Structure of an Atypical Orphan Response Regulator Protein Supports a New Phosphorylation-independent Regulatory Mechanism

Abstract

Two-component signal transduction systems, commonly found in prokaryotes, typically regulate cellular functions in response to environmental conditions through a phosphorylation-dependent process. A new type of response regulator, hp1043 (HP-RR) from Helicobacter pylori, has been recently identified. HP-RR is essential for cell growth and does not require the well known phosphorelay scheme. Unphosphorylated HP-RR binds specifically to its own promoter (P1043) and autoregulates the promoter of the tlpB gene (PtlpB). We have determined the structure of HP-RR by NMR and x-ray crystallography, revealing a symmetrical dimer with two functional domains. The molecular topology resembles that of the OmpR/PhoB subfamily, however, the symmetrical dimer is stable even in the unphosphorylated state. The dimer interface, formed by three secondary structure elements (α4-β5-α5), resembles that of the active, phosphorylated forms of ArcA and PhoB. Several conserved residues of the HP-RR dimeric interface deviate from the OmpR/PhoB subfamily, although there are similar salt bridges and hydrophobic patches within the interface. Our findings reveal how a new type of response regulator protein could function as a cell growth-associated regulator in the absence of post-translational modification. Two-component signal transduction systems, commonly found in prokaryotes, typically regulate cellular functions in response to environmental conditions through a phosphorylation-dependent process. A new type of response regulator, hp1043 (HP-RR) from Helicobacter pylori, has been recently identified. HP-RR is essential for cell growth and does not require the well known phosphorelay scheme. Unphosphorylated HP-RR binds specifically to its own promoter (P1043) and autoregulates the promoter of the tlpB gene (PtlpB). We have determined the structure of HP-RR by NMR and x-ray crystallography, revealing a symmetrical dimer with two functional domains. The molecular topology resembles that of the OmpR/PhoB subfamily, however, the symmetrical dimer is stable even in the unphosphorylated state. The dimer interface, formed by three secondary structure elements (α4-β5-α5), resembles that of the active, phosphorylated forms of ArcA and PhoB. Several conserved residues of the HP-RR dimeric interface deviate from the OmpR/PhoB subfamily, although there are similar salt bridges and hydrophobic patches within the interface. Our findings reveal how a new type of response regulator protein could function as a cell growth-associated regulator in the absence of post-translational modification. Two-component systems are the predominant signal transduction systems used by prokaryotes and are frequently involved in the regulation of cellular functions in response to variable environmental conditions. These systems exhibit a phosphorelay process from a sensor histidine kinase to an intracellular response regulator protein (RR), 4The abbreviations used are: RR, response regulator; HP, Helicobacter pylori; NOE, nuclear Overhauser effect. which typically acts as a transcription regulator (1Stock A.M. Robinson V.L. Goudreau P.N. Annu. Rev. Biochem. 2000; 69: 183-215Crossref PubMed Scopus (2533) Google Scholar). The environmental stimulus triggers the autophosphorylation of the transmitter domain of histidine kinase and then the phosphate group is transferred to an aspartate residue in the N-terminal regulatory domain of RR. Phosphorylation induces a conformational change resulting in dimerization of RR and binding to the promoter of target genes. RRs consist of a conserved N-terminal regulatory domain and a variable C-terminal transactivation domain. The transactivation domains can be divided into three major subfamilies based on the homology of their DNA-binding region: the OmpR/PhoB winged-helix domain (2Martinez-Hackert E. Stock A.M. Structure. 1997; 5: 109-124Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 3Kondo H. Nakagawa A. Nishihira J. Nishimura Y. Mizuno T. Tanaka I. Nat. Struct. Biol. 1997; 4: 28-31Crossref PubMed Scopus (85) Google Scholar, 4Okamura H. Hanaoka S. Nagadoi A. Makino K. Nishimura Y. J. Mol. Biol. 2000; 295: 1225-1236Crossref PubMed Scopus (84) Google Scholar), the NarL/FixJ four-helix domain (5Baikalov I. Schroder I. Kaczor-Grzeskowiak M. Grzeskowiak K. Gunsalus R.P. Dickerson R.E. Biochemistry. 1996; 35: 11053-11061Crossref PubMed Scopus (284) Google Scholar, 6Baikalov I. Schroder I. Kaczor-Grzeskowiak M. Cascio D. Gunsalus R.P. Dickerson R.E. Biochemistry. 1998; 37: 3665-3676Crossref PubMed Scopus (66) Google Scholar), and the NtrC ATPase-coupled transcription factors (7Pelton J.G. Kustu S. Wemmer D.E. J. Mol. Biol. 1999; 292: 1095-1110Crossref PubMed Scopus (69) Google Scholar). Other response regulator proteins, which are classified as a fourth subfamily, contain different effector domains, such as enzymes. In most RRs, phosphorylation induces conformational changes in a conserved region including the β4-α4 loop and the α4 helix. In CheY, the phosphate provides hydrogen bonds to the Thr87 O-γ and the Ala88 amide nitrogen, which then provide space for Tyr106 to access the interior, resulting in a stabilizing hydrogen bond between the Tyr106 hydroxyl and the Glu89 carbonyl oxygen in the loop. This conformational change drives the conformational change of the α4-β5-α5 face of the regulatory domain that in turn promotes dimerization. A conserved tyrosine (corresponding to Tyr106 of CheY) is the key residue indicating the status of an RR: the inward position, in which the hydroxyl group points toward the N terminus of helix α4, is found in the active state, whereas the outward position is found in the inactive state. It is well known that dimerization allows or increases DNA binding to the promoter recognition element (8Simonovic M. Volz K. J. Biol. Chem. 2001; 276: 28637-28640Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Recent analysis of the H. pylori genome sequence revealed the presence of four two-component systems and two orphan response regulators (9Tomb J.F. White O. Kerlavage A.R. Clayton R.A. Sutton G.G. Fleischmann R.D. Ketchum K.A. Klenk H.P. Gill S. Dougherty B.A. Nelson K. Quackenbush J. Zhou L. Kirkness E.F. Peterson S. Loftus B. Richardson D. Dodson R. Khalak H.G. Glodek A. McKenney K. Fitzegerald L.M. Lee N. Adams M.D. Hickey E.K. Berg D.E. Gocayne J.D. Utterback T.R. Peterson J.D. Kelley J.M. Cotton M.D. Weidman J.M. Fujii C. Bowman C. Watthey L. Wallin E. Hayes W.S. Borodovsky M. Karp P.D. Smith H.O. Fraser C.M. Venter J.C. Nature. 1997; 388: 539-547Crossref PubMed Scopus (3044) Google Scholar). Based on structural and functional homologies, one of these pairs (analogues of the CheA-CheY proteins) may play a role in regulating random tumbling motions and flagellar rotation in response to signals from chemotaxis receptors (10Armitage J.P. Adv. Microb. Physiol. 1999; 41: 229-289Crossref PubMed Google Scholar, 11Eisenbach M. Mol. Microbiol. 1996; 20: 903-910Crossref PubMed Scopus (112) Google Scholar). The other three pairs of histidine kinase and RR proteins (HP0165-HP0166, HP1364-HP1365, and HP0244-HP0703) and two RR proteins (HP1043 and HP1021) are predicted to be involved in transcriptional regulation (12Beier D. Frank R. J. Bacteriol. 2000; 182: 2068-2076Crossref PubMed Scopus (138) Google Scholar). The hp1043 and hp1021 genes encode orphan RRs, for which no histidine kinases have been identified (12Beier D. Frank R. J. Bacteriol. 2000; 182: 2068-2076Crossref PubMed Scopus (138) Google Scholar, 13Delany I. Spohn G. Rappuoli R. Scarlato V. J. Bacteriol. 2002; 184: 4800-4810Crossref PubMed Scopus (54) Google Scholar). The orphan response regulator HP1043 (HP-RR) has been shown to be essential for the growth of H. pylori (14Schar J. Sickmann A. Beier D. J. Bacteriol. 2005; 187: 3100-3109Crossref PubMed Scopus (71) Google Scholar). HP-RR binds to its own promoter and performs an autoregulatory function (13Delany I. Spohn G. Rappuoli R. Scarlato V. J. Bacteriol. 2002; 184: 4800-4810Crossref PubMed Scopus (54) Google Scholar, 15Muller S. Pflock M. Schar J. Kennard S. Beier D. Microbiol. Res. 2007; 162: 1-14Crossref PubMed Scopus (34) Google Scholar). Interestingly, a recent report revealed that the regulatory domain (HP-RRr) is not phosphorylated in vitro and phosphorylation is not necessary for its function, indicating that HP-RR differs from the well known two-component response regulators. HP-RR has thus been classified as belonging to a new response regulator family (14Schar J. Sickmann A. Beier D. J. Bacteriol. 2005; 187: 3100-3109Crossref PubMed Scopus (71) Google Scholar, 15Muller S. Pflock M. Schar J. Kennard S. Beier D. Microbiol. Res. 2007; 162: 1-14Crossref PubMed Scopus (34) Google Scholar). According to structural studies of the ArcA regulatory domain, it is suggested that the OmpR/PhoB family has a common dimerization mechanism mediated by the α4-β5-α5 interface with the participation of well conserved residues (16Toro-Roman A. Mack T.R. Stock A.M. J. Mol. Biol. 2005; 349: 11-26Crossref PubMed Scopus (109) Google Scholar). However, HP-RR has 5 residue substitutions of 11 conserved residues, which could affect the interactions of the dimer interface. A recent report showed that PhoB adopts two different dimerization modes: the inactive α1-α5 interface dimer and the active α4-β5-α5 interface dimer (17Bachhawat P. Swapna G.V. Montelione G.T. Stock A.M. Structure (Camb.). 2005; 13: 1353-1363Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). These reports raise questions about the structure-function relationships of HP-RR related to dimerization and activation. Previous studies proposed that HP-RR forms a dimer in vivo (13Delany I. Spohn G. Rappuoli R. Scarlato V. J. Bacteriol. 2002; 184: 4800-4810Crossref PubMed Scopus (54) Google Scholar). We have also reported preliminary NMR data showing that HP-RR is a symmetric dimer with two functional domains, an N-terminal regulatory domain (∼14 kDa) and C-terminal DNA-binding/transactivation domain (∼11 kDa) (18Hong E. Jung J.W. Shin J. Kim J.H. Jeon Y.H. Yamazaki T. Lee W. J. Biomol. NMR. 2004; 28: 85-86Crossref PubMed Scopus (5) Google Scholar). Additionally, ultracentrifugation data also support that HP-RR is a stable dimer in solution. 5E. Hong and W. Lee, unpublished results. In this report, we present a detailed three-dimensional structure of HP-RR, a new class of phosphorylation-independent response regulator, using NMR spectroscopy, x-ray crystallography, and site-directed mutagenesis. The structural information presented here will promote understanding of the molecular function of this new type of response regulator. Cloning, Expression, and Purification—Plasmid construction, protein expression, and purification of full-length HP-RR was performed as described (18Hong E. Jung J.W. Shin J. Kim J.H. Jeon Y.H. Yamazaki T. Lee W. J. Biomol. NMR. 2004; 28: 85-86Crossref PubMed Scopus (5) Google Scholar). DNA fragments encoding the N-terminal regulatory domain, HP-RRr (residues 1-119), and the C-terminal effector domain, HP-RRe (residues 120-223), were inserted into pET-21a(+) with a C-terminal (His)6 tag and into pET-21b3-2 (GE Healthcare) with an N-terminal (His)6 tag. These vectors were used to transform the Escherichia coli strain BL21(DE3) (Invitrogen) for fusion protein expression. HP-RRr and HP-RRe were overexpressed and purified according to the methods used for full-length HP-RR (18Hong E. Jung J.W. Shin J. Kim J.H. Jeon Y.H. Yamazaki T. Lee W. J. Biomol. NMR. 2004; 28: 85-86Crossref PubMed Scopus (5) Google Scholar). For x-ray crystallography, selenomethionine-substituted HP-RRr was prepared by transforming E. coli B834(DE3) methionine auxotroph cells (Novagen) with the vector containing HP-RRr and growing the cells in selenomethionine-containing minimal medium. Site-directed Mutagenesis—Mutations were introduced into both HP-RR and HP-RRe using an overlapping PCR strategy. Two PCRs were performed using pET21b3-2 as templates. The PCR products were purified with QIAquick (Qiagen), mixed together, and used as the templates in the proteins were and purified as described was with the DNA was with of kinase and of DNA was purified with a DNA purified HP-RR and HP-RRe were with DNA for in the presence of of DNA were on a in for and by NMR NMR were on a or a with an or a NMR were with the S. G. J. A. J. Biomol. NMR. PubMed Scopus Google and using of Scholar). data of HP-RR were by using K. R. G. K. S. A. 1997; PubMed Scopus Google Scholar). for HP-RRr and HP-RRe were by used for and For structural and were were from and using the G. A. J. Biomol. NMR. 1999; 13: PubMed Scopus Google Scholar). The hydrogen bond were determined from hydrogen on amide were by the in the in and and were using a or in a A.M. S. A. 1998; PubMed Scopus Google Scholar). NMR Structure structure were performed using J. P. J. Struct. 4: PubMed Scopus (84) Google Scholar, P. C. K. J. Mol. Biol. 1997; PubMed Scopus Google Scholar). The were with N. J. PubMed Scopus Google by a of and A of were and with the target function were for structural A of and of HP-RR, and HP-RRe is in The with the were by the R.A. R. J.M. J. Biomol. NMR. 1996; PubMed Scopus Google Scholar). were and using the J. 2001; PubMed Scopus (112) Google Scholar), Chem. S. 2004; Scholar), and R. M. K. J. Mol. 1996; Scopus Google Scholar). The was with the D. S. S. A. 2001; PubMed Scopus Google of bonds one hydrogen from the for residues for HP-RRr and for cell and are the and structure was with of the bond bond Two one hydrogen for residues for HP-RRr and for and are the and structure was with of the in a new and Structure purification and of HP-RRr were as described Hong E. Jeon Kim Lee W. PubMed Scopus Google Scholar). HP-RRr were also the conditions with the of 5 data the were in a which was the A data of the based on was and data were of the in using a The data were then and using the W. A. 1997; 276: Scopus Google Scholar). in the were and used for with the J. Biol. 1999; PubMed Scopus Google Scholar). The were by with the Biol. 2000; PubMed Scopus Google Scholar). The M. A. PubMed Scopus Google was used for the The data were and used for the of the with the Adams P.D. P. J. M. L.M. T. Biol. 1998; PubMed Scopus Google Scholar). for data are in were performed using the J. M. Biochemistry. PubMed Scopus Google Scholar). The of the regulatory domain were with of and The of the regulatory domain were with of and The of the effector domain were with of and The of the effector domain were with of and The was by with and A of was used in NMR data were with S. G. J. A. J. Biomol. NMR. PubMed Scopus Google and with of Scholar). of DNA of recent report proposed that unphosphorylated HP-RR specifically binds to a target DNA containing its own promoter sequence (13Delany I. Spohn G. Rappuoli R. Scarlato V. J. Bacteriol. 2002; 184: 4800-4810Crossref PubMed Scopus (54) Google Scholar), which is the growth the We have determined the promoter DNA in binding to HP-RR based on data from A and HP-RR has well conserved residues in its transactivation domain, (2Martinez-Hackert E. Stock A.M. Structure. 1997; 5: 109-124Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar), PhoB H. Hanaoka S. Nagadoi A. Makino K. Nishimura Y. J. Mol. Biol. 2000; 295: 1225-1236Crossref PubMed Scopus (84) Google Scholar, M. M. Structure (Camb.). 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), and the unphosphorylated HP-RR to its target DNA Interestingly, the protein for binding DNA the position Structure of HP-RR on and HP-RR is a symmetric A of the NMR the structure that it of and in The molecular topology of the HP-RR resembles that of the OmpR/PhoB with a in the absence of The N-terminal regulatory domain forms a dimer and the transactivation domain is to the regulatory domain by a and A of for the dimer interface are between two and However, no interactions between transactivation domains have been The molecular topology of the regulatory domain is an with a by The of HP-RR of two which differs from PhoB. of the NMR of the transactivation domain is in The transactivation domain of an N-terminal and a helix with three and The between and interactions with The two of and a DNA binding The recognition helix is also well The showed that the transactivation domain of two with The loop between and to as a transcription loop for both transcription and with the of the is identified. to family proteins, the DNA-binding domain is also by a conserved hydrophobic the structure of HP-RR with and the of the of the secondary structural are and data not of the from the transactivation and loop the DNA-binding and residues for DNA were by NMR with its The most changes were for the residues of a recognition helix as shown in and D. of structure that the symmetric dimeric interface is by the α4-β5-α5 interface that of The dimeric interface of the HP-RR regulatory domain is similar to that of the active, phosphorylated protein of ArcA (16Toro-Roman A. Mack T.R. Stock A.M. J. Mol. Biol. 2005; 349: 11-26Crossref PubMed Scopus (109) Google and PhoB (17Bachhawat P. Swapna G.V. Montelione G.T. Stock A.M. Structure (Camb.). 2005; 13: 1353-1363Abstract Full Text Full Text PDF PubMed Scopus (112) Google however, the interactions and the hydrophobic are different from that of active which is of four salt bridges and three hydrophobic This be to substitutions in the sequence of the dimeric interface. and in PhoB are by and in HP-RR, In structure of the regulatory domain based on NMR data that HP-RR forms a stable dimer through both and hydrophobic forms three salt and The interactions are by hydrogen bonds of the with the carbonyl oxygen of and the of with the carbonyl oxygen of In the hydrophobic interactions residues and as a major stabilizing for the HP-RR dimer it is that the interactions could as a for the dimerization process of HP-RR, whereas the hydrophobic interactions to dimer of data were within the of protein and an in for the residues in the interface These are by an in the of the residues in the interface These that the of the dimer the of and hydrophobic interactions between these interactions between the residues in the interface such as and in for these residues interactions between the residues such as and in for these residues The is for the regulatory domain, and for the transactivation domain, indicating that the regulatory domain forms a whereas the transactivation domain acts as a The from data of the transactivation domain for residues not The of the residues in the are that of in of secondary However, and indicating that the dimeric interface is of In the are the transactivation domain Site-directed of on structural and sequence analysis for both the regulatory and transactivation domains was performed proteins were prepared to both structural and DNA although are and in the dimeric interface is a or residue in other response regulator proteins, we and to a between The as a by and structure it the dimeric interface. However, the as an that the of the hydrophobic residues is of for hydrophobic which be for protein It is to that even is a it DNA binding whereas a conformational change allows DNA binding in the of the PhoB and response regulator proteins (17Bachhawat P. Swapna G.V. Montelione G.T. Stock A.M. Structure (Camb.). 2005; 13: 1353-1363Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, Kaczor-Grzeskowiak M. Schroder I. Gunsalus R.P. Dickerson R.E. Nat. Struct. Biol. 2002; PubMed Scopus Google Scholar). In of the DNA-binding domain does not affect dimerization of the regulatory domain. together, these data that the HP-RR regulatory domain does not DNA binding of the transactivation domain. The two domains of HP-RR could in The and as indicating that these residues are also for protein However, that be not for protein of the of the dimeric interface in the regulatory domain affect DNA binding and in the DNA recognition helix DNA binding with that of the Interestingly, and the DNA binding indicating that the two residues are for DNA binding Recent studies have proposed that the HP-RR is of binding to its target genes phosphorylation (14Schar J. Sickmann A. Beier D. J. Bacteriol. 2005; 187: 3100-3109Crossref PubMed Scopus (71) Google Scholar, 15Muller S. Pflock M. Schar J. Kennard S. Beier D. Microbiol. Res. 2007; 162: 1-14Crossref PubMed Scopus (34) Google Scholar). Other RRs, such as or can to DNA phosphorylation K. A. S. J. Bacteriol. 1997; PubMed Google Scholar, W. J. Bacteriol. 1997; PubMed Google Scholar, T. R. Rappuoli R. Scarlato V. Mol. Microbiol. 1996; PubMed Scopus Google however, the of DNA binding is by HP-RR does not have the conserved phosphorylation to in E. coli and binds its own promoter sequence Schar (14Schar J. Sickmann A. Beier D. J. Bacteriol. 2005; 187: 3100-3109Crossref PubMed Scopus (71) Google a phosphorylation and showed that it was not for HP-RR function in In showed that HP-RR could not be phosphorylated in For and the regulatory and DNA-binding domains are in space such that the α4-β5-α5 face of the regulatory domain the DNA-binding domain in the state. However, in the of the family protein the of helix of the regulatory domain with the of the DNA-binding domain. there are in the interface of the known RR proteins, the DNA-binding domains of RR proteins are by the regulatory domain. The of DNA binding is through a conformational change by phosphorylation of the regulatory domain that triggers dimerization of RR In we that the two domains of HP-RR no was from NMR This is by showing that the binds to This that dimerization of the regulatory domain in HP-RR does not affect the of the DNA-binding domain. Recent studies of regulatory domains have structural into their function as The unphosphorylated protein could not to DNA the regulatory domain is in with the recognition helix of the domain, resulting the of DNA binding Kaczor-Grzeskowiak M. Schroder I. Gunsalus R.P. Dickerson R.E. Nat. Struct. Biol. 2002; PubMed Scopus Google Scholar). The role of the regulatory domain was also for the protein from S. Goudreau P.N. Stock A.M. S. A. 1998; PubMed Scopus Google Scholar). In both phosphorylation drives a structural of the response regulator protein to DNA The of phosphorylation has been also for response regulator proteins including NtrC D. P. Kustu S. Wemmer D.E. Nature. 1999; PubMed Scopus Google Scholar), K. I. J. Mol. Biol. 1999; PubMed Scopus Google Scholar), and C. L. P. B. J. P. D. J.P. Structure 1999; Full Text Full Text PDF Scopus Google and in the phosphorylation of E. P. Volz K. Biochemistry. 2000; PubMed Scopus Google and J.G. D. Wemmer D.E. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). studies reported that conformational For the α4 helix of the phosphorylated provides a hydrophobic interactions with the other domain of the sequence of HP-RR with the OmpR/PhoB subfamily, it is also found that HP-RR does not have the sequence the that is for the However, the molecular topology of HP-RR resembles that of the OmpR/PhoB and the dimeric interface formed by the α4-β5-α5 face is also similar to the active, phosphorylated of ArcA and PhoB proteins In HP-RR, a major structural in the loop to a detailed with the phosphorylated of ArcA and the of the of the regulatory domain are and In the of key residues for the conformational change is also similar to that of the PhoB protein not to in in adopts an inward position, indicating that HP-RR is in an active This data the that the conserved phosphorylation is in HP-RR (14Schar J. Sickmann A. Beier D. J. Bacteriol. 2005; 187: 3100-3109Crossref PubMed Scopus (71) Google it is in an active The of the domain DNA binding is that a conformational change does not DNA binding in binding This is by the that the NMR of the HP-RRe are to of the full-length protein not showed that the of the binding is from the to Based on of HP-RR, both regulatory and transactivation domains not or a between two domains in solution. motions within residues also support that the transactivation domain a DNA Based on we a by molecular Our structure showed that HP-RR has a the two domains PhoB M. M. Structure (Camb.). 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). that the regulatory domains a symmetric dimer mediated by the α4-β5-α5 face with a it is that HP-RR to DNA in a which is with the that the DNA sequence of the binding is an we have a understanding about how this response regulator protein could function as a cell growth-associated regulator a phosphorylation A recent report showed that of HP-RR is both on the and post-translational S. Pflock M. Schar J. Kennard S. Beier D. Microbiol. Res. 2007; 162: 1-14Crossref PubMed Scopus (34) Google Scholar). However, the as to how the regulation of the protein can be We that HP-RR could its phosphorylation the structure of the regulatory domain resembles that of the active, phosphorylated of ArcA and PhoB. Our how HP-RR from the well known two-component systems functions in the absence of post-translational modification. We and Kim for and of