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• W2997652571 abstract "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. 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. Heme complexes play essential roles in many important physiological processes, including oxygen transport and storage, electron transport, oxidative stress protection, and signaling. They are also vital components of heme-containing sensor proteins, a large group of biologically significant proteins that are represented in almost all living organisms and exhibits great functional diversity (1Shimizu T. Huang D. Yan F. Stranava M. Bartosova M. Fojtíková V. Martínková M. Gaseous O2, NO, and CO in signal transduction: structure and function relationships of heme-based gas sensors and heme-redox sensors.Chem. Rev. 2015; 115 (26021768): 6491-653310.1021/acs.chemrev.5b00018Crossref PubMed Scopus (120) Google Scholar, 2Kühl T. Imhof D. Regulatory Fe(II/III) heme: the reconstruction of a molecule's biography.Chembiochem. 2014; 15 (25196849): 2024-203510.1002/cbic.201402218Crossref PubMed Scopus (44) Google Scholar, 3Tsiftsoglou A.S. Tsamadou A.I. Papadopoulou L.C. Heme as key regulator of major mammalian cellular functions: molecular, cellular, and pharmacological aspects.Pharmacol. Ther. 2006; 111 (16513178): 327-34510.1016/j.pharmthera.2005.10.017Crossref PubMed Scopus (217) Google Scholar, 4Ponka P. Sheftel A.D. English A.M. Scott Bohle D. Garcia-Santos D. Do mammalian cells really need to export and import heme?.Trends Biochem. Sci. 2017; 42 (28254242): 395-40610.1016/j.tibs.2017.01.006Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 5Roumenina L.T. Rayes J. Lacroix-Desmazes S. Dimitrov J.D. Heme: modulator of plasma systems in hemolytic diseases.Trends Mol. Med. 2016; 22 (26875449): 200-21310.1016/j.molmed.2016.01.004Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 6Shimizu T. Lengalova A. Martínek V. Martínková M. Heme: emergent roles of heme in signal transduction, functional regulation and as catalytic centres.Chem. Soc. Rev. 2019; 48: 5619-580810.1039/C9CS90100KCrossref Google Scholar). There is considerable interest in the structure-function relationships of heme-based oxygen sensors because the heme-iron complex serves as the signaling center (1Shimizu T. Huang D. Yan F. Stranava M. Bartosova M. Fojtíková V. Martínková M. Gaseous O2, NO, and CO in signal transduction: structure and function relationships of heme-based gas sensors and heme-redox sensors.Chem. Rev. 2015; 115 (26021768): 6491-653310.1021/acs.chemrev.5b00018Crossref PubMed Scopus (120) Google Scholar, 7Martínková M. Kitanishi K. Shimizu T. Heme-based globin-coupled oxygen sensors: linking oxygen binding to functional regulation of diguanylate cyclase, histidine kinase, and methyl-accepting chemotaxis.J. Biol. Chem. 2013; 288 (23928310): 27702-2771110.1074/jbc.R113.473249Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 8Rivera S. Young P.G. Hoffer E.D. Vansuch G.E. Metzler C.L. Dunham C.M. Weinert E.E. Structural insights into oxygen-dependent signal transduction within globin coupled sensors.Inorg. Chem. 2018; 57 (30378421): 14386-1439510.1021/acs.inorgchem.8b02584Crossref PubMed Scopus (11) Google Scholar, 9Lobão J.B.D.S. Gondim A.C.S. Guimarães W.G. Gilles-Gonzalez M.-A. Lopes L.G.F. Sousa E.H.S. Oxygen triggers signal transduction in the DevS (DosS) sensor of Mycobacterium tuberculosis by modulating the quaternary structure.FEBS J. 2019; 286 (30570222): 479-49410.1111/febs.14734Crossref PubMed Scopus (8) Google Scholar, 10Burns J.L. Rivera S. Deer D.D. Joynt S.C. Dvorak D. Weinert E.E. Oxygen and bis(3′,5′)-cyclic dimeric guanosine monophosphate binding control oligomerization state equilibria of diguanylate cyclase-containing globin coupled sensors.Biochemistry. 2016; 55 (27933792): 6642-665110.1021/acs.biochem.6b00526Crossref PubMed Scopus (14) Google Scholar, 11Rivera S. Burns J.L. Vansuch G.E. Chica B. Weinert E.E. Globin domain interactions control heme pocket conformation and oligomerization of globin coupled sensors.J. Inorg. Biochem. 2016; 164 (27614715): 70-7610.1016/j.jinorgbio.2016.08.016Crossref PubMed Scopus (9) Google Scholar, 12Burns J.L. Jariwala P.B. Rivera S. Fontaine B.M. Briggs L. Weinert E.E. Oxygen-dependent globin coupled sensor signaling modulates motility and virulence of the plant pathogen Pectobacterium carotovorum.ACS Chem. Biol. 2017; 12 (28612602): 2070-207710.1021/acschembio.7b00380Crossref PubMed Scopus (7) Google Scholar, 13Burns J.L. Deer D.D. Weinert E.E. Oligomeric state affects oxygen dissociation and diguanylate cyclase activity of globin coupled sensors.Mol. Biosyst. 2014; 10 (25174604): 2823-282610.1039/C4MB00366GCrossref PubMed Google Scholar, 14Walker J.A. Rivera S. Weinert E.E. Mechanism and role of globin-coupled sensor signalling.Adv. Microb. Physiol. 2017; 71 (28760321): 133-16910.1016/bs.ampbs.2017.05.003Crossref PubMed Scopus (16) Google Scholar). Proteins of this type often act as one component of a two-component signal transduction system. These systems are associated with biofilm formation and virulence in certain pathogenic bacteria but also fulfil many other functions (15Gushchin 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. Gordeliy V. Mechanism of transmembrane signaling by sensor histidine kinases.Science. 2017; 356 (28522691): eaah634510.1126/science.aah6345Crossref PubMed Scopus (93) Google Scholar, 16Abriata L.A. Albanesi D. Dal Peraro M. de Mendoza D. Signal sensing and transduction by histidine kinases as unveiled through studies on a temperature sensor.Acc. Chem. Res. 2017; 50 (28475313): 1359-136610.1021/acs.accounts.6b00593Crossref PubMed Scopus (36) Google Scholar, 17Zschiedrich C.P. Keidel V. Szurmant H. Molecular mechanisms of two-component signal transduction.J. Mol. Biol. 2016; 428 (27519796): 3752-377510.1016/j.jmb.2016.08.003Crossref PubMed Scopus (297) Google Scholar, 18Willett J.W. Crosson S. Atypical modes of bacterial histidine kinase signaling.Mol. Microbiol. 2017; 103 (27618209): 197-20210.1111/mmi.13525Crossref PubMed Scopus (21) Google Scholar). The heme-based oxygen sensor AfGcHK 4The abbreviations used are: AfGcHKglobin-coupled histidine kinase from the soil bacterium Anaeromyxobacter sp. Fw109-5AvGRegAzotobacter vinelandii globin-coupled oxygen sensorBpeGRegglobin-coupled oxygen sensor with diguanylate cyclase activity from Bordetella pertussisCusSE. coli copper and silver ions sensorDevS (DosS)heme-based oxygen sensor protein from Mycobacterium tuberculosisEcDOS or EcDosPheme-based oxygen sensor phosphodiesteraseHDX-MShydrogen deuterium exchange coupled with mass spectrometryHemATglobin-coupled oxygen sensor from B. subtilisheme-Fe(II)Fe(II)-protoporphyrin IX complexheme-Fe(III)Fe(III)-protoporphyrin IX complex or heminMPD2-methyl-2,4-pentanediolPccGCSglobin-coupled oxygen sensor with diguanylate cyclase activity from P. carotovorumPDBProtein Data BankTEVtobacco etch virusYddVglobin-coupled oxygen sensor diguanylate cyclase from E. coliRMSDroot mean square deviation. is a globin-coupled histidine kinase from the soil bacterium Anaeromyxobacter sp. Fw109-5 (19Kitanishi K. Kobayashi K. Uchida T. Ishimori K. Igarashi J. Shimizu T. Identification and functional and spectral characterization of a globin-coupled histidine kinase from Anaeromyxobacter sp. Fw109-5.J. Biol. Chem. 2011; 286 (21852234): 35522-3553410.1074/jbc.M111.274811Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). It has an N-terminal heme-bound globin domain and a C-terminal kinase domain. The binding of oxygen to a heme-Fe(II) complex in the oxygen-sensing globin domain induces autophosphorylation of its kinase domain via interdomain signal transduction (19Kitanishi K. Kobayashi K. Uchida T. Ishimori K. Igarashi J. Shimizu T. Identification and functional and spectral characterization of a globin-coupled histidine kinase from Anaeromyxobacter sp. Fw109-5.J. Biol. Chem. 2011; 286 (21852234): 35522-3553410.1074/jbc.M111.274811Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). AfGcHK proteins containing a heme-Fe(II)-O2 complex or an Fe(III)-OH− complex formed by autoxidation are both enzymatically fully active (20Fojtikova V. Stranava M. Vos M.H. Liebl U. Hranicek J. Kitanishi K. Shimizu T. Martinkova M. Kinetic analysis of a globin-coupled histidine kinase, AfGcHK: effects of the heme iron complex, response regulator, and metal cations on autophosphorylation activity.Biochemistry. 2015; 54 (26212354): 5017-502910.1021/acs.biochem.5b00517Crossref PubMed Scopus (16) Google Scholar). The kcat values of the heme-Fe(II)-O2 and Fe(III)-OH−-bound AfGcHK forms were 1.0–1.1 min−1, and their KmATP values were 18.9–23.0 μm (Table S1). However, the form with an Fe(II)-CO-heme complex had a kcat of 1.0 min−1 but a very high KmATP of 357 μm. This high KmATP suggests that in addition to causing pronounced changes in the structure of the sensing site, CO coordination dramatically altered the structure of the kinase domain (20Fojtikova V. Stranava M. Vos M.H. Liebl U. Hranicek J. Kitanishi K. Shimizu T. Martinkova M. Kinetic analysis of a globin-coupled histidine kinase, AfGcHK: effects of the heme iron complex, response regulator, and metal cations on autophosphorylation activity.Biochemistry. 2015; 54 (26212354): 5017-502910.1021/acs.biochem.5b00517Crossref PubMed Scopus (16) Google Scholar). The inactive AfGcHK forms either contain a heme-Fe(II) complex, lack the heme complex altogether (20Fojtikova V. Stranava M. Vos M.H. Liebl U. Hranicek J. Kitanishi K. Shimizu T. Martinkova M. Kinetic analysis of a globin-coupled histidine kinase, AfGcHK: effects of the heme iron complex, response regulator, and metal cations on autophosphorylation activity.Biochemistry. 2015; 54 (26212354): 5017-502910.1021/acs.biochem.5b00517Crossref PubMed Scopus (16) Google Scholar), or lack the whole sensing domain (21Stranava M. Man P. Skálová T. Kolenko P. Blaha J. Fojtikova V. Martínek V. Dohnálek J. Lengalova A. Rosůlek M. Shimizu T. Martínková M. Coordination and redox state-dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction.J. Biol. Chem. 2017; 292 (29092908): 20921-2093510.1074/jbc.M117.817023Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). globin-coupled histidine kinase from the soil bacterium Anaeromyxobacter sp. Fw109-5 Azotobacter vinelandii globin-coupled oxygen sensor globin-coupled oxygen sensor with diguanylate cyclase activity from Bordetella pertussis E. coli copper and silver ions sensor heme-based oxygen sensor protein from Mycobacterium tuberculosis heme-based oxygen sensor phosphodiesterase hydrogen deuterium exchange coupled with mass spectrometry globin-coupled oxygen sensor from B. subtilis Fe(II)-protoporphyrin IX complex Fe(III)-protoporphyrin IX complex or hemin 2-methyl-2,4-pentanediol globin-coupled oxygen sensor with diguanylate cyclase activity from P. carotovorum Protein Data Bank tobacco etch virus globin-coupled oxygen sensor diguanylate cyclase from E. coli root mean square deviation. Several studies have investigated the structure-function relationships of AfGcHK (19Kitanishi K. Kobayashi K. Uchida T. Ishimori K. Igarashi J. Shimizu T. Identification and functional and spectral characterization of a globin-coupled histidine kinase from Anaeromyxobacter sp. Fw109-5.J. Biol. Chem. 2011; 286 (21852234): 35522-3553410.1074/jbc.M111.274811Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 20Fojtikova V. Stranava M. Vos M.H. Liebl U. Hranicek J. Kitanishi K. Shimizu T. Martinkova M. Kinetic analysis of a globin-coupled histidine kinase, AfGcHK: effects of the heme iron complex, response regulator, and metal cations on autophosphorylation activity.Biochemistry. 2015; 54 (26212354): 5017-502910.1021/acs.biochem.5b00517Crossref PubMed Scopus (16) Google Scholar, 21Stranava M. Man P. Skálová T. Kolenko P. Blaha J. Fojtikova V. Martínek V. Dohnálek J. Lengalova A. Rosůlek M. Shimizu T. Martínková M. Coordination and redox state-dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction.J. Biol. Chem. 2017; 292 (29092908): 20921-2093510.1074/jbc.M117.817023Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 22Stranava M. Martínek V. Man P. Fojtikova V. Kavan D. Van[caron]ek O. Shimizu T. Martinkova M. 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. 2016; 84 (27273553): 1375-138910.1002/prot.25083Crossref PubMed Scopus (16) Google Scholar). As a result, the X-ray crystal structure of the isolated AfGcHK globin domain dimer has been solved and the full-length protein has been studied by hydrogen-deuterium exchange coupled with MS (HDX-MS) (21Stranava M. Man P. Skálová T. Kolenko P. Blaha J. Fojtikova V. Martínek V. Dohnálek J. Lengalova A. Rosůlek M. Shimizu T. Martínková M. Coordination and redox state-dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction.J. Biol. Chem. 2017; 292 (29092908): 20921-2093510.1074/jbc.M117.817023Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). These studies have revealed that changes in the heme redox state and axial ligand binding to the heme-iron complex induce large changes in the protein's structure in the vicinity of the heme, which in turn cause profound structural changes in the wider protein. It was suggested that these structural changes occur primarily within the dimerization interface of the globin domain and are linked to the regulation of the kinase domain's catalytic activity (21Stranava M. Man P. Skálová T. Kolenko P. Blaha J. Fojtikova V. Martínek V. Dohnálek J. Lengalova A. Rosůlek M. Shimizu T. Martínková M. Coordination and redox state-dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction.J. Biol. Chem. 2017; 292 (29092908): 20921-2093510.1074/jbc.M117.817023Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Here, we present X-ray crystallographic investigations and a thorough biophysical analysis including a kinase activity study that were conducted to determine how the oligomerization state of the globin (sensing) domain affects the autophosphorylation activity of AfGcHK. The crystal structure of the monomeric sensing domain co-crystallized with imidazole was solved, and inspection of this structure suggested that Tyr-15 is involved in the domain dimerization process and also forms important contacts with the neighboring heme group. Therefore, the oligomeric states and functions of Tyr-15 mutant proteins were investigated. Based on the results of these investigations, we suggest that the interaction of Tyr-15 with the heme-binding pocket of the neighboring globin domain in the AfGcHK dimer is critical for the sensing domain's dimerization, and that the sensing domain's dimerization is required to enable signal transduction between the sensing and functional domains and the functional domain's autophosphorylation activity. A new crystal structure of the globin (sensing) domain of AfGcHK in the presence of imidazole was solved at a resolution of 1.8 Å (Fig. 1 and Table 1). The globin domain is monomeric in this structure. It has a globin-fold and consists of seven helices and seven loops. Additionally, it contains a heme molecule and three small ligands: two imidazole molecules and one molecule of 2-methyl-2,4-pentanediol (MPD). The MPD molecule (which originates from the crystallization solution) is bound at a crystal contact of two protein molecules, forming hydrogen bonds to Glu-62 and Arg-139 residues from neighboring protein chains; it has no biological significance. The binding of imidazole (also originating from the crystallization solution) is discussed in detail below. The data processing and structure refinement parameters are shown in Table 1. All residues of the protein chain fit well to the observed electron density. 98.1% of the residues lie in the favored regions of the Ramachandran plot and there are no outliers. The atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB) under the code 6OTD.Table 1Data collection statistics and structure refinement parameters for the isolated AfGcHK globin domainData processing statisticsSpace groupP6122Unit cell parameters a, b, c (Å)69.4, 69.4, 113.4Resolution range (Å)41.25–1.80 (1.84–1.80)No. of observations213,525 (12,820)No. of unique reflections15,414 (902)Data completeness (%)99.2 (99.9)Average redundancy13.9 (14.2)Mosaicity (°)0.2Average I/σ(I)18.3 (2.0)Solvent content (%)43Matthews coefficient (Å3/Da)2.2Rmerge†Rmerge = ∑h∑i|Ihi − 〈Ih〉|/∑h∑iIhi,0.073 (1.320)Rpim$Rpim = ∑h∑i(nh − 1)−1/2|Ihi − 〈Ih〉|/∑h∑iIhi, and0.028 (0.506)CC1/20.999 (0.703)Structure refinement parametersRwork‡R = ∑h‖Fh,obs| − |Fh,calc‖/∑h|Fh,obs|, where Ihi is the observed intensity, 〈Ih〉 is the mean intensity of multiple observations of symmetry-related reflections, and Fh,obs and Fh,calc are the observed and calculated structure factor amplitudes. Rwork is the R factor calculated on 95% of reflections excluding a random subset of 5% of reflections marked as “free”. The final structure refinement was performed on all observed structure factors.0.182Rfree0.231Rall0.186Average B-factor (Å2)35RMSD bond lengths from ideal (Å)0.017RMSD bond angles from ideal (°)1.7Number of non-hydrogen atoms1,404Number of water molecules68Ramachandran statistics: residues in favored region (%)98.1PDB code6OTD† Rmerge = ∑h∑i|Ihi − 〈Ih〉|/∑h∑iIhi,§ Rpim = ∑h∑i(nh − 1)−1/2|Ihi − 〈Ih〉|/∑h∑iIhi, and‡ R = ∑h‖Fh,obs| − |Fh,calc‖/∑h|Fh,obs|, where Ihi is the observed intensity, 〈Ih〉 is the mean intensity of multiple observations of symmetry-related reflections, and Fh,obs and Fh,calc are the observed and calculated structure factor amplitudes. Rwork is the R factor calculated on 95% of reflections excluding a random subset of 5% of reflections marked as “free”. The final structure refinement was performed on all observed structure factors. Open table in a new tab The heme is located in its standard position and interacts directly with His-99 on the proximal side (Fig. 1A). On the distal side, an imidazole molecule (IMD203) occupies a typical ligand-binding position, binding to the heme-iron via N3; the Fe-N3 distance is 2.2 Å. The imidazole nitrogen N1 forms a hydrogen bond with the main chain carbonyl group of Leu-68 (2.7 Å). The His-67 side chain projects toward the heme molecule and forms a hydrogen bond to its propionate group (ND1 of His-67 and O2D of the heme are within a distance of 2.7 Å), making the heme inaccessible to the bulk solvent. This propionate group also makes a crystal contact with Phe-110 of an adjacent protein molecule. The heme's second propionate group makes a crystal contact with Arg-158, the last localized residue at the C terminus of the same crystal mate. Another imidazole molecule (IMD204) is bound approximately in the plane of the heme (Fig. 1A), with a distance of 4.1 Å between the imidazole's C2 carbon and the heme's CMC carbon. This is the position occupied by Tyr-15 from the neighboring chain in the dimeric globin structure (Fig. 1B) (21Stranava M. Man P. Skálová T. Kolenko P. Blaha J. Fojtikova V. Martínek V. Dohnálek J. Lengalova A. Rosůlek M. Shimizu T. Martínková M. Coordination and redox state-dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction.J. Biol. Chem. 2017; 292 (29092908): 20921-2093510.1074/jbc.M117.817023Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). IMD204 is positioned in a small cavity with direct access to the solvent; there is no protein-protein crystal contact in this area of monomeric protein. Although the first imidazole molecule, IMD203, is well-localized in the electron density, IMD204 is relatively free within its cavity and forms no hydrogen bonds, so it is much less well-localized. Its position was therefore verified by generating a Polder map (Fig. S1) (23Liebschner D. Afonine P.V. Moriarty N.W. Poon B.K. Sobolev O.V. Terwilliger T.C. Adams P.D. Polder maps: improving OMIT maps by excluding bulk solvent.Acta Crystallogr. D Struct. Biol. 2017; 73 (28177311): 148-15710.1107/S2059798316018210Crossref PubMed Scopus (364) Google Scholar). Two crystal structures of the AfGcHK globin domain (PDB codes 5OHE and 5OHF) have been solved previously (21Stranava M. Man P. Skálová T. Kolenko P. Blaha J. Fojtikova V. Martínek V. Dohnálek J. Lengalova A. Rosůlek M. Shimizu T. Martínková M. Coordination and redox state-dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction.J. Biol. Chem. 2017; 292 (29092908): 20921-2093510.1074/jbc.M117.817023Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Here we compare these structures to the new one (PDB code 6OTD). To this end, the globin domains (6OTD chain A, 5OHE chain G, and 5OHF chain G) were superimposed on the four nitrogen atoms of their heme groups. Structure 5OHE represents a dimeric CN-bound Fe(III) form of the AfGcHK globin domain, whereas structure 5OHF (in which chains G and H comprise the dimerization interface and exist in an alternative “B” configuration, denoted here as 5OHF-ligand-free) represents a dimeric form of the AfGcHK globin domain in the inactive state caused by treatment with sodium dithionite (21Stranava M. Man P. Skálová T. Kolenko P. Blaha J. Fojtikova V. Martínek V. Dohnálek J. Lengalova A. Rosůlek M. Shimizu T. Martínková M. Coordination and redox state-dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction.J. Biol. Chem. 2017; 292 (29092908): 20921-2093510.1074/jbc.M117.817023Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). The monomeric imidazole-bound structure has some unique structural features including relaxation of the dimer-forming helices (due to its monomeric form), a more accessible heme, and differences in the architecture of the heme-binding site (Fig. 1B). In the dimer (5OHE), Tyr-15 is immersed in the second chain's heme-binding cavity. This enables the residue to form part of the heme pocket and to protect the heme's equatorial region against interactions with the solvent, but requires the residue to adopt another conformation in which the torsion angle χ1 is −165°. As a result, the position of the Tyr-15 Cα is shifted by 2.5 Å relative to that in the monomeric structure, creating tension in the helix (H1) containing this residue. Conversely, in the monomeric structure 6OTD, the Tyr-15 side chain adopts the most common rotamer (χ1 = −65°) and helix H1 is straight and relaxed. Similar behavior is seen for the two long helices H6 and H7, which form the main part of the dimerization interface: both are straightened up and relaxed in the monomeric structure (Fig. 2). In the monomeric globin domain, the absence of Tyr-15 from a neighboring chain opens a new access tunnel to the heme pocket. This makes the heme more accessible to solvent molecules and oxygen, which is probably why the monomeric mutants Y15A and Y15G have high autoxidation rates (see below). In the monomeric crystal structure, this tunnel is occupied by the imidazole molecule IMD204, which is bound about 1 Å closer to the heme plane than the aromatic ring of Tyr-15 in the dimer. The Tyr-45 χ1 angles of the monomer and dimer differ by 113° (Fig. 1B). In the monomer, the bulky imidazole molecule IMD203 binds to the heme distal side, pushing away the side chain of Tyr-45, which forms a hydrogen bond to the cyanide ligand in the dimeric structure (21Stranava M. Man P. Skálová T. Kolenko P. Blaha J. Fojtikova V. Martínek V. Dohnálek J. Lengalova A. Rosůlek M. Shimizu T. Martínková M. Coordination and redox state-dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction.J. Biol. Chem. 2017; 292 (29092908): 20921-2093510.1074/jbc.M117.817023Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). No such hydrogen bond exists between Tyr-45 and IMD203 in the monomeric structure, which is unsurprising because Tyr-45 interacts with the signal molecule O2 (19Kitanishi K. Kobayashi K. Uchida T. Ishimori K. Igarashi J. Shimizu T. Identification and functional and spectral characterization of a globin-coupled histidine kinase from Anaeromyxobacter sp. Fw109-5.J. Biol. Chem. 2011; 286 (21852234): 35522-3553410.1074/jbc.M111.274811Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) and other small heme ligands; imidazole is much bulkier than these species. The space occupied by the Tyr-45 side chain in the dimeric structure is in the monomeric structure occupied by water (HOH347) and partially by Leu-68, which relaxes into a position closer to the axis above the heme center. This allows its main chain carbonyl oxygen to form hydrogen bonds with imidazole IMD203 (2.7 Å) and HOH347 (3.1 Å). In concert with the shift of Leu-68, His-67 changes its position and also changes its side chain rotamer (resulting in a χ1 difference of 115°), forming a hydrogen bond with one of the heme's propionate groups (Fig. 1A). The conformations of the distal helices of the globin domain in 5OHF ligand-free, which lacks an axial heme ligand, differ from those in the ligand-bound (CN− or imidazole) domains. The largest shift is observed for helix H2 (residues 20–50), which is located in similar positions in the CN− and imidazole-bound structures but is shifted away from the heme in the inactive ligand-free protein. The fine structure of the heme-binding pocket primarily depends on the volume of the heme distal ligand. Therefore, the architecture of the active CN-bound state represents something of an intermediate between those of the imidazole-bound monomer (6OTD) and the ligand-free dimer (5OHF-ligand-free). Because the crystal structure of the Fe(III)-imidazole complex of the isolated monomeric globin domain of AfGcHK showed that Tyr-15 plays an important role in the protein's architecture, we prepared Tyr-15 mutants (Y15A, Y15G, Y15F, and Y15W) of both the full-length protein and the isolated globin domain, and performed detailed functional (biophysical) studies on them. The WT full-length AfGcHK pro" @default.
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• W2997652571 title "Disruption of the dimerization interface of the sensing domain in the dimeric heme-based oxygen sensor AfGcHK abolishes bacterial signal transduction" @default.
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• W2997652571 doi "https://doi.org/10.1074/jbc.ra119.011574" @default.
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