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• W2026677195 abstract "The chief mechanism used by bacteria for sensing their environment is based on two conserved proteins: a sensor histidine kinase (HK) and an effector response regulator (RR). The signal transduction process involves highly conserved domains of both proteins that mediate autokinase, phosphotransfer, and phosphatase activities whose output is a finely tuned RR phosphorylation level. Here, we report the structure of the complex between the entire cytoplasmic portion of Thermotoga maritima class I HK853 and its cognate, RR468, as well as the structure of the isolated RR468, both free and BeF3− bound. Our results provide insight into partner specificity in two-component systems, recognition of the phosphorylation state of each partner, and the catalytic mechanism of the phosphatase reaction. Biochemical analysis shows that the HK853-catalyzed autokinase reaction proceeds by a cis autophosphorylation mechanism within the HK subunit. The results suggest a model for the signal transduction mechanism in two-component systems. The chief mechanism used by bacteria for sensing their environment is based on two conserved proteins: a sensor histidine kinase (HK) and an effector response regulator (RR). The signal transduction process involves highly conserved domains of both proteins that mediate autokinase, phosphotransfer, and phosphatase activities whose output is a finely tuned RR phosphorylation level. Here, we report the structure of the complex between the entire cytoplasmic portion of Thermotoga maritima class I HK853 and its cognate, RR468, as well as the structure of the isolated RR468, both free and BeF3− bound. Our results provide insight into partner specificity in two-component systems, recognition of the phosphorylation state of each partner, and the catalytic mechanism of the phosphatase reaction. Biochemical analysis shows that the HK853-catalyzed autokinase reaction proceeds by a cis autophosphorylation mechanism within the HK subunit. The results suggest a model for the signal transduction mechanism in two-component systems. The predominant prokaryotic signaling proteins are members of two-component signal transduction systems (TCS) (reviewed by Stock et al., 2000Stock A.M. Robinson V.L. Goudreau P.N. Two-component signal transduction.Annu. Rev. Biochem. 2000; 69: 183-215Crossref PubMed Scopus (2301) Google Scholar). Although very widespread in bacteria, where these systems were reported originally, TCSs are found in all domains of life except for animals. The signaling pathways defined by TCSs typically consist of four steps. First, a homodimeric sensor histidine kinase (HK) is autophosphorylated by ATP at a histidine residue. Second, the phosphoryl group is relayed to an aspartate on a cognate response regulator (RR). Third, the phosphorylated RR (P∼RR) interacts with genes or protein targets, triggering cellular responses. Finally, signaling is terminated by P∼RR dephosphorylation by an intrinsic or HK-induced P∼RR autophosphatase activity (Stock et al., 2000Stock A.M. Robinson V.L. Goudreau P.N. Two-component signal transduction.Annu. Rev. Biochem. 2000; 69: 183-215Crossref PubMed Scopus (2301) Google Scholar). The signal influences P∼RR levels by modulating autokinase and/or phosphatase activities (Russo and Silhavy, 1993Russo F.D. Silhavy T.J. The essential tension: opposed reactions in bacterial two-component regulatory systems.Trends Microbiol. 1993; 1: 306-310Abstract Full Text PDF PubMed Scopus (87) Google Scholar). A single bacterial cell can contain as many as 200–300 HK-RR pairs, but normally, each HK has only one or two cognate RRs, revealing exquisite partner specificity within each HK-RR pair (Laub and Goulian, 2007Laub M.T. Goulian M. Specificity in two-component signal transduction pathways.Annu. Rev. Genet. 2007; 41: 121-145Crossref PubMed Scopus (501) Google Scholar). Most HKs are homodimeric membrane receptors. Typically, they have a variable, N-terminal, extracellular sensor domain that is connected via transmembrane helices to the C-terminal, cytoplasmic portion, which contains the catalytic machinery. In the class I HKs, which predominate in prokaryotes (Dutta et al., 1999Dutta R. Qin L. Inouye M. Histidine kinases: diversity of domain organization.Mol. Microbiol. 1999; 34: 633-640Crossref PubMed Scopus (198) Google Scholar), the catalytic elements consist of an elongated two-helix dimerization and histidine-containing phosphotransfer (DHp) domain, which is connected to a C-terminal catalytic and ATP-binding (CA) globular domain that phosphorylates the histidine in the first step of the cascade (Stock et al., 2000Stock A.M. Robinson V.L. Goudreau P.N. Two-component signal transduction.Annu. Rev. Biochem. 2000; 69: 183-215Crossref PubMed Scopus (2301) Google Scholar). RRs typically consist of a highly conserved receiver domain that hosts the phosphoacceptor aspartate and an effector domain that interacts with targets. However, the effector domain may be missing and its functions taken over by the receiver domain, as in the RR studied here (Stock et al., 2000Stock A.M. Robinson V.L. Goudreau P.N. Two-component signal transduction.Annu. Rev. Biochem. 2000; 69: 183-215Crossref PubMed Scopus (2301) Google Scholar). Despite there being abundant biochemical and mutational information on TCS signaling, this process is not well understood in structural terms. The atomic resolution structure of the complete cytoplasmic portion of the class I HK that is the product of gene TM0853 of Thermotoga maritima, HK853CP, revealed the domain architecture of the intracellular part of the class I HK dimer (Marina et al., 2005Marina A. Waldburger C.D. Hendrickson W.A. Structure of the entire cytoplasmic portion of a sensor histidine-kinase protein.EMBO J. 2005; 24: 4247-4259Crossref PubMed Scopus (224) Google Scholar). In this architecture, the globular CA domains protrude on opposite sides of the HK dimer helical stem. This stem is composed of an N-terminal coiled coil and a four-helix bundle. The localization of the CA domains at the coiled coil-DHp domain junction, far from the phosphoacceptor His260, prevented any conclusions about how the autokinase reaction takes place in HKs from being drawn. More recently, the structure of the complex formed by the two-component pair ThkA-TrrA of T. maritima was determined (Yamada et al., 2006Yamada S. Akiyama S. Sugimoto H. Kumita H. Ito K. Fujisawa T. Nakamura H. Shiro Y. The signaling pathway in histidine kinase and the response regulator complex revealed by X-ray crystallography and solution scattering.J. Mol. Biol. 2006; 362: 123-139Crossref PubMed Scopus (25) Google Scholar). While the resolution (4.2 Å) of this structure allowed the mapping of the domains, it did not permit detailed analysis of protein-protein interactions and of the signaling process. The structure of the Spo0B-Spo0F complex of Bacillus subtilis was determined at atomic resolution, (Zapf et al., 2000Zapf J. Sen U. Madhusudan Hoch J.A. Varughese K.I. A transient interaction between two phosphorelay proteins trapped in a crystal lattice reveals the mechanism of molecular recognition and phosphotransfer in signal transduction.Structure. 2000; 8: 851-862Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), but Spo0B has no autokinase activity, and, therefore, the relevance of this complex for understanding HK-RR pairs is uncertain. High-resolution structures of functional complexes of typical class I HK-RR pairs must be determined to understand the signal transduction process. We now report the structure at 2.8 Å resolution of the complex formed by T. maritima HK853CP and its cognate response regulator RR468 (encoded by gene TM0468). This structure, which may represent a paradigm for the complexes formed by any class I HK with its cognate RR, is shown to be functionally relevant by mutational studies and has allowed clarification of the architecture of the complex, unveiling HK-RR interactions and explaining structurally the exquisite partner specificity of HK-RR pairs. We also determine here the structure of free RR468 either in the absence of ligands (fRR468) or bound to the phosphoryl group mimic BeF3− (BeF3−-RR468) (Lee et al., 2001Lee S.Y. Cho H.S. Pelton J.G. Yan D. Berry E.A. Wemmer D.E. Crystal structure of activated CheY. Comparison with other activated receiver domains.J. Biol. Chem. 2001; 276: 16425-16431Crossref PubMed Scopus (132) Google Scholar). The comparison of these structures and of that reported previously for isolated HK853CP (Marina et al., 2005Marina A. Waldburger C.D. Hendrickson W.A. Structure of the entire cytoplasmic portion of a sensor histidine-kinase protein.EMBO J. 2005; 24: 4247-4259Crossref PubMed Scopus (224) Google Scholar) with the structures of the RR and HK within the complex reveals important movements in both components and sheds light on phosphorylation state recognition and on the mechanisms of the autokinase and phosphatase reactions. One suggestion that derives from these movements is that the autokinase reaction takes place in cis (that is, ATP bound to one subunit phosphorylates the His residue of this same subunit). This mechanism is confirmed experimentally here for HK853 and for the Staphylococcus aureus HK, PhoR. Thus, the current thinking (Stock et al., 2000Stock A.M. Robinson V.L. Goudreau P.N. Two-component signal transduction.Annu. Rev. Biochem. 2000; 69: 183-215Crossref PubMed Scopus (2301) Google Scholar) that the autokinase reaction occurs universally in trans must be revised. We propose a working model for signal transduction that may guide further research into TCS function. For determination of the structure of the complex formed by T. maritima HK853CP and its cognate response regulator RR468, crystals of this complex prepared in the presence of the MgATP analog MgAMPPNP (Casino et al., 2007Casino P. Fernandez-Alvarez A. Alfonso C. Rivas G. Marina A. Identification of a novel two component system in Thermotoga maritima. Complex stoichiometry and crystallization.Biochim. Biophys. Acta. 2007; 1774: 603-609Crossref PubMed Scopus (7) Google Scholar) were subjected to X-ray synchrotron diffraction. A complete dataset was obtained at 2.8 Å resolution (Table 1). To determine the crystallographic phases, we exploited the anomalous diffraction of Se on crystals of the complex of SeMet-substituted proteins, using the multiwavelength anomalous diffraction approach (MAD) (Hendrickson et al., 1990Hendrickson W.A. Horton J.R. LeMaster D.M. Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure.EMBO J. 1990; 9: 1665-1672Crossref PubMed Scopus (986) Google Scholar). One molecule of the complex formed by one HK853CP dimer and two RR468 monomers was found in the asymmetric unit of the crystals (Figures 1A and 1B, and Figure S1A available online). The complex, with the expected (Casino et al., 2007Casino P. Fernandez-Alvarez A. Alfonso C. Rivas G. Marina A. Identification of a novel two component system in Thermotoga maritima. Complex stoichiometry and crystallization.Biochim. Biophys. Acta. 2007; 1774: 603-609Crossref PubMed Scopus (7) Google Scholar) equimolar stoichiometry, exhibits twofold symmetry. Each CA domain of the HK853CP dimer has one bound molecule of ADPβN (produced by AMPPNP hydrolysis [Marina et al., 2005Marina A. Waldburger C.D. Hendrickson W.A. Structure of the entire cytoplasmic portion of a sensor histidine-kinase protein.EMBO J. 2005; 24: 4247-4259Crossref PubMed Scopus (224) Google Scholar]), whereas each RR molecule binds one sulfate approximately at the position expected for the phosphate in P∼RR468 (Figure 1A). In an attempt to characterize the changes associated with complex formation and with RR phosphorylation, we determined crystal structures of free RR468 without ligands (fRR468), or of RR468 bound to the phosphoryl group mimic (Lee et al., 2001Lee S.Y. Cho H.S. Pelton J.G. Yan D. Berry E.A. Wemmer D.E. Crystal structure of activated CheY. Comparison with other activated receiver domains.J. Biol. Chem. 2001; 276: 16425-16431Crossref PubMed Scopus (132) Google Scholar) BeF3− (BeF3−-RR468), at 2.0 and 1.8 Å resolution, respectively (Table 1, Figure S2; see also the Supplemental Experimental Procedures). The BeF3− was bound at the expected position for the phosphoryl group at the phosphoacceptor aspartate, Asp53 (Figure S2). The analysis of these three protein structures has clarified the architecture of the complex and has shed light on some of the reactions occurring in TCSs.Table 1Data Collection and Refinement StatisticsfRR468BeF3−-RR468HK853CP-RR468Se-Met-labeled HK853CP-RR468Crystal ParametersSpace groupP212121P65C2C2C2C2Cell dimensions (Å)a = 34.6, b = 55.5, c = 58.4a = 155.2, b = 155.2, c = 34.5a = 184.0, b = 90.7, c = 69.0a = 185.6, b = 92.6, c = 70.2a = 185.6, b = 92.6, c = 70.2a = 185.6, b = 92.6, c = 70.2Angles (°)α = β = γ = 90°α = β = 90°, γ = 120°α = γ = 90, β = 108.4α = γ = 90, β = 109.0Data Collectionλ (Å)1.0080.9790.9310.979350.979500.95298Resolution (Å)22.1−2.0 (2.11−2.00)29.3−1.8 (1.9−1.8)43.64−2.8 (2.95−2.80)63.88−2.92 (3.00−2.92)55.90−2.92 (3.00−2.92)55.90−2.95 (3.03−2.95)Rmerge (%)aRmerge = (Σ|I−<I>|)/ΣI, where <I> is the average intensity over symmetry equivalent.8.8 (38.3)7.3 (33.3)7.2 (43.3)9.3 (47.1)10.8 (49.2)7.8 (42.7)I/δ(I)14.6 (3.9)23.6 (6.4)16.2 (2.8)13.7 (1.7)12.2 (1.5)15 (1.9)Number of reflections observed/unique40,193/7951 (5,812/1,143)479,700/44,647 (60,798/6,412)116,062/26,588 (17,132/3,875)98,845/23,723 (5,470/1,479)98,896/23,769 (5,738/1,503)95,472/22,826 (5,052/1,306)Completeness99.3 (100)99.7 (99.2)99.7 (100.0)96.9 (83.0)97 (84.4)96.4 (78.0)Redundancy5.1 (5.1)10.7 (9.5)4.4 (4.4)4.2 (3.7)4.2 (3.8)4.2 (3.9)Anomalous Completeness95.9 (74.6)96.3 (80.2)95.9 (73.6)Anomalous redundancy2.1 (2.0)2.1 (2.0)2.1 (2.0)RefinementRwork (%)bRwork = Σ ‖ Fo|−|Fc ‖ /Σ|Fo|. Rfree is equivalent to Rwork but calculated with 5% random data omitted from the refinement.21.0618.523.36Rfree (%)bRwork = Σ ‖ Fo|−|Fc ‖ /Σ|Fo|. Rfree is equivalent to Rwork but calculated with 5% random data omitted from the refinement.25.9523.728.44Number of protein atoms82838855714Number of ligand atoms2077Number of water molecules245653162Rmsd bond/angle (Å/°)0.02/1.600.01/1.20.018/1.55Media B factor (Å2), all atoms34.617.074.9Ramachandran map, favored/outlier (%)93.5/0.096.7/0.986.5/0.0Values in parentheses correspond to the highest-resolution shell.a Rmerge = (Σ|I−<I>|)/ΣI, where <I> is the average intensity over symmetry equivalent.b Rwork = Σ ‖ Fo|−|Fc ‖ /Σ|Fo|. Rfree is equivalent to Rwork but calculated with 5% random data omitted from the refinement. Open table in a new tab Values in parentheses correspond to the highest-resolution shell. The crystal structure of the complex shows that the RR468 molecules sit on opposite sides of the DHp domain dimer, below the phosphorylatable His260 (Figures 1A and 1B). In the orientation adopted by the RR468 molecules, the phosphoacceptor Asp53 is aligned with His260, favoring phosphoryl group transfer reactions involving both residues (Figure 1A). This alignment is possible because each RR molecule, which presents the characteristic αβα sandwich fold of the receiver domain of RRs (Stock et al., 2000Stock A.M. Robinson V.L. Goudreau P.N. Two-component signal transduction.Annu. Rev. Biochem. 2000; 69: 183-215Crossref PubMed Scopus (2301) Google Scholar), clings to the helical stem of one HK subunit by clamping the DHp domain α1 helix between first helix (α1) and last loop (β5-α5) of the RR molecule (Figure 1C). In this way, helix α1 of the RR is inserted between the two DHp domain helices (α1 and α2), thus extending the bundle of four helices formed by both DHp domains to six helices (Figure 1C). Each RR molecule also makes lateral contacts with both HK subunits, using two mobile loops that change their orientation depending on whether the RR is phosphorylated or not (see below). One of these loops (β3-α3 loop) protrudes laterally and contacts the CA domain of the same HK subunit that interacts with this RR molecule at the stem level (Figures 1A and 1B). The β3-α3 loop glues together two CA domain loops (α4-β4 loop and ATP lid) that flank the entry of the nucleotide site, locking in place the bound ADPβN molecule (Figure 1D). These RR molecule-CA domain contacts possibly account for the observation that ATP binding influences the affinity of the HK EnvZ for its partner RR, OmpR (Yoshida et al., 2002Yoshida T. Cai S. Inouye M. Interaction of EnvZ, a sensory histidine kinase, with phosphorylated OmpR, the cognate response regulator.Mol. Microbiol. 2002; 46: 1283-1294Crossref PubMed Scopus (45) Google Scholar). The other RR mobile loop involved in lateral contacts (β4-α4 loop) interacts with the C-end of the DHp domain and with the DHp-CA domain linker belonging to the other HK subunit (HK∗) of the dimer (Figures 1A and 1E). These interactions were inferred previously on the basis of biochemical and mutational data for EnvZ-OmpR (Hsing et al., 1998Hsing W. Russo F.D. Bernd K.K. Silhavy T.J. Mutations that alter the kinase and phosphatase activities of the two-component sensor EnvZ.J. Bacteriol. 1998; 180: 4538-4546Crossref PubMed Google Scholar, Zhu et al., 2000Zhu Y. Qin L. Yoshida T. Inouye M. Phosphatase activity of histidine kinase EnvZ without kinase catalytic domain.Proc. Natl. Acad. Sci. USA. 2000; 97: 7808-7813Crossref PubMed Scopus (100) Google Scholar). Collectively, the contacts between both proteins are not very extensive, as expected for a transient complex, burying ∼1175 Å2 of HK dimer surface per RR molecule. The largest part (885 Å2) of this surface corresponds to the contacts involving the DHp stem below His260. The lateral contacts of RR468 with the CA domain and the DHp-CA domain linker are smaller (150 and 140 Å2, respectively). The comparison of the HK853CP structures in the present complex and in the free form (Figure S1) (Marina et al., 2005Marina A. Waldburger C.D. Hendrickson W.A. Structure of the entire cytoplasmic portion of a sensor histidine-kinase protein.EMBO J. 2005; 24: 4247-4259Crossref PubMed Scopus (224) Google Scholar) suggests not only that HK autophosphorylation occurs in cis, but also possible ways for triggering this autophosphorylation. One key difference between these two structures is the position of the CA domains. In the complex, these domains are displaced toward the phosphoacceptor His260 of the same subunit (Figure 2A) because of a 37° rotation around an axis passing through the DHp domain-CA domain linker (Figures 2A and 2B). This close approach of the phosphoryl donor and acceptor of the same subunit suggests that His260 phosphorylation takes place in cis (Figure 2A and see below). Nevertheless, His260 phosphorylation should be precluded in the present complex because the nucleotide is buried under the ATP lid, which, as already indicated, is fixed in a closed position as a result of its interactions with bound RR468 (Figure 1D). In contrast, the ATP lid was disordered in the free HK853CP structure (Marina et al., 2005Marina A. Waldburger C.D. Hendrickson W.A. Structure of the entire cytoplasmic portion of a sensor histidine-kinase protein.EMBO J. 2005; 24: 4247-4259Crossref PubMed Scopus (224) Google Scholar). Since the RR468 molecules of this complex appear to represent P∼RR (see below), the RR-triggered ATP lid closure may be a way of preventing futile cycles of P∼RR dephosphorylation and immediate rephosphorylation by the HK. The structural comparisons also suggest possible ways by which extracellular signals can affect the position of the CA domain. The 12 residues at the N terminus of HK853CP are disordered in the complex, whereas they participate in the coiled coil formed by the initial three turns of helix α1 in the free HK (Figures 2B and S1) (Marina et al., 2005Marina A. Waldburger C.D. Hendrickson W.A. Structure of the entire cytoplasmic portion of a sensor histidine-kinase protein.EMBO J. 2005; 24: 4247-4259Crossref PubMed Scopus (224) Google Scholar). A rotation of these helices of up to 20° (value for the first turn of helix α1 that is visible in the complex) (Figure 2C) appears to underlie the unfolding of these initial three turns. A similar 26° cogwheel rotation was reported in HAMP domains in the transmission of the extracellular signal to the cell interior (Hulko et al., 2006Hulko M. Berndt F. Gruber M. Linder J.U. Truffault V. Schultz A. Martin J. Schultz J.E. Lupas A.N. Coles M. The HAMP domain structure implies helix rotation in transmembrane signaling.Cell. 2006; 126: 929-940Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). A concerted and inverse twist of the α2 helices is also observed in the complex, reflecting the required readjustments of DHp dimer packing to accommodate the helix α1 movements (Figure 2C). These packing changes expose parts of the hydrophobic core of the DHp dimer (particularly the side chains of residues F254, I255, and F312), promoting novel interactions with hydrophobic residues of the CA domain ATP lid (particularly F428 and L444) of the same subunit (Figure S3), which result in the changed position of the CA domain. These CA domain changes may be a prerequisite for autophosphorylation, and the cogwheel motion of the helices may connect the extracellular signal to the HK function (see Discussion). RRs exhibit different conformations depending on whether they are phosphorylated or not (Robinson et al., 2000Robinson V.L. Buckler D.R. Stock A.M. A tale of two components: a novel kinase and a regulatory switch.Nat. Struct. Biol. 2000; 7: 626-633Crossref PubMed Scopus (171) Google Scholar). RR468 in the present complex presents the P∼RR conformation (Figures 2D and S4). This is reflected in the excellent superimposition (Figure 2D) of its structure with that of BeF3−-RR468 (rmsd for the superimposition of all Cα atoms, 0.7 Å). In contrast, the superimposition with fRR468, which represents the non-phosphorylated form, is poorer (rmsd, 1.9 Å) (Figure 2D). The conformational traits that characterize P∼RRs include the hydrogen bonding of the phosphate to a conserved Thr/Ser, an associated characteristic bending of the β4-α4 loop, and the burying of a hydrophobic residue of strand β5 that is exposed in the non-phosphorylated form of the RR. All of these features are present in the RR468 molecules of the complex and in BeF3−-RR468. In the complex, the phosphate is replaced by a sulfate (Figures 2D, 2E, S4, and S5), and the residue involved in the hydrogen bond is T83, whereas the hydrophobic buried residue is V105 (Figure 2D). The sulfate is sandwiched between the γO of the phosphoacceptor Asp53 and the ɛN of His260 of HK853, occupying an essentially equivalent location and replicating the contacts of the BeF3− in BeF3−-RR468 (Figures 2D, 2E, and S5). These contacts include, in addition to the indicated hydrogen bond, the coordination to a Mg that is typically found in the active centers of P∼RRs (Lewis et al., 1999Lewis R.J. Brannigan J.A. Muchova K. Barak I. Wilkinson A.J. Phosphorylated aspartate in the structure of a response regulator protein.J. Mol. Biol. 1999; 294: 9-15Crossref PubMed Scopus (136) Google Scholar) (Figures 2D and 2E). In these RR468 molecules, the β3-α3 loop also has a bent conformation found in P∼RR molecules (Figures 2D and S4). This conformation is known to be important for P∼Asp stability (Zapf et al., 1998Zapf J. Madhusudan M. Grimshaw C.E. Hoch J.A. Varughese K.I. Whiteley J.M. A source of response regulator autophosphatase activity: the critical role of a residue adjacent to the Spo0F autophosphorylation active site.Biochemistry. 1998; 37: 7725-7732Crossref PubMed Scopus (50) Google Scholar) and allows the accommodation of the bound sulfate in RR468. In contrast, the sulfate cannot be accommodated in fRR468, given its different β3-α3 loop conformation (Figure S6). This characteristic bending of the β3-α3 and β4-α4 loops is essential for the RR468 molecules of the complex to come into contact with the DHp-CA linker and the CA domain of the HK (Figures 1A, 1D, and 1E). Therefore, it is clear that this complex must correspond to the binding of phosphorylated RR468 to HK853. In many TCSs like HK853, the HK can catalyze the dephosphorylation of the P∼RR, in a process that involves the phosphoacceptor His (Stock et al., 2000Stock A.M. Robinson V.L. Goudreau P.N. Two-component signal transduction.Annu. Rev. Biochem. 2000; 69: 183-215Crossref PubMed Scopus (2301) Google Scholar). The P∼Asp of P∼RR468 is highly stable in the absence of HK853 (t1/2 for dephosphorylation at 4°C, ∼19 hr) even at the elevated living temperature of T. maritima (t1/2 at 80°C, ∼2 min). However, P∼RR468 was dephosphorylated instantaneously in the presence of wild-type HK853, but not of in the presence of its H260A mutant (Figure S7 and the Supplemental Results). HK-activated dephosphorylation is not believed to be a simple reversal of RR phosphorylation (Stock et al., 2000Stock A.M. Robinson V.L. Goudreau P.N. Two-component signal transduction.Annu. Rev. Biochem. 2000; 69: 183-215Crossref PubMed Scopus (2301) Google Scholar). In the dephosphorylation reaction, the phosphorylatable His would act as an activating base, rendering one water molecule a strong nucleophile for attacking the phosphorus atom of the P∼Asp (Hsing and Silhavy, 1997Hsing W. Silhavy T.J. Function of conserved histidine-243 in phosphatase activity of EnvZ, the sensor for porin osmoregulation in Escherichia coli.J. Bacteriol. 1997; 179: 3729-3735Crossref PubMed Google Scholar, Lukat et al., 1991Lukat G.S. Lee B.H. Mottonen J.M. Stock A.M. Stock J.B. Roles of the highly conserved aspartate and lysine residues in the response regulator of bacterial chemotaxis.J. Biol. Chem. 1991; 266: 8348-8354Abstract Full Text PDF PubMed Google Scholar). In this mechanism, the water molecule would be interposed between the activating N atom of the imidazole ring and the targeted phosphorus atom. The active center of the present complex agrees with this mechanism of P∼RR dephosphorylation. One O atom of the sulfate sits between the sulfur center and the ɛN of His260 and is aligned with these two atoms. Thus, it may correspond to the proposed water molecule (Figure 2E). In the active center of the complex, the distance between the ɛN atom of His260 and the δO atom of Asp53 (7.9 Å) is 2.6 Å greater than the separation (5.3 Å) between the corresponding atoms in the BeF3−-containing Spo0B-Spo0F complex (Figure S8), which is believed to represent the His-Asp phosphoryl transfer (Varughese et al., 2006Varughese K.I. Tsigelny I. Zhao H. The crystal structure of beryllofluoride Spo0F in complex with the phosphotransferase Spo0B represents a phosphotransfer pretransition state.J. Bacteriol. 2006; 188: 4970-4977Crossref PubMed Scopus (50) Google Scholar). This extra space would be sufficient to accommodate the attacking water molecule. Other sulfate-interacting groups from RR468 (Figure 2E) provide positive charges (ξN of K105; Mg2+) and donate hydrogen bonds (N atoms of I54, M55, and A84 and γO of T83) that should neutralize or shield the negative charge on the phosphate, assisting phosphoryl transfer to the water molecule. Therefore, the entire active center of the complex appears well suited for dephosphorylation. To test the occurrence of the present complex in solution and to corroborate its functionality, we performed site-directed mutagenesis experiments in which we replaced residues predicted by the structure to be important for HK-RR interaction. The HK853 mutations T267D and Y272D and the RR468 mutations I17N and F20N affect residues of helix α1 of each partner (Figure 3A) that are involved in the six-helix bundle formed in the complex. These mutations were not detrimental to the stability or the purification of the proteins (data not shown). The mutant proteins were competent for HK853CP autophosphorylation and RR468 phosphorylation by acetylphosphate (Figure 3), consistent with their proper folding. However, native electrophoresis of HK853CP-RR468 mixtures revealed that Y272D, I17N, and F17N prevented HK-RR complex formation and that T267D drastically decreased complex stability (Figure 3B). In agreement with these effects, each of the four mutations abolished or greatly hampered both the phosphoryl transfer from the HK to the RR (determined as RR468-triggered dephosphorylation of 32P∼HK853CP) and the HK-catalyzed dephosphorylation of P∼RR468 (assessed by electrophoretic quantification of the phosphorylated fraction of RR468). Under the same conditions, these reactions were essentially complete when the wild-type proteins were used (Figures 3C and 3D). Furthermore, the residues identified in elegant mutagenesis and protein engineering experiments to determine the specificity of EnvZ for its cognate RR (Skerker et al., 2008Skerker J.M. Perchuk B.S. Siryaporn A. Lubin E.A. Ashenberg O. Goulian M. Laub M.T. Rewiring the specificity of two-component signal transduction systems.Cell. 2008; 133: 1043-1054Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar) are fully consistent with the structure of the present complex (see Figure 4 and the Discussion). In summary, the mutational data support the relevance of our present structure for understanding the functionality of complex formation in vivo, not only for the HK853-RR468 pair, but also for other TCSs.Figure 4Sequence and Structure of the HK853CP-RR468 InterfaceShow full caption(A) Structural alignment of HK853 with EnvZ (structures of its two domains given in PDB files 1JOY and IBXD) and with helices α1 and α2 of Spo0B (structure taken from the complex with Spo0F, PDB file 1F51).(B) Structural alignment of RR468 with Spo0F (PDB file 1F51). Secondary structure elements of HK853CP and RR468 are shown above" @default.
• W2026677195 created "2016-06-24" @default.
• W2026677195 creator A5058914818 @default.
• W2026677195 creator A5069419601 @default.
• W2026677195 creator A5073336560 @default.
• W2026677195 date "2009-10-01" @default.
• W2026677195 modified "2023-10-18" @default.
• W2026677195 title "Structural Insight into Partner Specificity and Phosphoryl Transfer in Two-Component Signal Transduction" @default.
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