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• W2000646546 abstract "Position 87 of the chemotaxis regulatory protein CheY is a highly conserved threonine/serine residue in the response regulator superfamily. A threonine 87 to isoleucine mutant in CheY, identified by its in vivo non-chemotactic phenotype, was also found to be phosphorylatable in vitro. These properties indicate that this mutant does not undergo activation upon phosphorylation.The x-ray crystallographic structure of the threonine to isoleucine CheY mutant has been solved and refined at 2.1-Å resolution, to an R factor of 15.6%. Comparison with the wild-type, Mg2+-free CheY structure shows that the active site structure is retained, but there are significant localized differences in the backbone conformation distal from the substitution. The presence of the isoleucine side chain also restricts the rotational conformation of another conserved residue in the molecule, tyrosine at position 106. These results provide further evidence for a signaling surface remote from the phosphorylation site of the CheY molecule and implicate threonine 87 and other residues in the post-phosphorylation signaling events. Position 87 of the chemotaxis regulatory protein CheY is a highly conserved threonine/serine residue in the response regulator superfamily. A threonine 87 to isoleucine mutant in CheY, identified by its in vivo non-chemotactic phenotype, was also found to be phosphorylatable in vitro. These properties indicate that this mutant does not undergo activation upon phosphorylation. The x-ray crystallographic structure of the threonine to isoleucine CheY mutant has been solved and refined at 2.1-Å resolution, to an R factor of 15.6%. Comparison with the wild-type, Mg2+-free CheY structure shows that the active site structure is retained, but there are significant localized differences in the backbone conformation distal from the substitution. The presence of the isoleucine side chain also restricts the rotational conformation of another conserved residue in the molecule, tyrosine at position 106. These results provide further evidence for a signaling surface remote from the phosphorylation site of the CheY molecule and implicate threonine 87 and other residues in the post-phosphorylation signaling events. The CheY protein is the regulator of the chemotactic response in bacteria (for reviews, see (1Stewart R.C. Dahlquist F.W. Chem. Rev. 1987; 87: 997-1025Crossref Scopus (96) Google Scholar, 2Macnab R.M. Neidhardt F.C. Ingraham J.L. Low K.B. Magasanik B. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella Typhimurium: Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1987: 732-759Google Scholar, 3Berg H.C. Cold Spring Harbor Symp. Quant. Biol. 1988; 53: 1-9Crossref PubMed Google Scholar, 4Parkinson J.S. Cell. 1993; 73: 857-871Abstract Full Text PDF PubMed Scopus (606) Google Scholar)) and is the prototype regulatory domain of two-component signal transduction systems(5Volz K. Biochemistry. 1993; 32: 11741-11753Crossref PubMed Scopus (253) Google Scholar). This 14-kDa monomeric, cytoplasmic protein links the chemosensing machinery of the cell with the flagellar motors. Rotational bias at the flagellar motor is changed from a counter-clockwise to clockwise state in the presence of activated CheY, and this change in bias leads to an increased tumbling frequency of the bacterial cell(6Clegg D.O. Koshland Jr., D.E. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 5056-5060Crossref PubMed Scopus (54) Google Scholar, 7Ravid S. Matsumura P. Eisenbach M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7157-7161Crossref PubMed Scopus (67) Google Scholar, 8Wolfe A.J. Conley P. Kramer T. Berg H.C. J. Bacteriol. 1987; 169: 1878-1885Crossref PubMed Scopus (149) Google Scholar). CheY's regulatory activity is accomplished through post-translational modifications. CheY can be acetylated (9Barak R. Welch M. Yanovsky A. Oosawa K. Eisenbach M. Biochemistry. 1992; 31: 10099-10107Crossref PubMed Scopus (65) Google Scholar) and is phosphorylated by phospho-CheAL(10Hess J.F. Oosawa K. Matsumura P. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7609-7613Crossref PubMed Scopus (121) Google Scholar, 11Hess J.F. Oosawa K. Kaplan N. Simon M.I. Cell. 1988; 53: 79-87Abstract Full Text PDF PubMed Scopus (394) Google Scholar, 12Wylie D. Stock A. Wong C.-Y. Stock J. Biochem. Biophys. Res. Commun. 1988; 151: 891-896Crossref PubMed Scopus (118) Google Scholar). Transfer of the phosphoryl group from CheA to the aspartate residue 57 in CheY results in an increased concentration of the phosphorylated, activated form(13Sanders D.A. 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). Phosphorylated CheY binds to the flagellar switch proteins (14Welch M. Oosawa K. Aizawa S.-I. Eisenbach M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8757-8791Crossref Scopus (346) Google Scholar) and enhances clockwise bias at the switch(15Barak R. Eisenbach M. Biochemistry. 1992; 31: 1821-1826Crossref PubMed Scopus (125) Google Scholar), resulting in the increased frequency of tumbles. The fast decay of phosphorylated CheY, accelerated by another protein called CheZ, ensures a short life-time for the tumble signal (t ∼ 10 s)(11Hess J.F. Oosawa K. Kaplan N. Simon M.I. Cell. 1988; 53: 79-87Abstract Full Text PDF PubMed Scopus (394) Google Scholar, 25Lukat 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). The other modification of CheY acetylation increases the clockwise bias at the flagellar switch apparatus by a factor of 40,000(9Barak R. Welch M. Yanovsky A. Oosawa K. Eisenbach M. Biochemistry. 1992; 31: 10099-10107Crossref PubMed Scopus (65) Google Scholar), but acetylation of CheY may not be required for chemotaxis under normal physiological conditions(16Daily F.E. Berg H.C. J. Bacteriol. 1993; 175: 3236-3239Crossref PubMed Google Scholar). Phosphorylation-induced conformational changes in CheY are believed to be the physiological signal for tumble generation(17Bourret R.B. Hess J.F. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 41-45Crossref PubMed Scopus (200) Google Scholar). The lability of phosphorylated CheY has precluded direct crystallographic investigations of the active conformation of CheY. However, the structure of the unphosphorylated wild-type CheY protein has been described at high resolution from Escherichia coli(18Volz K. Matsumura P. J. Biol. Chem. 1991; 266: 15511-15519Abstract Full Text PDF PubMed Google Scholar) and Salmonella typhimurium(19Stock A.M. Martinez-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), as well as two unphosphorylated, Mg2+-bound forms(19Stock A.M. Martinez-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, 20Bellsolell L. Prieto J. Serrano L. Coll M. J. Mol. Biol. 1994; 238: 489-495Crossref PubMed Scopus (118) Google Scholar). CheY is a single domain protein that folds into a (β/α)5 topology with five parallel β-strands forming the hydrophobic core, surrounded by five α-helices. Three aspartate residues, Asp12, Asp13, and Asp57, are highly conserved within the superfamily of bacterial response regulators, and form the phosphorylation site near the carboxyl termini of the β strands. The side chain of another conserved residue Lys109 reaches into the active site and forms a strong hydrogen bond with the Asp57 side chain in the Mg2+-free, inactive form(18Volz K. Matsumura P. J. Biol. Chem. 1991; 266: 15511-15519Abstract Full Text PDF PubMed Google Scholar). The active site binds monophosphates (21Kar L. De Croos P.Z. Roman S.J. Matsumura P. Johnson M.E. Biochem. J. 1992; 287: 533-543Crossref PubMed Scopus (5) Google Scholar) and a Mg2+ ion, required for catalysis(20Bellsolell L. Prieto J. Serrano L. Coll M. J. Mol. Biol. 1994; 238: 489-495Crossref PubMed Scopus (118) Google Scholar, 22Lukat G.S. Stock A.M. Stock J.B. Biochemistry. 1990; 29: 5436-5442Crossref PubMed Scopus (155) Google Scholar, 23Kar L. Matsumura P. Johnson M.E. Biochem. J. 1992; 287: 521-531Crossref PubMed Scopus (18) Google Scholar). Two other conserved sites near the active site, Thr87 and Ala88, located at the COOH terminus of the β-4 strand, lead into the 90's loop connecting β-4 to α-4. The conserved aromatic position on β-5, 106, is a tyrosine residue which assumes two widely different rotameric environments in the 1.7-Å structure of wild-type, Mg2+-free E. coli CheY. The functional roles of most of these conserved residues are known(17Bourret R.B. Hess J.F. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 41-45Crossref PubMed Scopus (200) Google Scholar, 24Bourret R.B. Drake S.K. Chervitz S.A. Simon M.I. Falke J.J. J. Biol. Chem. 1993; 268: 13089-13096Abstract Full Text PDF PubMed Google Scholar, 25Lukat 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), but there is as yet no definitive assignment of biological importance for residues Thr87, Ala88, and Tyr106 in CheY. In this study we characterize the phenotypic effect of a threonine to isoleucine mutation at position 87 in CheY identified by random mutagenesis screening and describe the refined crystal structure of this mutant at 2.1-Å resolution. Replacement of the γ-hydroxyl group of threonine by the ethyl moiety of isoleucine increases the volume and hydrophobicity of the side chain at this site. Our in vitro and in vivo results show that the T87I mutant is phosphorylatable but non-chemotactic. Since this mutant CheY protein cannot convey the chemotactic signal, the biological activity of this molecule is presumably blocked at a step after the phosphorylation event. The crystal structure of the T87I CheY mutant reveals significant structural rearrangements distal from the site of mutation, in addition to conformational changes and solvent structure rearrangement in the immediate vicinity. By comparing the mutant structure with the wild-type protein, we identify conformational rearrangements which may be involved in the biological functionalities of this region of the CheY protein. The bacterial strains and plasmids used in this study are listed in Table 1. Cultures were routinely grown in Luria Broth (1% Tryptone, 1% NaCl, and 0.5% yeast extract). Wild-type CheY was over-produced from plasmid pRL22 in E. coli-K12 strain CY15040 by inducing expression at 42°C(26Matsumura P. Rydel J.J. Linzmeier R. Vacante D. J. Bacteriol. 1984; 160: 36-41Crossref PubMed Google Scholar). The T87I mutant was expressed from a similar plasmid construct pYM31, except that strain SG2 (devoid of chemotaxis proteins) was used to eliminate contamination by wild-type CheY.TABLE I Open table in a new tab Genetic phenotypes of the mutants were characterized following established procedures. Non-chemotactic mutants were screened on motility agar plates (1% NaCl, 1% tryptone, and 0.3% agar) by measuring swarm sizes after growth for 8-10 h at 30°C. Phosphorylation assays were done as described previously by Hess, et al.(11Hess J.F. Oosawa K. Kaplan N. Simon M.I. Cell. 1988; 53: 79-87Abstract Full Text PDF PubMed Scopus (394) Google Scholar) Reactions were carried out in a phosphorylation buffer (50 mM Tris, pH 7.9, 5 mM MgCl2, 50 mM KCl, and 0.5 mM [γ-32P]ATP (specific activity of 0.25 μCi/nmol) in a total reaction volume of 20 μl at room temperature. The reaction was stopped by addition of 20 μl of 2 × SDS-PAGE1 1The abbreviation used is: PAGEpolyacrylamide gel electrophoresis. sample buffer, and the reaction products were separated on SDS-PAGE (15% polyacrylamide) and autoradiographed. polyacrylamide gel electrophoresis. The stability of phosphorylated CheY was determined from the kinetics of CheY's intrinsic dephosphorylation activity. This was accomplished by permitting autophosphorylation of CheA coupled to Sepharose beads, and then allowing the phosphoryl group transfer reaction to free CheY. The reaction mixture for CheA phosphorylation contained 30 μl of beads (approximately 1 μg CheA/μl bead) suspended in 20 μl of phosphorylation buffer, supplemented with 1 mM [γ32P]ATP (specific activity 0.75 μCi/nmol). The reaction was carried out at room temperature for 45 min on a rotating shaker and was stopped by washing the beads with excess phosphorylation buffer. Purified CheY protein was added to the phospho-CheA beads, and phosphorylation was allowed to proceed for 30 s. Phosphorylated CheY was then separated from the CheA beads by centrifugation. Samples of phosphorylated CheY were aliquotted at different time points, where the autodephosphorylation reaction was stopped by adding 2 × SDS-PAGE sample buffer, and the samples were run on SDS-PAGE (15% polyacrylamide). The gels were dried, autoradiographed, and scanned on a β-particle scanner (Ambis Inc.) to quantitate the 32P-label attached to CheY. T87I mutant CheY was purified to homogeneity by dye-ligand chromatography and gel filtration chromatography, as described for the wild-type protein(26Matsumura P. Rydel J.J. Linzmeier R. Vacante D. J. Bacteriol. 1984; 160: 36-41Crossref PubMed Google Scholar). Protein concentration was estimated by a Coomassie dye binding assay kit (Pierce), with bovine serum albumin as the standard(27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar). Purified protein stock was concentrated to ∼10-15 mg/ml by ammonium sulfate precipitation and then loaded into 20- or 30-μl microdialysis chambers which were then sealed with dialysis membranes and soaked in different solution conditions inside 20-ml glass vials. Ammonium sulfate was used as the major precipitant in acetate or Tris-HCl buffered solutions. Sodium azide was routinely added at 0.05% concentrations in all crystallization trials to suppress fungal contamination. The mutant protein could not be crystallized at acidic pH; repeated crystallization trials around pH 4.5 led to amorphous precipitates. Multiple bunches of needles and plates appeared in a few days at pH 8.3 in 50 mM Tris-HCl buffer at 4°C. Effects of pH, temperature, ethanol, and polyethylene glycol were explored to improve the quality of crystals. Though the crystallization yield was very low, diffraction quality crystals grew as single trapezoidal plates from a mother liquor with an initial ammonium sulfate concentration of 2.7 M in Tris-HCl buffered at pH 8.3, at 4°C (these conditions are identical to those used for crystallization of wild-type CheY(18Volz K. Matsumura P. J. Biol. Chem. 1991; 266: 15511-15519Abstract Full Text PDF PubMed Google Scholar)). The crystals used for data collection grew to dimensions of 0.45 × 0.10 × 0.08 mm over a period of months. The crystals of the T87I mutant were found to be of the monoclinic space group P21. Unit cell dimensions of a = 53.59 Å, b = 71.81 Å, c = 35.83 Å, and β = 109.07° were determined from a least-squares fit of 23 intense reflections measured on an Enraf Nonius CAD4 diffractometer with 2θ settings in the range of 13.98°≤ 2θ≤ 27.36°. Assumption of 2 molecules/asymmetric unit yields a reasonable Matthews' coefficient (28Matthews B.M. J. Mol. Biol. 1968; 33: 491-497Crossref PubMed Scopus (7894) Google Scholar) of 2.39 Å3/dalton. Diffraction data were collected from a single crystal at the area detector facility at Argonne National Laboratory. The crystal was exposed for a total of ∼80 h in graphite monochromatized Cu Ka radiation from a rotating anode generator (50 kV, 100 mA) passing through a 0.3-mm collimator. Data were recorded at a crystal to detector distance of 100 mm on a Siemens multiwire area detector, with a swing angle of 18.5° in 2θ for a theoretical upper resolution limit of 2.04 Å. Data merging and reduction were done using the XENGEN program package(29Howard A. Gilliland G.L. Finzel B.C. Poulos T.L. Ohlendorf D.H. Salemme F.R. J. Appl. Crystallogr. 1987; 20: 383-390Crossref Scopus (571) Google Scholar). The unit cell parameters of the crystal used for data collection were within 0.12% of those previously determined from CAD4 diffractometer measurements. The final data set included a total of 30,935 observations of 15,093 unique reflections ranging from ∞♦ to 2.04 Å. The unweighted absolute value R factor on intensity for merging all observations was 4.57%. Statistics for the final data set used in the structural solutions are given in Table 2.TABLE II Open table in a new tab Phasing of the structure was initiated by the results of molecular replacement calculations (30Rossmann M.G. The Molecular Replacement Method. Gordon and Breach, New York1972Google Scholar) using the software package MERLOT(31Fitzgerald P.M.D. J. Appl. Crystallogr. 1988; 21: 273-278Crossref Scopus (377) Google Scholar). The search model comprised 891 atoms (90%) of the refined 1.7-Å structure of the wild-type CheY protein. All solvent molecules and the side chain atoms of polar, solvent accessible residues were excluded from the search model, and residue 87 was modeled as an alanine. Diffraction data greater than 2σI in the resolution range 10.0-4.0 Å were used. Interpretation of the rotation function search was straightforward, with two distinct peaks confirming 2 molecules/asymmetric unit. Satisfactory solutions from the translation searches were followed by 20 cycles of R value minimization refinement of Euler angles and translation parameters. The R factor at that stage was 40.4%. The molecular replacement solutions were used as the starting model for further refinement of atomic positions using the restrained least-squares method(32Hendrickson W.A. Methods Enzymol. 1985; 115: 252-270Crossref PubMed Scopus (585) Google Scholar), with a total of six stages of resolution extensions and 16 rounds of manual rebuilding of the atomic models on an interactive graphics terminal. The two molecules in the asymmetric unit were refined independently and simultaneously. All atoms were refined with unit occupancies. The R factor dropped rapidly during the initial cycles of refinement as the resolution limit was increased from 3.5 to 2.5 Å. Reflections were initially given resolution dependent weightings ranging from −12.5 to −0.75, in addition to a constant weight of ∼0.5〈ΔF〉. The average temperature factor for the two molecules at this stage was 21.9 Å2. Constraints on temperature factors were released after cycle 80. The quality of the Sim-weighted electron density maps improved significantly after including data to the upper resolution limit and modeling solvent molecules. At that stage, the resolution-dependent weighting factor was set to zero, and the R factor smoothly converged to a final value of 15.6% after 245 cycles of refinement and 12 rebuilds. Two side chains of the COOH termini and the 3 residues of the N termini were not visible until very near the end. 13,198 data in the resolution range 10-2.04 Å greater than 2σI were used in the final refinement. Calculation of electron density maps and other data processing were carried out with the XTAL software package (33Hall S.R. Stewart J.M. XTAL 2.4 User's Manual. Universities of Western Australia and Maryland, Perth, Australia1988Google Scholar) and several locally developed programs. Least-squares refinement was done with the software packages PROTIN and PROFFT(32Hendrickson W.A. Methods Enzymol. 1985; 115: 252-270Crossref PubMed Scopus (585) Google Scholar, 34Finzel B.F. J. Appl. Crystallogr. 1987; 20: 53-55Crossref Google Scholar). Visualization of electron density maps and model rebuilding were done using the graphics package FRODO (35Jones T.A. Methods Enzymol. 1985; 115: 157-171Crossref PubMed Scopus (934) Google Scholar) on an Evans and Sutherland PS300 system. Inspection of results was done with the program insightII on a Silicon Graphics Personal Iris. Plasmid pRL22ΔZ was mutagenized in vitro with hydroxylamine, and the mutants were screened for Penr and Che− (non-chemotactic) phenotypes in a ΔcheY mutant strain RP4079. One of these mutants carried a mutation at position 87 of the CheY amino acid sequence, with threonine substituted by isoleucine. Plasmid pYM31 carrying the T87I allele of CheY was not able to complement the ΔcheY mutant strain RP4079. This T87I mutant protein was further characterized functionally and structurally. The non-chemotactic phenotype of the T87I mutant would be easily explained had it lost its ability to be phosphorylated. However, T87I was found to be phosphorylatable in vitro (Fig. 1). Under the described reaction conditions, the CheA kinase was phosphorylated, and the phosphoryl group was transferred to the CheY protein in the presence of excess of CheY. Wild-type CheY can completely remove the phosphoryl group from phospho-CheA (lane 2versuslane 1). The lower amount of total radioactivity remaining in lane 2 is due to the rapid dephosphorylation activity of wild-type CheY. The T87I CheY mutant can also dephosphorylate phospho-CheA (lane 3). The band corresponding to phosphorylated T87I is more intense than that for wild-type (lane 2versuslane 3), due to T87I's slower rate of dephosphorylation (see below). Its reduced dephosphorylation activity is also responsible for the higher residual CheA-phosphate. Since the results described in Fig. 1 suggested a defect in dephosphorylation activity of the mutant protein, the stability of phosphorylated T87I was compared to that of wild-type. Wild-type- and T87I-CheY-phosphate were purified as described under “Materials and Methods.” The kinetics of dephosphorylation were monitored to obtain their first-order rate constants and half-lives (Fig. 2). These measurements show that phosphorylated T87I is approximately six times more stable than phosphorylated wild-type CheY. Other experiments show that the T87I mutant is also less susceptible to the phosphatase activity of CheZ (data not shown). The position corresponding to 87 in CheY is occupied by a hydroxyamino acid-threonine or serine in 96% of the known two-component response regulators(5Volz K. Biochemistry. 1993; 32: 11741-11753Crossref PubMed Scopus (253) Google Scholar). Such a high degree of conservation within the superfamily implies an important functional role for Thr87. The possibility of Thr87 being an alternate site of phosphorylation has been previously suggested(18Volz K. Matsumura P. J. Biol. Chem. 1991; 266: 15511-15519Abstract Full Text PDF PubMed Google Scholar), as well as a role in facilitating hydrolysis of the acylphosphate on Asp57(13Sanders D.A. 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). Our data demonstrate that Thr87 is not required for phosphorylation of CheY, since the T87I mutant is phosphorylated to a significant extent (Fig. 1). Moreover, the reduction of the dephosphorylation activity of the T87I mutant (Fig. 2) does not appear to be large enough to assign a catalytic role for Thr87 in that process. These results show that the T87I mutant cannot be activated by phosphorylation. A different CheY mutant from S. typhimurium, K109R, is also phosphorylatable in vitro, and also fails to generate tumbles when expressed in vivo from a multicopy plasmid(25Lukat 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). Both of these mutants are non-chemotactic despite their ability to be phosphorylated. They presumably are blocked in event(s) subsequent to phosphorylation during the activation pathway of the CheY molecule. A similar phenotypic effect had been found in a mutation at the Thr83 position of OmpR(37Brissette R.E. Tsung K. Inouye M. J. Bacteriol. 1991; 173: 3749-3755Crossref PubMed Google Scholar), corresponding to Thr87 of CheY. Since the T83A mutation of OmpR can intragenically suppress the effect of non-phosphorylatable D55Q mutant in OmpR,2 2Asp55 is the site of phosphorylation in OmpR, although a D11A mutation also lost the ability to be phosphorylated (36). the Thr83 residue of OmpR could also be important in event(s) subsequent to OmpR phosphorylation. These phenotypic similarities between equivalent site mutations in homologous proteins support our functional interpretations of Thr87 in CheY. The final electron density maps from the |2Fo - Fc|αcalc and |Fo - Fj|αcalc difference Fourier calculations are clear and well defined for the entire backbone of both molecules in the asymmetric unit, except for the amino-terminal residue of molecule B. There are a total of 14 amino acid side chains for which electron density was not interpretable past Cβ: residues 19, 89, 91, 92, 122, and 125 of molecule A, and residues 3, 26, 89, 91, 92, 93, 118, and 125 of molecule B. These are all charged, solvent exposed residues (Asp, Glu, Lys, and Arg) that possessed high temperature factors in the high resolution, wild-type CheY structure. The average thermal parameters for all atoms are 14.2 and 18.4 Å2, respectively, for molecules A and B (Fig. 3). The final coordinate set consists of 2070 atoms of which 1898 are protein, and 172 are solvent molecules with unit occupancies. Molecule A is modeled with 954 protein atoms and 91 associated solvent molecules and molecule B with 944 protein atoms and 81 solvent molecules. Clearly defined electron density for the Cγ-Cδ ethyl moieties at position 87 confirms the threonine to isoleucine substitution. No rotameric side chains were reliably detected in the mutant structures. Of the 7 residues that were modeled as two-state rotamers in the high resolution wild-type CheY structure, one of them, Tyr106, occurs at a conserved site in the two-component response regulator superfamily(5Volz K. Biochemistry. 1993; 32: 11741-11753Crossref PubMed Scopus (253) Google Scholar). In both of the T87I mutant molecules, this residue is well ordered, exclusively occupying the external, solvated position. The significance of this restriction of rotameric arrangement of the Tyr106 side chain is discussed below. The overall structure of the central β-sheet and the topology of the surrounding α-helices are unaltered in the mutant. Positional shifts in the hydrophobic core of the mutant are minimal when compared to the wild-type structure, with root mean square differences in the backbone atoms (N, Cα, C, and O) of the β-sheet of 0.133 and 0.143 Å for molecules A and B, respectively. Shifts in atomic positions are most pronounced in the vicinity of the turn leading into the 90's loop. Details of the conformational changes associated with this region are described below. Superpositions of the wild-type and mutant structures reveal significant shifts in the backbone conformation of the protein adjacent to the site of mutation. These shifts are not due to chemical differences, since the crystallization conditions for T87I and wild-type CheY were identical. The root mean squaredifferences in positions of equivalent α carbons of the least-squares superimposed mutant and wild-type structures are summarized in Table 3. A Luzzati analysis (38Luzzati V. Acta Crystallogr. 1952; 5: 802-810Crossref Google Scholar) suggests errors on the order of 0.20- 0.40 Å for structures refined to this resolution and R factor(39Ohlendorf D.H. Acta Crystallogr. 1994; D50: 808-812Google Scholar). The root mean square differences in corresponding α-carbon positions of molecule A and molecule B range within 0.32-0.40 Å. This compares well with the 0.31-Å root mean square differences for main chain atoms of another protein, myoglobin, in two crystal forms(40Phillips Jr., G.N. Arduini R.M. Springer B.A. Sligar S.G. Proteins: Struct. Funct. & Genet. 1990; 7: 358-365Crossref PubMed Scopus (98) Google Scholar). This range of values may thus be viewed as a lower practical limit in comparing different structures in different crystal packing environments. Since a visual inspection of the structural superpositions revealed significant backbone differences for only the COOH-terminal portion of the molecule past the site of mutation, root mean square errors for corresponding positions were calculated separately for the NH2-terminal (residues 3-86) and COOH-terminal portions (residues 87-129) (Table 3). The overall differences among the wild-type and the mutant molecules in the NH2-terminal two-thirds is in the lower limit of this range (root mean square deviations of 0.32 Å), but the COOH-terminal regions of molecule A and B differ from the wild-type by 0.90 and 0.68 Å, respectively. Thus the COOH-terminal portion of the T87I molecule is not just rotated as a rigid body as a result of the mutation, but there are localized distortions within this region as well. Figs. 4 and 5 illustrate these differences.TABLE III Open table in a new tab Interpretations of positional shifts of macromolecules from different crystal forms (in this case, P212121 for the wild-type and P21 for T87I) can be complicated by distortions caused by different packing environments. However, the crystal form of the T87I mutant with 2 molecules/asymmetric unit provides an “internal control.” Molecular distortions due to packing alone are detectable in the two chemically equivalent but crystallographically independent mutant molecules, and those differences can be used to separate at least some of the packing effects from the true structural consequences of the mutation. This type of analysis is presented graphically in the δ distance plots of Fig. 5, where comparisons of the interatomic distances of equivalent Cα atoms were done in pairwise fashion for all three molecules (" @default.
• W2000646546 created "2016-06-24" @default.
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• W2000646546 date "1995-07-01" @default.
• W2000646546 modified "2023-10-18" @default.
• W2000646546 title "Uncoupled Phosphorylation and Activation in Bacterial Chemotaxis" @default.
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