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• W1991438329 abstract "The crystal structure of BeF3−-activated CheY, with manganese in the magnesium binding site, was determined at 2.4-Å resolution. BeF3− bonds to Asp57, the normal site of phosphorylation, forming a hydrogen bond and salt bridge with Thr87 and Lys109, respectively. The six coordination sites for manganese are satisfied by a fluorine of BeF3−, the side chain oxygens of Asp13 and Asp57, the carbonyl oxygen of Asn59, and two water molecules. All of the active site interactions seen for BeF3−-CheY are also observed in P-Spo0Ar. Thus, BeF3− activates CheY as well as other receiver domains by mimicking both the tetrahedral geometry and electrostatic potential of a phosphoryl group. The aromatic ring of Tyr106 is found buried within a hydrophobic pocket formed by β-strand β4 and helix H4. The tyrosine side chain is stabilized in this conformation by a hydrogen bond between the hydroxyl group and the backbone carbonyl oxygen of Glu89. This hydrogen bond appears to stabilize the active conformation of the β4/H4 loop. Comparison of the backbone coordinates for the active and inactive states of CheY reveals that only modest changes occur upon activation, except in the loops, with the largest changes occurring in the β4/H4 loop. This region is known to be conformationally flexible in inactive CheY and is part of the surface used by activated CheY for binding its target, FliM. The pattern of activation-induced backbone coordinate changes is similar to that seen in FixJr. A common feature in the active sites of BeF3−-CheY, P-Spo0Ar, P-FixJr, and phosphono-CheY is a salt bridge between Lys109 Nζ and the phosphate or its equivalent, beryllofluoride. This suggests that, in addition to the concerted movements of Thr87 and Tyr106(Thr-Tyr coupling), formation of the Lys109-PO3− salt bridge is directly involved in the activation of receiver domains generally.1FQW The crystal structure of BeF3−-activated CheY, with manganese in the magnesium binding site, was determined at 2.4-Å resolution. BeF3− bonds to Asp57, the normal site of phosphorylation, forming a hydrogen bond and salt bridge with Thr87 and Lys109, respectively. The six coordination sites for manganese are satisfied by a fluorine of BeF3−, the side chain oxygens of Asp13 and Asp57, the carbonyl oxygen of Asn59, and two water molecules. All of the active site interactions seen for BeF3−-CheY are also observed in P-Spo0Ar. Thus, BeF3− activates CheY as well as other receiver domains by mimicking both the tetrahedral geometry and electrostatic potential of a phosphoryl group. The aromatic ring of Tyr106 is found buried within a hydrophobic pocket formed by β-strand β4 and helix H4. The tyrosine side chain is stabilized in this conformation by a hydrogen bond between the hydroxyl group and the backbone carbonyl oxygen of Glu89. This hydrogen bond appears to stabilize the active conformation of the β4/H4 loop. Comparison of the backbone coordinates for the active and inactive states of CheY reveals that only modest changes occur upon activation, except in the loops, with the largest changes occurring in the β4/H4 loop. This region is known to be conformationally flexible in inactive CheY and is part of the surface used by activated CheY for binding its target, FliM. The pattern of activation-induced backbone coordinate changes is similar to that seen in FixJr. A common feature in the active sites of BeF3−-CheY, P-Spo0Ar, P-FixJr, and phosphono-CheY is a salt bridge between Lys109 Nζ and the phosphate or its equivalent, beryllofluoride. This suggests that, in addition to the concerted movements of Thr87 and Tyr106(Thr-Tyr coupling), formation of the Lys109-PO3− salt bridge is directly involved in the activation of receiver domains generally.1FQW molecular replacement Two-component signal transduction systems control a variety of cellular processes, including chemotaxis and expression of some genes in bacteria and lower eukaryotes (1Nixon B.T. Ronson C.W. Ausubel F.M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7850-7854Crossref PubMed Scopus (254) Google Scholar, 2Ota I.M. Varshavsky A. Science. 1993; 262: 566-569Crossref PubMed Scopus (362) Google Scholar, 3Parkinson J.S. Kofoid E.C. Annu. Rev. Genet. 1992; 26: 71-112Crossref PubMed Scopus (1239) Google Scholar, 4Stock A.M. Robinson V.L. Goudreau P.N. Annu. Rev. Biochem. 2000; 69: 183-215Crossref PubMed Scopus (2416) Google Scholar). Signal transduction is mediated by phosphotransfer from a histidine kinase to a conserved aspartyl residue of a response regulator. To date, more than 300 response regulators have been identified based on homology in a domain of ∼120 residues, commonly referred to as a receiver or regulatory domain (5Grebe T.W. Stock J.B. Adv. Microb. Physiol. 1999; 41: 139-227Crossref PubMed Google Scholar). The structures of several receiver domains have been solved (4Stock A.M. Robinson V.L. Goudreau P.N. Annu. Rev. Biochem. 2000; 69: 183-215Crossref PubMed Scopus (2416) Google Scholar). They all have a similar (β/α)5 fold with an active site comprised of five highly conserved residues, including three aspartates, a lysine, and either threonine or serine (6Djordjevic S. Stock A.M. J. Struct. Biol. 1998; 124: 189-200Crossref PubMed Scopus (79) Google Scholar).CheY, the response regulator of bacterial chemotaxis, has served as the model for understanding phosphorylation-induced activation of response regulators (7Stock J.B. Stock A.M. Mottonen J.M. Nature. 1990; 344: 395-400Crossref PubMed Scopus (481) Google Scholar, 8Volz K. Biochemistry. 1993; 32: 11741-11753Crossref PubMed Scopus (253) Google Scholar). Early biochemical, genetic, and structural studies on CheY indicate that phosphorylation induces a structural change from an inactive to an active conformation (for a review, see Ref. 6Djordjevic S. Stock A.M. J. Struct. Biol. 1998; 124: 189-200Crossref PubMed Scopus (79) Google Scholar). The five conserved active site residues were shown to be important for phosphorylation and/or conformational changes subsequent to phosphorylation. Asp57 was established as the site of phosphorylation (9Sanders D. Gillece-Castro B.L. Stock A.M. Burlingame A.L. Koshland Jr., D.E. J. Biol. Chem. 1989; 264: 21770-21778Abstract Full Text PDF PubMed Google Scholar) and, along with Asp12 and Asp13, is required for magnesium binding (10Lukat G.S. Lee B.H. Mottonen J.M. Stock A.M. Stock J.B. J. Biol. Chem. 1991; 266: 8348-8354Abstract Full Text PDF PubMed Google Scholar, 11Lukat G.S. Stock A.M. Stock J.B. Biochemistry. 1990; 29: 5436-5442Crossref PubMed Scopus (155) Google Scholar, 12Needham J.V. Chen T.Y. Falke J.J. Biochemistry. 1993; 32: 3363-3367Crossref PubMed Scopus (74) Google Scholar). While Thr87 and Lys109 were implicated in post-phosphorylation events (10Lukat G.S. Lee B.H. Mottonen J.M. Stock A.M. Stock J.B. J. Biol. Chem. 1991; 266: 8348-8354Abstract Full Text PDF PubMed Google Scholar, 13Appleby J.L. Bourret R.B. J. Bacteriol. 1998; 180: 3563-3569Crossref PubMed Google Scholar, 14Volz K. Matsumura P. J. Biol. Chem. 1991; 266: 15511-15519Abstract Full Text PDF PubMed Google Scholar, 15Zhu X. Rebello J. Matsumura P. Volz K. J. Biol. Chem. 1997; 272: 5000-5006Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), it is only recently that their roles have been defined (see below). Finally, the rotameric position of Tyr106, another conserved residue adjacent to the active site, was thought to be correlated with the signaling state of CheY (16Zhu X.Y. Amsler C.D. Volz K. Matsumura P. J. Bacteriol. 1996; 178: 4208-4215Crossref PubMed Google Scholar). Although the importance of these residues has been noted in numerous studies of inactive and mutant forms of CheY as well as other response regulators (17Feher V.A. Cavanagh J. Nature. 1999; 400: 289-293Crossref PubMed Scopus (203) Google Scholar, 18Weinstein M. Lois A.F. Monson E.K. Ditta G.S. Helinski D.R. Mol. Microbiol. 1992; 6: 2041-2049Crossref PubMed Scopus (13) Google Scholar), a detailed understanding of the mechanism of the phosphorylation-induced transformation from an inactive to active conformation could not be reached because of the short half-life of the aspartyl-phosphate linkage (half-life of a few seconds to hours).We have shown through biochemical and structural studies that BeF3− forms persistent complexes with receiver domains, mimicking the phosphorylation-activated states (19Yan D. Cho H.S. Hastings C.A. Igo M.M. Lee S.-Y. Pelton J.G. Stewart V. Wemmer D.E. Kustu S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14789-14794Crossref PubMed Scopus (121) Google Scholar). For example, similar to phosphorylated CheY (P-CheY), BeF3−-CheY shows enhanced binding to the N-terminal 16 residues of its target, FliM, enhanced affinity for CheZ, and decreased affinity for CheA (19Yan D. Cho H.S. Hastings C.A. Igo M.M. Lee S.-Y. Pelton J.G. Stewart V. Wemmer D.E. Kustu S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14789-14794Crossref PubMed Scopus (121) Google Scholar, 20Cho H.S. Lee S.-Y. Yan D. Pan X. Parkinson J.S. Kustu S. Wemmer D.E. Pelton J.G. J. Mol. Biol. 2000; 297: 543-551Crossref PubMed Scopus (118) Google Scholar). Our recent NMR structure of BeF3−-CheY (20Cho H.S. Lee S.-Y. Yan D. Pan X. Parkinson J.S. Kustu S. Wemmer D.E. Pelton J.G. J. Mol. Biol. 2000; 297: 543-551Crossref PubMed Scopus (118) Google Scholar) revealed many aspects of the structural changes induced upon activation. The hydroxyl group of Thr87 forms a hydrogen bond with an active site acceptor, presumably BeF3−-Asp57, and the side chain of Tyr106 is restrained in a buried conformation. Unfortunately, the positions of the BeF3− moiety, magnesium cation, and the side chain conformation of Lys109could not be defined by the NMR data.Recently, the crystal structures of the phosphorylated forms of two other response regulators, Spo0Ar (21Lewis R.J. Brannigan J.A. Muchova K. Barak I. Wilkinson A.J. J. Mol. Biol. 1999; 294: 9-15Crossref PubMed Scopus (140) Google Scholar) and FixJr (22Birck C. Mourey L. Gouet P. Fabry B. Schumacher J. Rousseau P. Kahn D. Samama J.-P. Structure ( Lond. ). 1999; 7: 1505-1515Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), have been reported (superscript r denotes receiver domain). Spo0Ar was unknowingly crystallized in the phosphorylated state with calcium as the divalent metal rather than magnesium, and FixJr was crystallized in the absence of a divalent metal ion to circumvent problems associated with hydrolysis of the phospho-aspartate. It is known that removal of magnesium from phosphorylated CheY does not alter its enhanced affinity for FliM (23Welch M. Oosawa K. Aizawa S.-I. Eisenbach M. Biochemistry. 1994; 33: 10470-10476Crossref PubMed Scopus (79) Google Scholar). This suggests that, while magnesium is important in the chemistry of phosphorylation of receiver domains, it is probably not required for stabilizing the active conformations. Thus, the structures of P-Spo0Ar and P-FixJr do likely represent the phosphorylation-activated states, although the activities of these two proteins in the conditions used for crystallization cannot be directly assessed. Importantly, the residues homologous to Thr87 and Tyr106 in both structures adopt similar conformations to those seen the NMR structure of BeF3−-CheY.We have recently determined the crystal structures of BeF3−-CheY and BeF3−-CheY complexed with a 16-residue peptide derived from the N terminus of FliM. The binding interactions of the CheY-peptide complex have been discussed (24Lee S.-Y. Cho H.S. Pelton J.G. Yan D. Henderson R.K. King D.S. Huang L. Kustu S. Berry E.A. Wemmer D.E. Nature Struct. Biol. 2001; 8: 52-56Crossref PubMed Scopus (147) Google Scholar). Herein we report the crystal structure of BeF3−-CheY complexed with the divalent cation manganese solved at 2.4-Å resolution. A detailed comparison of the active sites of BeF3−-CheY and P-Spo0Ar(21Lewis R.J. Brannigan J.A. Muchova K. Barak I. Wilkinson A.J. J. Mol. Biol. 1999; 294: 9-15Crossref PubMed Scopus (140) Google Scholar) clearly shows that BeF3−-aspartate activates receiver domains by reproducing the geometry and electrostatic potential of a phospho-aspartate. Indeed, all of the active site interactions in BeF3−-CheY are identical to those in P-Spo0Ar, indicating that the structural changes induced by BeF3− activation of response regulators are the same as those induced by phosphorylation. We also show, through a comparison of backbone coordinates of BeF3−-CheY with inactive magnesium-bound CheY, that activation results in only relatively small structural differences, except in loops, and that these differences are similar in magnitude to those observed between inactive and phosphorylated FixJr (22Birck C. Mourey L. Gouet P. Fabry B. Schumacher J. Rousseau P. Kahn D. Samama J.-P. Structure ( Lond. ). 1999; 7: 1505-1515Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar).RESULTS AND DISCUSSIONBeF3−-CheY crystals were grown in the presence of manganese (Mn2+). Although in vivo CheY is complexed with magnesium (Mg2+), NMR studies have shown that the active site readily accommodates larger divalent cations (12Needham J.V. Chen T.Y. Falke J.J. Biochemistry. 1993; 32: 3363-3367Crossref PubMed Scopus (74) Google Scholar,35Kar L. Matsumura P. Johnson M.E. Biochem. J. 1992; 287: 521-531Crossref PubMed Scopus (18) Google Scholar). Given that Mn2+ has the same coordination geometry as Mg2+ and supports phospho-transfer from CheA-P to CheY (11Lukat G.S. Stock A.M. Stock J.B. Biochemistry. 1990; 29: 5436-5442Crossref PubMed Scopus (155) Google Scholar), we expect that it does not perturb the active structure significantly.BeF3−-CheY complexed with Mn2+crystallized in the space group P212121 and diffracted to 2.4 Å. The two molecules in the asymmetric unit form a noncrystallographic symmetric dimer (Fig. 1) similar to that seen for P-FixJr (22Birck C. Mourey L. Gouet P. Fabry B. Schumacher J. Rousseau P. Kahn D. Samama J.-P. Structure ( Lond. ). 1999; 7: 1505-1515Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), with helix H4 of one molecule packing against the H4-β5-H5 face of the second molecule. For CheY, dimer formation in the crystal must be due to lattice packing forces and is not biologically relevant, because in solution BeF3−-CheY remains monomeric even at 3 mmprotein concentrations. Refinement statistics are summarized in TableI.Table IData collection and refinement statisticsCrystal:Space groupP212121Cell dimensions a, b, c53.3, 53.8, 161.2 ÅData collection (cryogenic):Resolution limit (Å)29.2–2.37Measured reflections89,177Unique reflections17,703Rsym (%) (overall/last shell)1-aThe resolution range in the highest bin is 2.41–2.37 Å.7.5/23.0Completeness (%)/last shell89.1/52.2AverageB-factor (Wilson 2.4–3.0 Å)43.4Refinement:No. of molecules in AU2No. of amino acids256No. of solvent molecules173Resolution used (Å)15.0–2.37Sigma cutoff0.0No. of reflections work/test16748/846FinalR-factor/Rfree (‖F‖ > 0 ς)21.0/24.0Average B-factor (Å2)44.8Root mean square deviations from ideal geometryBond lengths (Å)0.011Bond angles (degree)1.51-a The resolution range in the highest bin is 2.41–2.37 Å. Open table in a new tab The overall crystal structure of BeF3−-CheY retains the (β/α)5 fold of receiver domains (6Djordjevic S. Stock A.M. J. Struct. Biol. 1998; 124: 189-200Crossref PubMed Scopus (79) Google Scholar) (Fig. 1) and is very similar to the NMR structure of BeF3−-CheY (20Cho H.S. Lee S.-Y. Yan D. Pan X. Parkinson J.S. Kustu S. Wemmer D.E. Pelton J.G. J. Mol. Biol. 2000; 297: 543-551Crossref PubMed Scopus (118) Google Scholar) as well as the crystal structure of inactive Mg2+-bound CheY (36Stock A.M. Martinex-Hackert E. Rasmussen B.F. West A.H. Stock J.B. Ringe D. Petsko G.A. Biochemistry. 1993; 32: 13375-13380Crossref PubMed Scopus (194) Google Scholar). Superposition of Cα coordinates (residues 6–125) of the BeF3−-CheY x-ray tructure with the mean BeF3−-CheY NMR structure and the Mg2+-bound CheY x-ray structure yielded root mean square deviations of only 1.2 and 0.8 Å, respectively. Differences in Cα coordinates between BeF3−-CheY and the crystal structure of inactive Mg2+-bound CheY, based on a superposition of the residues least affected by activation, are shown in Fig. 2a. The biggest changes are observed in the β4/H4 loop, the β5/H5 loop, and the N terminus of helix H5. The significance of the changes in the β4/H4 loop are particularly hard to interpret, because it adopts different conformations in the various crystal structures of inactive CheY (6Djordjevic S. Stock A.M. J. Struct. Biol. 1998; 124: 189-200Crossref PubMed Scopus (79) Google Scholar). Indeed, dynamics studies of Mg2+-bound CheY showed that this region is flexible in solution (37Moy F.J. Lowry D.F. Matsumura P. Dahlquist F.W. Krywko J.E. Domaille P.J. Biochemistry. 1994; 33: 10731-10742Crossref PubMed Scopus (73) Google Scholar), and a superposition of the x-ray structure of BeF3−-CheY with the NMR structures of Mg2+-bound CheY shows that the β4/H4 loop of the active (x-ray) structure falls on the edge of the bundle formed by the inactive (NMR, Mg2+-bound) structures. Rather than a conformational change, we prefer to view the activation-induced changes in the β4/H4 loop as a stabilization of the active conformation that may be sampled by the inactive protein. Unfortunately, it is hard to make similar conclusions for the β5/H5 loop, because the relaxation data for residues in this loop could not be reliably interpreted due to complications caused by chemical exchange of Mg2+ in the active site (37Moy F.J. Lowry D.F. Matsumura P. Dahlquist F.W. Krywko J.E. Domaille P.J. Biochemistry. 1994; 33: 10731-10742Crossref PubMed Scopus (73) Google Scholar).Figure 2Activation-induced Cα coordinate changes (active-inactive) for CheY (a), FixJr (b), and phosphono-CheY (c). For CheY, a δ-distance plot comparing crystal structures of Mg2+ and BeF3−-CheY showed that residues 5–55 and 65–84 were the least affected by activation. These residues were used to superimpose the Mg2+ and BeF3−-CheY structures from which changes in Cα positions were calculated. For FixJr, residues least influenced by phosphorylation (residues 1–8, 16–52, and 106–122) were used to superimpose Mn2+-bound and phosphorylated (no metal) structures from which changes in Cα positions were calculated. For phosphono-CheY, residues least influenced by the phosphono group (residues 5–50) were used to superimpose Mg2+-CheY and phosphono-CheY (no metal) structures. The horizontal line in each plot denotes the overall backbone root mean square deviation for each pair of structures.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Active Site of BeF3−-CheYFrom the NMR structure of BeF3−-CheY we determined that the switch from an inactive to an active conformation involves hydrogen bond formation between the hydroxyl group of Thr87 and an active site residue, presumably BeF3−-Asp57. As a consequence of, or in conjunction with, formation of this hydrogen bond, β-strand β4 (along with Thr87) is displaced, and the aromatic ring of Tyr106 becomes buried in a hydrophobic pocket between helix H4 and β5. However, the NMR data did not define the positions of either the BeF3− moiety or the divalent cation. In addition, the NMR data for Lys109, a residue known to be critical for switching to the active conformation (10Lukat G.S. Lee B.H. Mottonen J.M. Stock A.M. Stock J.B. J. Biol. Chem. 1991; 266: 8348-8354Abstract Full Text PDF PubMed Google Scholar), were insufficient to define the position of the side chain in the active site accurately. The BeF3−-CheY crystal structure verifies the previous conclusions and extends the detail in the active site (Fig. 3). The hydroxyl group of Thr87 does hydrogen-bond with one of the fluorine atoms of the BeF3− moiety that is bonded to Asp57 Oδ (Oδ-Be distance 1.5 Å) in a tetrahedral configuration. The aromatic ring of Tyr106 is seen exclusively in the buried position, stabilized in this rotameric conformation by a hydrogen bond that was not previously identified in the NMR studies between the tyrosine hydroxyl group and the backbone carbonyl oxygen of Glu89 as well as hydrophobic interactions. The divalent cation (Mn2+) is located adjacent to Asp57-BeF3− in the crystal structure and is coordinated by Asp13 Oδ, Asp57 Oδ, backbone carbonyl oxygen of Asn59, a fluorine atom, and two water molecules. Finally, the side chain of Lys109 forms a salt bridge with BeF3− and Asp12 Oδ.Figure 3Stereo view of the active site of BeF 3− -CheY. Carbon, nitrogen, oxygen beryllofluoride, and manganese atoms are colored gray,dark blue, red, yellow, andgreen, respectively. a, omit map contoured at 3.0 ς covering Asp12, Asp13, BeF3−-Asp57, Thr87, Lys109, and two water molecules. This map was calculated with the occupancies for these residues set to zero. For clarity, the density for manganese is not shown. b,ball-and-stick diagram of the BeF3−-activated CheY active site. Dashed lines and numbers denote active site interactions defined in Table II. c, stereo view of active site residues for BeF3−-CheY(Mn2+) (blue), phosphorylated FixJr(no metal) (lime), and phosphorylated Spo0Ar(Ca2+) (copper). Mn2+ and Ca2+ are shown asred and green balls, respectively. Residue numbers are based on E. coli CheY. For clarity, phosphono-CheY was not included.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Active Site Comparisons with P-Spo0Ar,P-FixJr, and Phosphono-CheYComparison of the BeF3−-activated CheY active site with those of phosphorylated receiver domains determined to high resolution, including P-Spo0Ar (21Lewis R.J. Brannigan J.A. Muchova K. Barak I. Wilkinson A.J. J. Mol. Biol. 1999; 294: 9-15Crossref PubMed Scopus (140) Google Scholar), P-FixJr (22Birck C. Mourey L. Gouet P. Fabry B. Schumacher J. Rousseau P. Kahn D. Samama J.-P. Structure ( Lond. ). 1999; 7: 1505-1515Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), and phosphono-CheY (38Halkides C.J. McEvoy M.M. Casper E. Matsumura P. Volz K. Dahlquist F.W. Biochemistry. 2000; 39: 5280-5286Crossref PubMed Scopus (58) Google Scholar), provides a structural basis by which BeF3− mimics the phosphoryl group (Fig. 3). Of these, BeF3−-CheY is best compared with P-Spo0Arbecause both contain a divalent cation, hexavalent Mn2+, and heptavalent Ca2+, respectively, in the active site. Similar to the phospho-aspartate in P-Spo0Ar, BeF3−-aspartate acts as a ligand to the divalent metal atom, forms a salt bridge with Lys109 Nζ, and hydrogen bonds with Thr87 Oγ and the backbone amides of Trp58, Asn59, and Ala88. The measured distances for the common interactions in the structural models of BeF3−-CheY and P-Spo0Ar are within coordinate uncertainties (∼0.3 Å) (TableII). It appears that, although calcium has an extra coordination site relative to manganese (and magnesium), which is occupied by a water molecule in P-Spo0Ar, the extra ligand is accommodated without requiring a significantly different active site geometry.Table IIActive sites distances (Å)BeF3−-CheYP-Spo0ArP-FixJrPhospono-CheY1. PO3—Thr87 Oγ2.52.52.76.42. PO3—Ala88 NH2.92.83.04.33. PO3—Lys109 NH2.93.03.13.04. Lys109 NH—Asp12Oδ2.82.64.15.85. Asp12Oδ—H2O2.62.5N/AN/A6. M2+—H2O2.42.6N/AN/A7. M2+—PO32.22.4N/AN/A8. M2+—Asp13Oδ2.32.6N/AN/A9. M2+—Asn59CO2.32.5N/AN/A10. PO3—Asn59 NH3.02.92.95.511. PO3—Trp58 NH2.92.93.45.912. M2+—Asp57Oδ2.22.6N/AN/AM2+ denotes divalent cation; N/A denotes not applicable. Residue numbers correspond to those of E. coli CheY. Open table in a new tab Although lacking a divalent cation, the distances for the analogous interactions in P-FixJr are also similar to those seen for BeF3−-CheY and P-Spo0Ar (Table II). The only exception is the large distance (4.1 Å) between Lys109 Nζ and Asp12 Oδ in P-FixJr, indicating that this salt bridge is broken in the absence of metal. It is interesting to note that, although a divalent cation is necessary for the chemistry of phosphorylation and dephosphorylation of CheY, removal of the metal after phosphorylation apparently does not alter the affinity of P-CheY for FliM (23Welch M. Oosawa K. Aizawa S.-I. Eisenbach M. Biochemistry. 1994; 33: 10470-10476Crossref PubMed Scopus (79) Google Scholar). Similarly, the fact that P-FixJr purifies as a dimer in the absence of metal, consistent with its activated state, suggests that the metal is not required for inducing the active conformation of FixJr. This may be a general feature of receiver domains.In phosphono-CheY, except for the salt bridge between Lys109 Nζ and a phosphonate oxygen, the distances measured for the analogous hydrogen bonds are outside of the acceptable range (2.5–3.1 Å) (Table II). The absence of these interactions leads to much smaller changes in the β4/H4 and β5/H5 loops (Fig.2c). The modest structural differences relative to inactive CheY appear to be consistent with the partial activity of phosphono-CheY, which shows an 8-fold increase in affinity for N16-FliM (38Halkides C.J. McEvoy M.M. Casper E. Matsumura P. Volz K. Dahlquist F.W. Biochemistry. 2000; 39: 5280-5286Crossref PubMed Scopus (58) Google Scholar), whereas BeF3−- and phosphorylation-activated CheY show a 25-fold increase in affinity (19Yan D. Cho H.S. Hastings C.A. Igo M.M. Lee S.-Y. Pelton J.G. Stewart V. Wemmer D.E. Kustu S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14789-14794Crossref PubMed Scopus (121) Google Scholar, 39McEvoy M.M. Bren A. Eisenbach M. Dahlquist F.W. J. Mol. Biol. 1999; 289: 1423-1433Crossref PubMed Scopus (87) Google Scholar). Considering that the Sγ–Cδ bond in phosphono-cysteine is only 0.5 Å longer than the Cγ–Oδ bond in phospho-aspartate, it is surprising that the phosphonate analog does not better activate CheY. Since the salt bridge formed by Lys109 Nζ and an active site partner (BeF3−, PO3−, phosphonate) is the only common interaction in P-Spo0Ar, P-FixJr, BeF3−-CheY, and phosphono-CheY, it appears to be an important part of the active site interactions that together induce the fully active conformation. Based on crystal structures of mutant forms of CheY, it was previously suggested that Lys109 plays a role in positioning the β5/H5 loop (40Bellsolell L. Cronet P. Majolero M. Serrano L. Coll M. J. Mol. Biol. 1996; 257: 116-128Crossref PubMed Scopus (25) Google Scholar, 41Sola M. Lopez-Hernandez E. Cronet P. Lacroix E. Serrano L. Coll M. Parraga A. J. Mol. Biol. 2000; 303: 213-225Crossref PubMed Scopus (26) Google Scholar).Activation-induced Conformational ChangesCheY and FixJr are the only receiver domains that have been solved with sufficient resolution in both active (22Birck C. Mourey L. Gouet P. Fabry B. Schumacher J. Rousseau P. Kahn D. Samama J.-P. Structure ( Lond. ). 1999; 7: 1505-1515Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar) and inactive (36Stock A.M. Martinex-Hackert E. Rasmussen B.F. West A.H. Stock J.B. Ringe D. Petsko G.A. Biochemistry. 1993; 32: 13375-13380Crossref PubMed Scopus (194) Google Scholar, 42Gouet P. Fabry B. Guillet V. Birck C. Mourey L. Kahn D. Samama J.-P. Structure ( Lond. ). 1999; 7: 1517-1526Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) states to allow a detailed comparison of activation-induced structural changes. The largest activation-induced Cα coordinate changes for both proteins occur in loop regions, particularly the β4/H4 loop. In addition, the β5/H5 loop shows significant displacement in CheY, potentially due to the Lys109 Nζ-BeF3−salt bridge, but the analogous conformational change is not seen in FixJr. As stated previously, for inactive CheY the β4/H4 loop is conformationally flexible according to solution NMR studies (37Moy F.J. Lowry D.F. Matsumura P. Dahlquist F.W. Krywko J.E. Domaille P.J. Biochemistry. 1994; 33: 10731-10742Crossref PubMed Scopus (73) Google Scholar). Activation results in the formation of a new hydrogen bond between the hydroxyl of Tyr106 and the backbone carbonyl of Glu89. This likely helps to stabilize the active conformation of this loop. Thus, a comparison of just the active and inactive CheY crystal structures could lead one to conclude that activation induced dramatic conformational changes in the β4/H4 loop. However, in light of the NMR data, the exact magnitude of this change is hard to quantify. In FixJr the residue homologous to Tyr106 is a phenylalanine, which cannot stabilize the β4/H4 loop through a side chain-backbone hydrogen bond. It would be interesting to determine whether this loop in FixJr is also conformationally flexible in the inactive state and becomes stabilized upon activation.Even though the loops show significant activation-induced changes, activation of CheY and FixJr does not result in any major structural rearrangements. Whereas some β-strands and α-helices are slightly displaced, the actual residues that define these elements of secondary structure remain unchanged in both proteins. In both BeF3−-CheY and P-FixJr the N terminus of H4 moves slightly upon activation, and in CheY there is also a small displacement of the N terminus of H5. Indeed, even when compared as a group (Fig. 4), including P-Spo0Ar, there are no dramatic structural differences among either the active or inactive forms of the receiver domains. Although there are small differences in the tilt and inclination of the helices, these differences do not give rise to changes in atomic coordinates of more than a few Ångstroms.Figure 4Stereo view of Cα overlays of inactive and active response regulators.In" @default.
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• W1991438329 title "Crystal Structure of Activated CheY" @default.
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