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• W2149188360 abstract "Pathogenesis by Bacillus anthracis requires coordination between two distinct activities: plasmid-encoded virulence factor expression (which protects vegetative cells from immune surveillance during outgrowth and replication) and chromosomally encoded sporulation (required only during the final stages of infection). Sporulation is regulated by at least five sensor histidine kinases that are activated in response to various environmental cues. One of these kinases, BA2291, harbors a sensor domain that has ∼35% sequence identity with two plasmid proteins, pXO1-118 and pXO2-61. Because overexpression of pXO2-61 (or pXO1-118) inhibits sporulation of B. anthracis in a BA2291-dependent manner, and pXO2-61 expression is strongly up-regulated by the major virulence gene regulator, AtxA, it was suggested that their function is to titrate out an environmental signal that would otherwise promote untimely sporulation. To explore this hypothesis, we determined crystal structures of both plasmid-encoded proteins. We found that they adopt a dimeric globin fold but, most unusually, do not bind heme. Instead, they house a hydrophobic tunnel and hydrophilic chamber that are occupied by fatty acid, which engages a conserved arginine and chloride ion via its carboxyl head group. In vivo, these domains may therefore recognize changes in fatty acid synthesis, chloride ion concentration, and/or pH. Structure-based comparisons with BA2291 suggest that it binds ligand and dimerizes in an analogous fashion, consistent with the titration hypothesis. Analysis of newly sequenced bacterial genomes points to the existence of a much broader family of non-heme, globin-based sensor domains, with related but distinct functionalities, that may have evolved from an ancestral heme-linked globin. Pathogenesis by Bacillus anthracis requires coordination between two distinct activities: plasmid-encoded virulence factor expression (which protects vegetative cells from immune surveillance during outgrowth and replication) and chromosomally encoded sporulation (required only during the final stages of infection). Sporulation is regulated by at least five sensor histidine kinases that are activated in response to various environmental cues. One of these kinases, BA2291, harbors a sensor domain that has ∼35% sequence identity with two plasmid proteins, pXO1-118 and pXO2-61. Because overexpression of pXO2-61 (or pXO1-118) inhibits sporulation of B. anthracis in a BA2291-dependent manner, and pXO2-61 expression is strongly up-regulated by the major virulence gene regulator, AtxA, it was suggested that their function is to titrate out an environmental signal that would otherwise promote untimely sporulation. To explore this hypothesis, we determined crystal structures of both plasmid-encoded proteins. We found that they adopt a dimeric globin fold but, most unusually, do not bind heme. Instead, they house a hydrophobic tunnel and hydrophilic chamber that are occupied by fatty acid, which engages a conserved arginine and chloride ion via its carboxyl head group. In vivo, these domains may therefore recognize changes in fatty acid synthesis, chloride ion concentration, and/or pH. Structure-based comparisons with BA2291 suggest that it binds ligand and dimerizes in an analogous fashion, consistent with the titration hypothesis. Analysis of newly sequenced bacterial genomes points to the existence of a much broader family of non-heme, globin-based sensor domains, with related but distinct functionalities, that may have evolved from an ancestral heme-linked globin. Fully virulent Bacillus anthracis carries two large plasmids, pXO1 and pXO2, that are responsible for the production of its major virulence factors, anthrax toxin and the poly-γ-d-glutamic acid capsule (1Okinaka R.T. Cloud K. Hampton O. Hoffmaster A.R. Hill K.K. Keim P. Koehler T.M. Lamke G. Kumano S. Mahillon J. Manter D. Martinez Y. Ricke D. Svensson R. Jackson P.J. J. Bacteriol. 1999; 181: 6509-6515Crossref PubMed Google Scholar, 2Ezzell J.W. Welkos S.L. J. Appl. Microbiol. 1999; 87: 250Crossref PubMed Google Scholar, 3Candela T. Mock M. Fouet A. J. Bacteriol. 2005; 187: 7765-7772Crossref PubMed Scopus (84) Google Scholar). Following host-triggered germination of B. anthracis spores, toxin and capsule enable the bacilli in their vegetative form to evade the host's immune system and replicate rapidly in the lymphatic system and bloodstream. If the infection is not treated at an early stage, toxemia and septicemia leading to host death may rapidly follow (4Mock M. Fouet A. Annu. Rev. Microbiol. 2001; 55: 647-671Crossref PubMed Scopus (866) Google Scholar, 5Kang T.J. Fenton M.J. Weiner M.A. Hibbs S. Basu S. Baillie L. Cross A.S. Infect. Immun. 2005; 73: 7495-7501Crossref PubMed Scopus (100) Google Scholar, 6Drysdale M. Heninger S. Hutt J. Chen Y. Lyons C.R. Koehler T.M. EMBO J. 2005; 24: 221-227Crossref PubMed Scopus (143) Google Scholar, 7Hu H. Sa Q. Koehler T.M. Aronson A.I. Zhou D. Cell. Microbiol. 2006; 8: 1634-1642Crossref PubMed Scopus (66) Google Scholar). Sporulation is required for long term survival of B. anthracis following host death, but the process must be carefully coordinated with toxin expression and the progress of the infection because sporulating cells (in contrast to encapsulated vegetative cells) are susceptible to host defenses. Given that sporulation is directed primarily by chromosomal genes, close coordination with the regulatory elements encoded by plasmid genes is therefore required for effective pathogenesis. There is much evidence for such chromosome-plasmid “cross-talk,” although the overall picture is far from clear (for review, see Ref. 8Perego M. Hoch J.A. Trends Microbiol. 2008; 16: 215-221Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). For example, the pXO1-encoded protein, AtxA, regulates synthesis of toxin (also pXO1-encoded) as well as capsule (pXO2-encoded) (9Dai Z. Sirard J.C. Mock M. Koehler T.M. Mol. Microbiol. 1995; 16: 1171-1181Crossref PubMed Scopus (133) Google Scholar, 10Uchida I. Hornung J.M. Thorne C.B. Klimpel K.R. Leppla S.H. J. Bacteriol. 1993; 175: 5329-5338Crossref PubMed Google Scholar, 11Drysdale M. Bourgogne A. Hilsenbeck S.G. Koehler T.M. J. Bacteriol. 2004; 186: 307-315Crossref PubMed Scopus (81) Google Scholar, 12Bourgogne A. Drysdale M. Hilsenbeck S.G. Peterson S.N. Koehler T.M. Infect. Immun. 2003; 71: 2736-2743Crossref PubMed Scopus (117) Google Scholar). AtxA is also part of a regulatory network for S-layer synthesis (a further defensive layer distinct from the capsule and peptidoglycan cell wall), a task performed by chromosomal proteins (13Mignot T. Mock M. Fouet A. Mol. Microbiol. 2003; 47: 917-927Crossref PubMed Scopus (76) Google Scholar). In turn, the synthesis of AtxA is regulated by the chromosomally encoded transcription factor Spo0A, the master regulator of sporulation (14Hadjifrangiskou M. Chen Y. Koehler T.M. J. Bacteriol. 2007; 189: 1874-1883Crossref PubMed Scopus (36) Google Scholar, 15Saile E. Koehler T.M. J. Bacteriol. 2002; 184: 370-380Crossref PubMed Scopus (115) Google Scholar), which completes a regulatory link between sporulation and virulence factor expression. Spo0A is activated by phosphorylation to induce or repress transcription of genes required or not required for sporulation, respectively (16Stephenson K. Hoch J.A. Mol. Microbiol. 2002; 46: 297-304Crossref PubMed Scopus (107) Google Scholar). The regulatory pathway controlling Spo0A is more complex than most two-component signal transduction systems. In B. anthracis, this pathway includes at least five (chromosomally encoded) sensor histidine kinases that are capable of inducing sporulation (17Brunsing R.L. La Clair C. Tang S. Chiang C. Hancock L.E. Perego M. Hoch J.A. J. Bacteriol. 2005; 187: 6972-6981Crossref PubMed Scopus (56) Google Scholar) as well as several aspartyl phosphatases, one of them encoded by the pXO1 virulence plasmid, that inhibit sporulation (18Bongiorni C. Stoessel R. Shoemaker D. Perego M. J. Bacteriol. 2006; 188: 487-498Crossref PubMed Scopus (57) Google Scholar, 19Bongiorni C. Stoessel R. Perego M. J. Bacteriol. 2007; 189: 2637-2645Crossref PubMed Scopus (23) Google Scholar). Sensor histidine kinases are the primary sensors of environmental cues and form a large family of signaling proteins in both Gram-positive and Gram-negative bacteria. They have a modular architecture comprising at least a “sensor” domain and a catalytic domain (which includes the phosphorylatable domain, DHp, and the ATP-binding domain) that autophosphorylates on a histidine residue in response to sensor domain activation. Phosphohistidine is a high energy species that transfers its phosphoryl group to an aspartic acid residue on the downstream effector (for review, see Ref. 20Szurmant H. Hoch J.A. Curr. Opin. Microbiol. 2010; 13: 190-197Crossref PubMed Scopus (50) Google Scholar). BA2291 is one of the most active kinases in promoting sporulation in B. anthracis (17Brunsing R.L. La Clair C. Tang S. Chiang C. Hancock L.E. Perego M. Hoch J.A. J. Bacteriol. 2005; 187: 6972-6981Crossref PubMed Scopus (56) Google Scholar). It also appears to be unique in using GTP rather than ATP as its energy source for phosphorylation (21Scaramozzino F. White A. Perego M. Hoch J.A. J. Bacteriol. 2009; 191: 687-692Crossref PubMed Scopus (15) Google Scholar), and orthologs are found in most members of the Bacillus cereus group (a subfamily of the genus Bacillae, which includes B. anthracis and B. thuringiensis (an insect pathogen), but not Bacillus subtilis). Two plasmid-encoded proteins, pXO1-118 and pXO2-61, express “sensor-only” domains that share ∼35% sequence identity with BA2291 (22White A.K. Hoch J.A. Grynberg M. Godzik A. Perego M. J. Bacteriol. 2006; 188: 6354-6360Crossref PubMed Scopus (27) Google Scholar) and are only found in B. anthracis and certain strains of B. cereus that harbor similar plasmids. The pXO1-118 gene lies next to and is divergently transcribed from the atxA gene, whereas pXO2-61 lies within the region directing capsule synthesis. A microarray study found that transcription of pXO2-61 was strongly up-regulated when the atxA gene was present (12Bourgogne A. Drysdale M. Hilsenbeck S.G. Peterson S.N. Koehler T.M. Infect. Immun. 2003; 71: 2736-2743Crossref PubMed Scopus (117) Google Scholar); and overexpression of pXO2-61 was found to reduce sporulation of B. anthracis in a BA2291-dependent manner (22White A.K. Hoch J.A. Grynberg M. Godzik A. Perego M. J. Bacteriol. 2006; 188: 6354-6360Crossref PubMed Scopus (27) Google Scholar). Expression of BA2291 in a B. subtilis model induced sporulation when expressed at lower copy levels, and this was repressed by co-expression of either pXO1-118 or pXO2-61. However, higher levels of BA2291 expression led to repression of sporulation, suggesting that when the activating signal is in limited supply, the kinase activity is reversed, and BA2291 acts as a phosphatase. These observations led to a model in which the plasmid-encoded sensor domains modulate BA2291 activity by titrating the sporulation signal, thereby preventing premature sporulation (22White A.K. Hoch J.A. Grynberg M. Godzik A. Perego M. J. Bacteriol. 2006; 188: 6354-6360Crossref PubMed Scopus (27) Google Scholar). To explore the molecular mechanisms of BA2291 regulation and the nature of the sporulation signal, we determined the crystal structures of pXO1-118 and pXO2-61 at high resolution. We show that they adopt a dimeric globin fold but, most unusually, do not bind heme. We demonstrate that they bind fatty acid and a halide ion (most likely chloride) in a central cavity, and we show that the key structural features of ligand recognition and dimerization are conserved in a large family of kinases found in bacilli and related species. This suggests that they recognize the same environmental cue(s) and support the titration mechanism for the sensor-only domains. Recognizable homologs are also found in a number of distinct bacterial phyla, and our analysis points to an evolution of the globin family into a versatile group of “heme-free” environmental sensors. The plasmid for Escherichia coli overexpression of B. anthracis ORF118 was obtained by cloning the PCR-amplified coding sequence using oligonucleotides BaORF1185′Nde (5′-GAGTGGACATATGGAAGCAACAAAACG-3′) and BaORF1183′Bam (5′-CTATAGGATCCAAAAATTTCAAGGTG-3′) into plasmid pET28a (Stratagene) digested with NdeI and BamHI. The coding sequence for residues 1–146 of BA2291 was amplified using oligonucleotides BaKin5′Nde (5′-TATTCGTCATATGGAAATGGAGGGAATG-3′) and BaKinXho2 (5′-TTTCCCTCGAGTTTTATAATATAATTTCCGAGTAT-3′), and the fragment cloned into pET28a digested with NdeI and XhoI. For pXO2-61, a synthetic gene was purchased from GenScript Co. and subcloned into pET28 as described for pXO1-118. Expression was obtained in E. coli BL21(DE3) cells grown in LB medium after induction with 0.1 mm isopropyl β-d-1-thiogalactopyranoside for 4 h at 32 °C. All proteins included a His6 tag and were purified by nickel affinity chromatography on a His-trap chelating column (Pharmacia), followed by His tag removal by thrombin and size exclusion on Superdex 75 or Superdex 200 columns (Amersham Biosciences), the apparent molecular mass in each case was consistent with a dimer. pXO1-118 was stored at −20 °C in 20 mm Tris-HCl, pH 7.4, 1 m NaCl, 50 μm KCl, 5 mm DTT. pXO2-61 and BA2291 sensor domains required 500 mm NaCl to keep the proteins stable for long term storage. For pXO1-118, selenomethionine-labeled protein was purified using a similar protocol, except that cells were grown in minimal medium supplemented with selenomethionine (23Harrison C.J. Bohm A.A. Nelson H.C. Science. 1994; 263: 224-227Crossref PubMed Scopus (223) Google Scholar). The molecular mass of all proteins was confirmed by SDS-PAGE and MALDI-TOF mass spectrometry. Native and selenomethionine-labeled pXO1-118 were crystallized by sitting or hanging drop vapor diffusion at room temperature by mixing 3 μl of precipitant solution (40% (v/v) PEG 300, 100 mm Tris-HCl, pH 5.4, 5% (w/v) PEG 1000), and 3 μl of protein solution at 14 mg/ml. Rod-shaped crystals grew within 3 days in space group P3221 with cell dimensions a = 89.9 Å, c = 35.3 Å. One native and one selenomethionine data set at the selenium absorption edge were collected at the Stanford Synchrotron Research Lightsource beamline 9-2, and the Brookhaven National Synchrotron Light Source beamline X26C, respectively, at 100 K. Diffraction images were processed and scaled with the HKL package (24Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). SOLVE (25Terwilliger T.C. Berendzen J. Acta Crystallogr. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar) located four selenium sites, leading to initial phases with a figure of merit of 0.32. Density modification increased the figure of merit to 0.60; and automatic model building in RESOLVE (26Terwilliger T.C. Acta Crystallogr. D Biol Crystallogr. 2001; 57: 1755-1762Crossref PubMed Scopus (166) Google Scholar) generated a model that was 77% complete. Manual model building was carried out in O (27Kleywegt G.J. Zou J.Y. Kjeldgaard M. Jones T.A. Rossmann M.G. Arnold E. International Tables for Crystallography: Crystallography of Biological Macromolecules. Kluwer Academic Publishers, Dordrecht, The Netherlands2001: 353-356Google Scholar), and the structure refined with REFMAC5 (28Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar) and simulated-annealing using CNS (29Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). The final model for pXO1-118 contains a single domain (residues 1–150), plus 3 nonnative N-terminal residues, 1 molecule of undecanoic acid, 95 water molecules, and 1 Cl− ion; and has Rwork = 0.181 and Rfree = 0.225 for data from 80 to 1.76 Å resolution. A dimer is generated by rotation of the monomer about a crystallographic dyad. pXO2-61 was crystallized by the microbatch method under paraffin oil. Crystals were obtained in 2 days from a buffer containing 1 m NaI, 20% (v/v) PEG 3350, 100 mm Tris-HCl, pH 7.5. Crystals belong to space group P212121 with unit cell dimensions a = 44.1, b = 62.6 and c = 124.7 Å. Data were collected at the Stanford Synchrotron Research Lightsource to 1.49 Å resolution and processed with the HKL package. The structure was solved by molecular replacement using the refined pXO1-118 structure as the search model. Model building and refinement were carried out in O and REFMAC5. The final model has Rwork = 0.177 and Rfree = 0.209 for data from 60 to 1.49 Å resolution. The asymmetric unit contains 2 molecules forming a dimer (residues 5–136 for each molecule), 364 water molecules, 26 I− ions, and 8 Na+ ions. The solvent content is 56.7%. Data collection and refinement statistics are summarized in Table 1. The stereochemical quality of both models was assessed using PROCHECK (30Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar).TABLE 1X-ray data collection, phasing and refinementParameterPhasing λpeakModel refinementpXO1-118pXO2-61Space groupP3221P212121Cell dimensions (Å)a = 89.9a = 44.1c = 35.3b = 62.6c = 124.7Wavelength (Å)0.97810.979230.97923Resolution range (Å)50–2.580–1.7660–1.49Observations70,890173,637408,633Unique reflections5,88816,46156,717CompletenessaNumbers in parentheses are for highest resolution shell. (%)99.8 (100)99.5 (95.0)98.9 (94.5)RsymaNumbers in parentheses are for highest resolution shell.,bRsym = Σ|Ih − <Ih>|/ΣIh, where <Ih> is the average intensity over symmetry equivalent reflection. (%)6.9 (25)5.7 (46)6.8 (32)RworkcR-factor = Σ|Fobs − Fcalc| − Fobs, where summation is over the data used for refinement./RfreedRfree was calculated using 5% of data excluded from refinement. (%)0.185/0.2410.177/0.209Protein atoms1,4082,628Water molecules95364Other ions134Ligand1Root mean square deviationBonds (Å)0.0270.012Angles (°)1.701.37Average B-factor (Å2)Main chain25.018.9Side chain32.724.0Water34.635.9Ligands/Ions37.439.7a Numbers in parentheses are for highest resolution shell.b Rsym = Σ|Ih − <Ih>|/ΣIh, where <Ih> is the average intensity over symmetry equivalent reflection.c R-factor = Σ|Fobs − Fcalc| − Fobs, where summation is over the data used for refinement.d Rfree was calculated using 5% of data excluded from refinement. Open table in a new tab 200 μl of chloroform was added to 0.1–1 ml of 10 mg/ml sensor domain. The resulting two-phase system was sonicated for 10 min, incubated at 70 °C for 1 h, and then centrifuged. The organic phase was separated by syringe. For pXO2-61, the carboxylic acid group of the fatty acid was verified by using 20 μl of bis-trimethylsyliltrifluoroacetamide and 20 μl of pyridine incubated for 1.5 h at 65 °C. Samples were evaporated to dryness under a stream of N2 and reconstituted in 100 μl of methylene chloride, prior to analysis by GC-MS (Scripps Center for Mass Spectrometry). ITC 5The abbreviations used are: ITCisothermal titration calorimetryAUCanalytical ultracentrifugation. was performed using a VP-ITC calorimeter from Microcal (Northampton, MA). 8 μl of fatty acid solution (1.6–2.6 mm) was injected into cells containing 100 μm protein (pXO1-118 or pXO2-61) in 20 mm Tris, pH 7.4, and either 500 mm or 1 m NaCl, respectively. All titrations were performed at 23 °C, and each experiment involved 37 injections. Myristic acid (n-C14:0) and palmitic acid (n-C16:0) were purchased from Sigma-Aldrich. 12-Methyltetradecanoic acid (anteiso-C15:0) and 13-methyltetradecanoic acid (iso-C15:0) were purchased from Indofine Chemical Co. Palmitoleic acid was purchased from Fluka. Data were analyzed using Microcal Origin software provided by the manufacturer. isothermal titration calorimetry analytical ultracentrifugation. Sedimentation equilibrium experiments were performed in a ProteomeLab XL-I (BeckmanCoulter) AUC. Protein samples (pXO2-61 or BA2291 sensor domain) in 20 mm Pipes, pH 7.5, 500 mm NaCl, were loaded at concentrations of 0.5, 0.167, and 0.056 mg/ml in 6-channel equilibrium cells and spun in an An-50 Ti 8-place rotor at 25,000 rpm for 24 h at 20 °C. Data were analyzed with HeteroAnalysis software (J. L. Cole and J. W. Lary, University of Connecticut). In each case, an ideal equilibrium model gave a convincing fit for a monomer-dimer equilibrium in solution, with dimer molecular mass values of 32.5 kDa (pXO2-61) and 33.8 kDa (BA2291 sensor domain) and no evidence for higher oligomers. The yeast two-hybrid system (Clontech) was used to explore interactions between pXO1-118 and AtxA. Coding genes were singly cloned into the bait plasmid (pGBT9) and prey plasmid (pGAD424). Assays were performed in the yeast strain AH109. We detected interaction when pXO1-118 was present on both pGBT9 and pGAD424, consistent with homodimer formation in yeast cells. There was no evidence for AtxA-pXO1-118 interactions (data not shown). Homologs of sensor domains were found using CS-BLAST (31Biegert A. Söding J. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 3770-3775Crossref PubMed Scopus (134) Google Scholar). We solved the crystal structures of pXO1-118 and pXO2-61 at 1.76 Å and 1.49 Å resolution, respectively (see “Experimental Procedures,” Table 1, and FIGURE 1, FIGURE 2). The asymmetric unit of pXO1-118 contains a single sensor domain that adopts the globin fold. Although it does not bind heme, we follow the standard nomenclature for hemoglobins: helices A, B, E, F, G, and H are present in pXO1-118, whereas helices C and D are replaced by an ordered loop (BE). A dimer is formed across a crystallographic dyad, mediated by the packing of the G and H helices from apposing monomers, forming an antiparallel, left-handed four-helix bundle (Fig. 1) that buries a large interface (∼3,500 Å2).FIGURE 1Structure of the B. anthracis pXO1-118 sensor domain. A, ribbon representation of the dimer (side view), with termini and helices labeled (according to standard globin nomenclature). Helices are colored in spectral order (blue → orange) for each monomer. Labels for second monomer are primed. The KIAXER motif within helix F is colored red. Fatty acid is shown as cyan (methylene carbons) and red (carboxyl oxygens) spheres. B, same as in A, but orthogonal view looking down the 2-fold axis of the dimer. C, same view as in A, highlighting the dimer interface, with water molecules found only in a central region. D, sequence alignments of pXO1-118, pXO2-61, and the sensor domain of the B. anthracis sporulation kinase, BA2291. Secondary structure elements for pXO1-118 are indicated, as is the KIAXER motif.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As expected, the structure of pXO2-61 (Fig. 2) is very similar to that of pXO1-118 in both tertiary fold and dimer organization. The asymmetric unit in this case contains a dimer, and the monomers superpose with a root mean square deviation of 0.43 Å for Cα carbons. The most obvious external difference arises from a shorter C-terminal helix, leading to a more compact shape and a decrease in the dimerization interface, to 2,200 Å2. The dimers of pXO1-118 and pXO2-61 superpose with a root mean square difference of 1.2 Å for 264 Cα carbons (0.85 Å for pairwise comparisons of single domains). By AUC, we found that pXO2-61 forms dimers in solution at concentrations greater than or equal to micromolar (dimer Kd = 0.6 μm). We also purified the sensor domain from BA2291 and found that it also forms dimers in solution, with a similar Kd = 0.8 μm (supplemental Fig. 1). In addition, we demonstrated that pXO1-118 forms dimers in cells, as evidenced in a yeast two-hybrid system (see “Experimental Procedures”). The dimer interfaces of pXO1-118 and pXO2-61 include upper and lower hydrophobic regions that are more closely packed; whereas in the central section the helices diverge, and the interface is chiefly hydrophilic, comprising a large number of direct and water-mediated hydrophilic interactions (Fig. 1C). We found a total of 34 (pXO1-118) and 20 (pXO2-61) solvent molecules buried at distinct locations in this space. pXO1-118 has an additional interface formed by the ends of the longer C-terminal helices, which form a short antiparallel cross-over β-sheet with clusters of Tyr and Lys residues. Given the high homology between the BA2291 sensor domain and the plasmid domains (35% identity, 59% similarity) and the existence of a dimer in all cases, it is reasonable to assume that BA2291 will share a very similar tertiary and quaternary organization. Three-dimensional comparisons of the plasmid-encoded sensor domains using the DALI server (32Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3566) Google Scholar) identified many structures with the globin fold that superpose with root mean square differences in the 2.3–2.7 Å range for ∼150 Cαs (supplemental Table I). However, the Protein Data Bank contains no close sequence homologs (identities range from 8 to 14%). Moreover, most proteins with the globin fold have an Fe-heme co-factor sandwiched between the E and F helices. The most similar structures (in both tertiary and quaternary organization) are two bacterial (B. subtilis and Geobacter) heme-containing oxygen sensor domains, which form dimers via a similar pairing of the G and H helices. There is one globin structure that lacks co-factor altogether: the B. subtilis stress response regulator RsbR (33Murray J.W. Delumeau O. Lewis R.J. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 17320-17325Crossref PubMed Scopus (59) Google Scholar); in this case, the dimerization helices (G and H) bend inward, eliminating the co-factor cavity. Mammalian hemoglobins have closely related tertiary folds, but only one, the recently discovered cytoglobin, has a related dimeric organization (34de Sanctis D. Dewilde S. Pesce A. Moens L. Ascenzi P. Hankeln T. Burmester T. Bolognesi M. J. Mol. Biol. 2004; 336: 917-927Crossref PubMed Scopus (152) Google Scholar). The cyanobacter phycocyanins also adopt a globin-like fold (35Contreras-Martel C. Matamala A. Bruna C. Poo-Caamaño G. Almonacid D. Figueroa M. Martínez-Oyanedel J. Bunster M. Biophys. Chem. 2007; 125: 388-396Crossref PubMed Scopus (40) Google Scholar); they are electron transfer proteins involved in photosynthesis and bind porphyrin-like moieties via cysteine-mediated thioether bonds. However, they have large N-terminal extensions, and their quaternary organization is quite distinct from any of the hemoglobins. An overlay of pXO1-118 and the B. subtilis oxygen-sensor domain (36Zhang W. Phillips Jr., G.N. Structure. 2003; 11: 1097-1110Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) illustrates the overall similarity in secondary and tertiary folds (Fig. 3A) and shows how helix E of pXO1-118 rotates and bends to pack more closely against helix F, partially filling the space that is occupied by heme in the oxygen sensor. The short rigid BE loop (which replaces the CD helix/turn) packs against the FG turn, stabilizing this conformation (see below). Oxygen-binding hemoglobins are linked to the heme via a “proximal histidine” at the eighth position of helix F (F8). In pXO1-118 (and pXO2-61), there is no similarly located histidine, and, as expected, attempts to reconstitute the proteins with hemin were unsuccessful (data not shown). Remarkably, DALI-based structural alignment places pXO1-118 residue Arg-74 at the F8 heme location (Fig. 3B). A cross-section through the central cavity (Fig. 3C) further demonstrates the steric mismatch between heme and the pXO1-118 cavity. As discussed below, Arg-74 is an invariant residue that appears to play a key role in binding an alternative co-factor (fatty acid; see Fig. 3B) and thus may be considered a structural and functional analog of the proximal histidine. Note that in RsbR, DALI-based structural alignment indicates that helix F is truncated such that there is no analog of F8 (Fig. 3D), consistent with its being co-factor-free. The central cavity runs roughly parallel with helices E and G, with a contour length of ∼20 Å (Fig. 4). It comprises a narrow (∼6-Å diameter) tunnel lined with hydrophobic and aromatic residues, capped at one end by residue Phe-19, which appears to act as a gate, adopting conformations that either open or close the hydrophobic entrance to the tunnel. The other end of the tunnel opens into a hydrophilic chamber, which is sealed from bulk solvent by a “canopy” created by the packing of the BE and FG turns. It includes water molecules as well as a heavier anion, which we believe to be chloride (see below). In pXO1-118, the cavity is also occupied by continuous worm-like electron density that ends in a symmetric bifurcation, consistent with the presence of a fatty acid (Fig. 4A). Using mass spectrometry (GC-MS), we determined that the major chloroform-soluble nonprotein component in the crystals is palmitic (hexadecanoic) acid, presumably derived from the cell wall of E. coli (37Song Y. Gunner M.R. J. Mol. Biol. 2009; 387: 840-856Crossref PubMed Scopus (37) Google Scholar) during expression or cell lysis (supplemental Fig. 2). The tunnel is long enough to completely bury tetradecanoic acid (with the Phe-19 gate in the open position), and additional methylene groups would presumably extrude into solvent. We searched for an authentic Bacillus co" @default.
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