases.Microbiol. Mol. Biol. Rev. 2006; 70: 910-938Crossref PubMed Scopus (480) Google Scholar, 2Krell T. Lacal J. Busch A. Silva-Jiménez H. Guazzaroni M.-E. Ramos J.L. Bacterial sensor kinases: diversity in the recognition of environmental signals.Annu. Rev. Microbiol. 2010; 64: 539-559Crossref PubMed Scopus (226) Google Scholar). Intracellularly, the signal is forwarded via cytoplasmic PAS, HAMP, or GAF domains to the kinase domain. 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Kleefeld A. Kim O.B. C4-Dicarboxylate utilization in aerobic and anaerobic growth.EcoSal Plus. 2016; 7Crossref PubMed Scopus (24) Google Scholar). DcuS forms a homodimer in bacterial membranes (27Scheu P.D. Liao Y.-F. Bauer J. Kneuper H. Basché T. Unden G. Erker W. Oligomeric sensor kinase DcuS in the membrane of Escherichia coli and in proteoliposomes: chemical cross-linking and FRET spectroscopy.J. Bacteriol. 2010; 192: 3474-3483Crossref PubMed Scopus (29) Google Scholar), and the two α-helices TM1 and TM2 of the DcuS monomer are supposed to facilitate DcuS homodimerization (18Monzel C. Unden G. Transmembrane signaling in the sensor kinase DcuS of Escherichia coli: a long-range piston-type displacement of transmembrane helix 2.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 11042-11047Crossref PubMed Scopus (18) Google Scholar). DcuS TM signaling involves TM2, which is linked on the periplasmic side to the PASP domain and on the cytoplasmic side to the PASC domain. Binding of a C4DC ligand to the sensor domain (28Kneuper H. Janausch I.G. Vijayan V. Zweckstetter M. Bock V. Griesinger C. Unden G. The nature of the stimulus and of the fumarate binding site of the fumarate sensor DcuS of Escherichia coli.J. Biol. Chem. 2005; 280: 20596-20603Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 29Pappalardo L. Janausch I.G. Vijayan V. Zientz E. Junker J. Peti W. Zweckstetter M. Unden G. Griesinger C. The NMR structure of the sensory domain of the membranous two-component fumarate sensor (histidine protein kinase) DcuS of Escherichia coli.J. Biol. Chem. 2003; 278: 39185-39188Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 30Krämer J. Fischer J.D. Zientz E. Vijayan V. Griesinger C. Lupas A. Unden G. Citrate sensing by the C4-dicarboxylate/citrate sensor kinase DcuS of Escherichia coli: binding site and conversion of DcuS to a C4-dicarboxylate- or citrate-specific sensor.J. Bacteriol. 2007; 189: 4290-4298Crossref PubMed Scopus (34) Google Scholar) results in compaction of the ligand binding site in PASP and uplifting of the C-terminal α6-helix of PASp. The uplift and structurally related arrangements upon citrate binding have been shown directly for homologous CitA in a membrane context (6Sevvana M. Vijayan V. Zweckstetter M. Reinelt S. Madden D.R. Herbst-Irmer R. Sheldrick G.M. Bott M. Griesinger C. Becker S. A ligand-induced switch in the periplasmic domain of sensor histidine kinase CitA.J. Mol. Biol. 2008; 377: 512-523Crossref PubMed Scopus (88) Google Scholar, 21Salvi M. Schomburg B. Giller K. Graf S. Unden G. Becker S. Lange A. Griesinger C. Sensory domain contraction in histidine kinase CitA triggers transmembrane signaling in the membrane-bound sensor.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 3115-3120Crossref PubMed Scopus (12) Google Scholar). This uplift pulls the periplasmic part of TM2 of DcuS in the direction of the periplasm in a piston-like movement (18Monzel C. Unden G. Transmembrane signaling in the sensor kinase DcuS of Escherichia coli: a long-range piston-type displacement of transmembrane helix 2.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 11042-11047Crossref PubMed Scopus (18) Google Scholar). TM signaling by sensor kinases is, however, complex and can change in different segments of the TM region or combine different modes of conformational changes (31Gushchin I. Orekhov P. Melnikov I. Polovinkin V. Yuzhakova A. Gordeliy V. Sensor histidine kinase NarQ activates via helical rotation, diagonal scissoring, and eventually piston-like shifts.Int. J. Mol. Sci. 2020; 21: 3110Crossref Scopus (5) Google Scholar). In contrast, TM1 of DcuS exhibits no piston movement and revealed no contribution to TM signaling (18Monzel C. Unden G. Transmembrane signaling in the sensor kinase DcuS of Escherichia coli: a long-range piston-type displacement of transmembrane helix 2.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 11042-11047Crossref PubMed Scopus (18) Google Scholar). To test for piston-type movement or indications for additional modes of TM signaling upon fumarate activation of DcuS, we studied the α-helical arrangement and dimerization of TM2 for the full-length and for the adjacent cytoplasmic region by Cys scanning and oxidative cross-linking (CL). This should define the extension of the α-helix from the periplasmic side across the membrane and its connection to the cytoplasmic PASC domain, with an emphasis on structural properties that could be relevant for the signal transfer across the membrane. Cysteine scanning and oxidative CL were applied as major methods to study TM2 interactions, as well as structural alterations due to receptor activation by fumarate. All studies were performed in bacteria containing DctA, which is a functional determinant of DcuS function (25Witan J. Bauer J. Wittig I. Steinmetz P.A. Erker W. Unden G. Interaction of the Escherichia coli transporter DctA with the sensor kinase DcuS: presence of functional DctA/DcuS sensor units.Mol. Microbiol. 2012; 85: 846-861Crossref PubMed Scopus (33) Google Scholar). The role of TM2 and the adjacent cytoplasmic region in DcuS homodimerization was analyzed in aerobically grown Escherichia coli cells, i.e., in the native context with DctA, by Cys CL using membrane-permeant copper(II)-(1,10-phenanthroline)3, or "Cu2+ phenanthroline," as an oxidant (18Monzel C. Unden G. Transmembrane signaling in the sensor kinase DcuS of Escherichia coli: a long-range piston-type displacement of transmembrane helix 2.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 11042-11047Crossref PubMed Scopus (18) Google Scholar, 32Lee G.F. Lebert M.R. Lilly A.A. Hazelbauer G.L. 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Here, we studied the involvement of every single residue of the complete TM2 helix in DcuS dimerization up to residue Glu213 (Fig. 1), which is located on the cytoplasmic side in the N terminus of the α1-helix in PASC (9Etzkorn M. Kneuper H. Dünnwald P. Vijayan V. Krämer J. Griesinger C. Becker S. Unden G. Baldus M. Plasticity of the PAS domain and a potential role for signal transduction in the histidine kinase DcuS.Nat. Struct. Mol. Biol. 2008; 15: 1031-1039Crossref PubMed Scopus (72) Google Scholar, 34Weisenburger S. Boening D. Schomburg B. Giller K. Becker S. Griesinger C. Sandoghdar V. Cryogenic optical localization provides 3D protein structure data with Angstrom resolution.Nat. Methods. 2017; 14: 141-144Crossref PubMed Scopus (38) Google Scholar). Each amino acid was individually replaced genetically by a Cys residue in the plasmid-encoded Cys-less variant DcuSCys0. In DcuSCys0, the natural Cys199 and Cys471 are replaced by Ser, which only moderately decreases fumarate stimulation of DcuS (18Monzel C. Unden G. Transmembrane signaling in the sensor kinase DcuS of Escherichia coli: a long-range piston-type displacement of transmembrane helix 2.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 11042-11047Crossref PubMed Scopus (18) Google Scholar, 27Scheu P.D. Liao Y.-F. Bauer J. Kneuper H. Basché T. Unden G. Erker W. Oligomeric sensor kinase DcuS in the membrane of Escherichia coli and in proteoliposomes: chemical cross-linking and FRET spectroscopy.J. Bacteriol. 2010; 192: 3474-3483Crossref PubMed Scopus (29) Google Scholar). The Cys variants retained all 87%–118% of DcuSCys0 activity in dcuB-lacZ expression and induced dcuB-lacZ in a fumarate-dependent manner as the wild-type (Fig. 2). However, there was a striking drop in dcuB-lacZ expression in a coherent stretch of seven amino acids (F189–L195).Figure 2Effect of DcuS cysteine substitutions in the TM2-PASC linker region on dcuB-lacZ expression. Expression of dcuB and effect of the substitutions were tested in the dcuS negative strain IMW260 (DcuS–) complemented with plasmid (pMW336)-encoded Cys-less DcuS (DcuSCys0) and derivatives of DcuS with single Cys substitutions. Growth was performed under anaerobic conditions in eM9 medium with glycerol plus 20 mM DMSO with or without 20 mM disodium fumarate. All activities were normalized to the wild-type control of DcuS in the fumarate-activated state. Variants 172–188 and 196–213 have been tested earlier (18Monzel C. Unden G. Transmembrane signaling in the sensor kinase DcuS of Escherichia coli: a long-range piston-type displacement of transmembrane helix 2.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 11042-11047Crossref PubMed Scopus (18) Google Scholar) without presenting the data.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cys-mediated CL of the DcuS variants was visualized by nonreducing SDS-PAGE and Western blotting with DcuS specific antisera (18Monzel C. Unden G. Transmembrane signaling in the sensor kinase DcuS of Escherichia coli: a long-range piston-type displacement of transmembrane helix 2.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 11042-11047Crossref PubMed Scopus (18) Google Scholar), which allowed us to separate DcuS monomers (calculated mass 62 kDa; apparent mass ∼55 kDa for His6-DcuS) from cross-linked DcuS homodimers (calculated mass 124 kDa; apparent mass ∼170 kDa; Fig. 1A). Reaction of the antiserum with DcuS and the cross-link product was verified by comparing the reaction with purified DcuS (Fig. S1) (35Janausch I.G. Garcia-Moreno I. Unden G. Function of DcuS from Escherichia coli as a fumarate-stimulated histidine protein kinase in vitro.J. Biol. Chem. 2002; 277: 39809-39814Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The CL efficiency was calculated from the levels of cross-linked versus free DcuS (Fig. 1A) and ranged from 0 to 74%. We ensured that the band intensity was in the linear range of detection to allow a comparison of band intensities (18Monzel C. Unden G. Transmembrane signaling in the sensor kinase DcuS of Escherichia coli: a long-range piston-type displacement of transmembrane helix 2.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 11042-11047Crossref PubMed Scopus (18) Google Scholar). The CL efficiencies were high at several positions within TM2 (approx. Ser182 to Leu201), the periplasmic α6 of PASP, and the cytoplasmic α1-PASC helices (see Fig. 1B). Lower CL efficiency was observed at the membrane/water interfaces on the periplasmic (residues Trp181–Trp185) and cytoplasmic (Cys199–Lys207) sites. For most regions, a +3/+4 periodicity of high CL efficiency was observed (Fig. 1B), which is characteristic of the interaction between two α-helices. Notably, the CL efficiency and the +3/+4 periodicity were retained in the region from F189 to L195, demonstrating that the region has a supposed α-helicity, which is retained in the Cys replacement mutations. Remarkably, the CL pattern and yield were essentially identical for fumarate activated versus nonactivated DcuS, with a maximal 12% difference in the CL efficiency. The clearest differences were observed in the Cys199–Lys207 region, corresponding to the TM2-PASC interdomain or linker region. In this region, a generally low CL efficiency was observed with partial nonhelical spacing of the CL maxima, and relatively large fumarate-induced differences, especially for residues Leu201, Val202, Val204, Leu205, and Lys206. The CL results suggest that the region from α6 in PASP to α1 in PASC forms a continuous α-helix, including the complete TM2 region. The sensitivity of DcuS activity to Cys replacement mutation in the F189–L195 region coincides with the presence of a GxxxG motif in this region. GxxxG motifs frequently mediate the interactions of TM α-helices via "ridge into groove" tight packing of two interacting TM helices (36Senes A. Gerstein M. Engelman D.M. Statistical analysis of amino acid patterns in transmembrane helices: the GxxxG motif occurs frequently and in association with beta-branched residues at neighboring positions.J. Mol. Biol. 2000; 296: 921-936Crossref PubMed Scopus (494) Google Scholar, 37Kleiger G. Grothe R. Mallick P. Eisenberg D. 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A. 2005; 102: 14278-14283Crossref PubMed Scopus (190) Google Scholar). The F189–L195 region in TM2 is part of such a glycine-zipper-type motif S186xxxGxxxG194 (Fig. S2) (41Bailey T.L. Williams N. Misleh C. Li W.W. MEME: discovering and analyzing DNA and protein sequence motifs.Nucleic Acids Res. 2006; 34: W369-W373Crossref PubMed Scopus (1537) Google Scholar, 42Huerta-Cepas J. Szklarczyk D. Forslund K. Cook H. Heller D. Walter M.C. Rattei T. Mende D.R. Sunagawa S. Kuhn M. Jensen L.J. Mering C. von Bork P. eggNOG 4.5: a hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences.Nucleic Acids Res. 2016; 44: D286-D293Crossref PubMed Scopus (987) Google Scholar). A role of the motif for DcuS function and activity was tested by substituting the motif residues and adjacent residues with Ala or Cys (for Ser), which avoids gross structural rearrangements. Replacement of Gly with Ala in GxxxG motifs disrupts helix–helix interactions, as the Gly residues are important for TM helix dimerization (38Dawson J.P. Weinger J.S. Engelman D.M. Motifs of serine and threonine can drive association of transmembrane helices.J. Mol. Biol. 2002; 316: 799-805Crossref PubMed Scopus (211) Google Scholar, 43Lemmon M.A. Flanagan J.M. Treutlein H.R. Zhang J. Engelman D.M. Sequence specificity in the dimerization of transmembrane alpha-helices.Biochemistry. 1992; 31: 12719-12725Crossref PubMed Scopus (455) Google Scholar, 44Fleming K.G. Engelman D.M. Specificity in transmembrane helix-helix interactions can define a hierarchy of stability for sequence variants.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14340-14344Crossref PubMed Scopus (151) Google Scholar). The impact of the mutations on DcuS function was assayed using the variants in a DcuS-less background by monitoring DcuS-dependent dcuB-lacZ reporter gene expression (Fig. 3). Individual replacements in the signature residues Ser186 and Gly190 of the motif did not significantly affect dcuB-lacZ expression, whereas the activity decreased to 44% of wild-type after replacement of Gly194 by Ala. Mutation of other residues in that region had no effect as well. However, mutation of the core pair of the motif (Gly190 residue together with Gly194) dramatically reduced the activity to ∼16% of wild-type. The effect is specific since mutation of neighboring residues (W185A together with F189A) had no effect. Remarkably, mutation of Ser186 together with Gly190 showed the same reduction in activity as for the Gly190 plus Gly194 pair. Taken together, the data suggest that the S186xxxGxxxG194 motif in TM2 (particularly Gly190 or the Gly190 + G194 pair) is crucial for DcuS function. Notably, the level of DcuS protein in membranes was similar for wild-type and all variants after expression (Fig. S3A), confirming that the observed differences were caused by the mutations rather than by altered DcuS levels. As Thr residues can also be a part of (small)xxx(small) motifs (38Dawson J.P. Weinger J.S. Engelman D.M. Motifs of serine and threonine can drive association of transmembrane helices.J. Mol. Biol. 2002; 316: 799-805Crossref PubMed Scopus (211) Google Scholar), we analyzed the involvement of Thr198. However, when both the Thr198 and the Gly194 residues were replaced by Ala residues (Fig. 3), dcuB-lacZ expression was not decreased compared with DcuS(G194A) alone, indicating that Thr198 is not important for DcuS activity. The role of the S186xxxGxxxG194 motif in DcuS homodimerization was tested using bacterial two-hybrid (BACTH) systems. For the full-length DcuS protein, we applied the BACTH system that relies on the restoration of adenylate cyclase activity and cAMP production by fusing the individual domains (T18 or T25) of Bordetella pertussis adenylate cyclase to interacting proteins. Interaction restores the adenylate cyclase activity, which can be monitored as β-galactosidase expression (49Karimova G. Pidoux J. Ullmann A. Ladant D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5752-5756Crossref PubMed Scopus (1069) Google Scholar, 50Karimova G. Ullmann A. Ladant D. Bordetella pertussis adenylate cyclase toxin as a tool to analyze molecular interactions in a bacterial two-hybrid system.Int. J. Med. Microbiol. 2000; 290: 441-445Crossref PubMed Scopus (14) Google Scholar). The T18 and T25 domains were genetically fused to the DcuS N terminus. When T18-DcuS was coexpressed with T25-DcuS, high β-galactosidase activity was observed for wild-type DcuS (Fig. 4A). When the signature residues Gly190 and Gly194 of the S186xxxGxxxG194 motif were individually muta
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