The heme-based oxygen sensor protein AfGcHK is a globin-coupled histidine kinase in the soil bacterium Anaeromyxobacter sp. Fw109-5. Its C-terminal functional domain exhibits autophosphorylation activity induced by oxygen binding to the heme-Fe(II) complex located in the oxygen-sensing N-terminal globin domain. A detailed understanding of the signal transduction mechanisms in heme-containing sensor proteins remains elusive. Here, we investigated the role of the globin domain's dimerization interface in signal transduction in AfGcHK. We present a crystal structure of a monomeric imidazole-bound AfGcHK globin domain at 1.8 Å resolution, revealing that the helices of the WT globin dimer are under tension and suggesting that Tyr-15 plays a role in both this tension and the globin domain's dimerization. Biophysical experiments revealed that whereas the isolated WT globin domain is dimeric in solution, the Y15A and Y15G variants in which Tyr-15 is replaced with Ala or Gly, respectively, are monomeric. Additionally, we found that although the dimerization of the full-length protein is preserved via the kinase domain dimerization interface in all variants, full-length AfGcHK variants bearing the Y15A or Y15G substitutions lack enzymatic activity. The combined structural and biophysical results presented here indicate that Tyr-15 plays a key role in the dimerization of the globin domain of AfGcHK and that globin domain dimerization is essential for internal signal transduction and autophosphorylation in this protein. These findings provide critical insights into the signal transduction mechanism of the histidine kinase AfGcHK from Anaeromyxobacter.

Department of Biochemistry Faculty of Science Charles University Prague 2 128 43 Czech Republic

Division of Structural Biology The Wellcome Trust Centre for Human Genetics University of Oxford OX3 7BN Oxford United Kingdom

Institute of Biotechnology of the Czech Academy of Sciences v v i Biocev Vestec 252 50 Czech Republic

Institute of Biotechnology of the Czech Academy of Sciences v v i Biocev Vestec 252 50 Czech Republic; FNSPE Czech Technical University Prague Brehova 7 Prague 1 115 19 Czech Republic

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Shimizu T., Huang D., Yan F., Stranava M., Bartosova M., Fojtíková V., and Martínková M. (2015) Gaseous O2, NO, and CO in signal transduction: structure and function relationships of heme-based gas sensors and heme-redox sensors. Chem. Rev. 115, 6491–6533 10.1021/acs.chemrev.5b00018 PubMed DOI

Kühl T., and Imhof D. (2014) Regulatory Fe(II/III) heme: the reconstruction of a molecule's biography. Chembiochem 15, 2024–2035 10.1002/cbic.201402218 PubMed DOI

Tsiftsoglou A. S., Tsamadou A. I., and Papadopoulou L. C. (2006) Heme as key regulator of major mammalian cellular functions: molecular, cellular, and pharmacological aspects. Pharmacol. Ther. 111, 327–345 10.1016/j.pharmthera.2005.10.017 PubMed DOI

Ponka P., Sheftel A. D., English A. M., Scott Bohle D., and Garcia-Santos D. (2017) Do mammalian cells really need to export and import heme? Trends Biochem. Sci. 42, 395–406 10.1016/j.tibs.2017.01.006 PubMed DOI

Roumenina L. T., Rayes J., Lacroix-Desmazes S., and Dimitrov J. D. (2016) Heme: modulator of plasma systems in hemolytic diseases. Trends Mol. Med. 22, 200–213 10.1016/j.molmed.2016.01.004 PubMed DOI

Shimizu T., Lengalova A., Martínek V., and Martínková M. (2019) Heme: emergent roles of heme in signal transduction, functional regulation and as catalytic centres. Chem. Soc. Rev. 48, 5619–5808 10.1039/C9CS90100K PubMed DOI

Martínková M., Kitanishi K., and Shimizu T. (2013) Heme-based globin-coupled oxygen sensors: linking oxygen binding to functional regulation of diguanylate cyclase, histidine kinase, and methyl-accepting chemotaxis. J. Biol. Chem. 288, 27702–27711 10.1074/jbc.R113.473249 PubMed DOI PMC

Rivera S., Young P. G., Hoffer E. D., Vansuch G. E., Metzler C. L., Dunham C. M., and Weinert E. E. (2018) Structural insights into oxygen-dependent signal transduction within globin coupled sensors. Inorg. Chem. 57, 14386–14395 10.1021/acs.inorgchem.8b02584 PubMed DOI

Lobão J. B. D. S., Gondim A. C. S., Guimarães W. G., Gilles-Gonzalez M.-A., Lopes L. G. F., and Sousa E. H. S. (2019) Oxygen triggers signal transduction in the DevS (DosS) sensor of Mycobacterium tuberculosis by modulating the quaternary structure. FEBS J. 286, 479–494 10.1111/febs.14734 PubMed DOI

Burns J. L., Rivera S., Deer D. D., Joynt S. C., Dvorak D., and Weinert E. E. (2016) Oxygen and bis(3′,5′)-cyclic dimeric guanosine monophosphate binding control oligomerization state equilibria of diguanylate cyclase-containing globin coupled sensors. Biochemistry 55, 6642–6651 10.1021/acs.biochem.6b00526 PubMed DOI

Rivera S., Burns J. L., Vansuch G. E., Chica B., and Weinert E. E. (2016) Globin domain interactions control heme pocket conformation and oligomerization of globin coupled sensors. J. Inorg. Biochem. 164, 70–76 10.1016/j.jinorgbio.2016.08.016 PubMed DOI

Burns J. L., Jariwala P. B., Rivera S., Fontaine B. M., Briggs L., and Weinert E. E. (2017) Oxygen-dependent globin coupled sensor signaling modulates motility and virulence of the plant pathogen Pectobacterium carotovorum. ACS Chem. Biol. 12, 2070–2077 10.1021/acschembio.7b00380 PubMed DOI

Burns J. L., Deer D. D., and Weinert E. E. (2014) Oligomeric state affects oxygen dissociation and diguanylate cyclase activity of globin coupled sensors. Mol. Biosyst. 10, 2823–2826 10.1039/C4MB00366G PubMed DOI

Walker J. A., Rivera S., and Weinert E. E. (2017) Mechanism and role of globin-coupled sensor signalling. Adv. Microb. Physiol. 71, 133–169 10.1016/bs.ampbs.2017.05.003 PubMed DOI PMC

Gushchin I., Melnikov I., Polovinkin V., Ishchenko A., Yuzhakova A., Buslaev P., Bourenkov G., Grudinin S., Round E., Balandin T., Borshchevskiy V., Willbold D., Leonard G., Büldt G., Popov A., and Gordeliy V. (2017) Mechanism of transmembrane signaling by sensor histidine kinases. Science 356, eaah6345 10.1126/science.aah6345 PubMed DOI

Abriata L. A., Albanesi D., Dal Peraro M., and de Mendoza D. (2017) Signal sensing and transduction by histidine kinases as unveiled through studies on a temperature sensor. Acc. Chem. Res. 50, 1359–1366 10.1021/acs.accounts.6b00593 PubMed DOI

Zschiedrich C. P., Keidel V., and Szurmant H. (2016) Molecular mechanisms of two-component signal transduction. J. Mol. Biol. 428, 3752–3775 10.1016/j.jmb.2016.08.003 PubMed DOI PMC

Willett J. W., and Crosson S. (2017) Atypical modes of bacterial histidine kinase signaling. Mol. Microbiol. 103, 197–202 10.1111/mmi.13525 PubMed DOI PMC

Kitanishi K., Kobayashi K., Uchida T., Ishimori K., Igarashi J., and Shimizu T. (2011) Identification and functional and spectral characterization of a globin-coupled histidine kinase from Anaeromyxobacter sp. Fw109-5. J. Biol. Chem. 286, 35522–35534 10.1074/jbc.M111.274811 PubMed DOI PMC

Fojtikova V., Stranava M., Vos M. H., Liebl U., Hranicek J., Kitanishi K., Shimizu T., and Martinkova M. (2015) Kinetic analysis of a globin-coupled histidine kinase, AfGcHK: effects of the heme iron complex, response regulator, and metal cations on autophosphorylation activity. Biochemistry 54, 5017–5029 10.1021/acs.biochem.5b00517 PubMed DOI

Stranava M., Man P., Skálová T., Kolenko P., Blaha J., Fojtikova V., Martínek V., Dohnálek J., Lengalova A., Rosůlek M., Shimizu T., and Martínková M. (2017) Coordination and redox state-dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction. J. Biol. Chem. 292, 20921–20935 10.1074/jbc.M117.817023 PubMed DOI PMC

Stranava M., Martínek V., Man P., Fojtikova V., Kavan D., Van[caron]ek O., Shimizu T., and Martinkova M. (2016) Structural characterization of the heme-based oxygen sensor, AfGcHK, its interactions with the cognate response regulator, and their combined mechanism of action in a bacterial two-component signaling system. Proteins 84, 1375–1389 10.1002/prot.25083 PubMed DOI

Liebschner D., Afonine P. V., Moriarty N. W., Poon B. K., Sobolev O. V., Terwilliger T. C., and Adams P. D. (2017) Polder maps: improving OMIT maps by excluding bulk solvent. Acta Crystallogr. D Struct. Biol. 73, 148–157 10.1107/S2059798316018210 PubMed DOI PMC

Pesce A., Thijs L., Nardini M., Desmet F., Sisinni L., Gourlay L., Bolli A., Coletta M., Van Doorslaer S., Wan X., Alam M., Ascenzi P., Moens L., Bolognesi M., and Dewilde S. (2009) HisE11 and HisF8 provide bis-histidyl heme hexa-coordination in the globin domain of Geobacter sulfurreducens globin-coupled sensor. J. Mol. Biol. 386, 246–260 10.1016/j.jmb.2008.12.023 PubMed DOI

Zhang W., and Phillips G. N. (2003) Structure of the oxygen sensor in Bacillus subtilis: signal transduction of chemotaxis by control of symmetry. Structure 11, 1097–1110 10.1016/S0969-2126(03)00169-2 PubMed DOI

Tarnawski M., Barends T. R., and Schlichting I. (2015) Structural analysis of an oxygen-regulated diguanylate cyclase. Acta Crystallogr. D Biol. Crystallogr. 71, 2158–2177 10.1107/S139900471501545X PubMed DOI

Lionetti C., Guanziroli M. G., Frigerio F., Ascenzi P., and Bolognesi M. (1991) X-ray crystal structure of the ferric sperm whale myoglobin: imidazole complex at 2.0 Å resolution. J. Mol. Biol. 217, 409–412 10.1016/0022-2836(91)90744-Q PubMed DOI

Pesce A., Tilleman L., Donné J., Aste E., Ascenzi P., Ciaccio C., Coletta M., Moens L., Viappiani C., Dewilde S., Bolognesi M., and Nardini M. (2013) Structure and haem-distal site plasticity in Methanosarcina acetivorans protoglobin. PLoS ONE 8, e66144 10.1371/journal.pone.0066144 PubMed DOI PMC

Cunningham K. A., and Burkholder W. F. (2009) The histidine kinase inhibitor Sda binds near the site of autophosphorylation and may sterically hinder autophosphorylation and phosphotransfer to Spo0F. Mol. Microbiol. 71, 659–677 10.1111/j.1365-2958.2008.06554.x PubMed DOI

Rao M., Herzik M. A. Jr., Iavarone A. T., and Marletta M. A. (2017) Nitric oxide-induced conformational changes govern H-NOX and histidine kinase interaction and regulation in Shewanella oneidensis. Biochemistry 56, 1274–1284 10.1021/acs.biochem.6b01133 PubMed DOI

Dikiy I., Edupuganti U. R., Abzalimov R. R., Borbat P. P., Srivastava M., Freed J. H., and Gardner K. H. (2019) Insights into histidine kinase activation mechanisms from the monomeric blue light sensor EL346. Proc. Natl. Acad. Sci. U.S.A. 116, 4963–4972 10.1073/pnas.1813586116 PubMed DOI PMC

Jacob-Dubuisson F., Mechaly A., Betton J.-M., and Antoine R. (2018) Structural insights into the signalling mechanisms of two-component systems. Nat. Rev. Microbiol. 16, 585–593 10.1038/s41579-018-0055-7 PubMed DOI

Affandi T., and McEvoy M. M. (2019) Mechanism of metal ion-induced activation of a two-component sensor kinase. Biochem. J. 476, 115–135 10.1042/BCJ20180577 PubMed DOI PMC

Kurokawa H., Lee D.-S., Watanabe M., Sagami I., Mikami B., Raman C. S., and Shimizu T. (2004) A redox-controlled molecular switch revealed by the crystal structure of a bacterial heme PAS sensor. J. Biol. Chem. 279, 20186–20193 10.1074/jbc.M314199200 PubMed DOI

Igarashi J., Murase M., Iizuka A., Pichierri F., Martinkova M., and Shimizu T. (2008) Elucidation of the heme binding site of heme-regulated eukaryotic initiation factor 2α kinase and the role of the regulatory motif in heme sensing by spectroscopic and catalytic studies of mutant proteins. J. Biol. Chem. 283, 18782–18791 10.1074/jbc.M801400200 PubMed DOI

Yamada S., Nakamura H., Kinoshita E., Kinoshita-Kikuta E., Koike T., and Shiro Y. (2007) Separation of a phosphorylated histidine protein using phosphate affinity polyacrylamide gel electrophoresis. Anal. Biochem. 360, 160–162 10.1016/j.ab.2006.10.005 PubMed DOI

Kabsch W. (2010) XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 10.1107/S0907444909047337 PubMed DOI PMC

Winn M. D., Ballard C. C., Cowtan K. D., Dodson E. J., Emsley P., Evans P. R., Keegan R. M., Krissinel E. B., Leslie A. G., McCoy A., McNicholas S. J., Murshudov G. N., Pannu N. S., Potterton E. A., Powell H. R., Read R. J., Vagin A., and Wilson K. S. (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 10.1107/S0907444910045749 PubMed DOI PMC

Vagin A., and Teplyakov A. (2010) Molecular replacement with MOLREP. Acta Crystallogr. D Biol. Crystallogr. 66, 22–25 10.1107/S0907444909042589 PubMed DOI

Murshudov G. N., Skubák P., Lebedev A. A., Pannu N. S., Steiner R. A., Nicholls R. A., Winn M. D., Long F., and Vagin A. A. (2011) REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 10.1107/S0907444911001314 PubMed DOI PMC

Emsley P., Lohkamp B., Scott W. G., and Cowtan K. (2010) Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 10.1107/S0907444910007493 PubMed DOI PMC

Chen V. B., Arendall W. B. 3rd, Headd J. J., Keedy D. A., Immormino R. M., Kapral G. J., Murray L. W., Richardson J. S., and Richardson D. C. (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 10.1107/S0907444909042073 PubMed DOI PMC

Monitoring the Kinase Activity of Heme-Based Oxygen Sensors and Its Dependence on O2 and Other Ligands Using Phos-Tag Electrophoresis

Hydrogen/Deuterium Exchange Mass Spectrometry of Heme-Based Oxygen Sensor Proteins

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PDB
6OTD, 5OHE, 5OHF, 2W31, 1OR4, 1OR6, 4UII, 4ZVA-4ZVH, 1MBI, 4UIQ, 3ZJP