©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Uncoupled Phosphorylation and Activation in Bacterial Chemotaxis
THE 2.1-Å STRUCTURE OF A THREONINE TO ISOLEUCINE MUTANT AT POSITION 87 OF CheY (*)

(Received for publication, January 27, 1995; and in revised form, May 3, 1995)

Subrata Ganguli , Hui Wang , Philip Matsumura , Karl Volz (§)

From the Department of Microbiology and Immunology, University of Illinois, Chicago, Illinois 60612

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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, Mg-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.


INTRODUCTION

The CheY protein is the regulator of the chemotactic response in bacteria (for reviews, see (1, 2, 3, 4) ) and is the prototype regulatory domain of two-component signal transduction systems(5) . 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(6, 7, 8) . CheY's regulatory activity is accomplished through post-translational modifications. CheY can be acetylated (9) and is phosphorylated by phospho-CheA(10, 11, 12) . Transfer of the phosphoryl group from CheA to the aspartate residue 57 in CheY results in an increased concentration of the phosphorylated, activated form(13) . Phosphorylated CheY binds to the flagellar switch proteins (14) and enhances clockwise bias at the switch(15) , 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)(11, 25) . The other modification of CheY acetylation increases the clockwise bias at the flagellar switch apparatus by a factor of 40,000(9) , but acetylation of CheY may not be required for chemotaxis under normal physiological conditions(16) . Phosphorylation-induced conformational changes in CheY are believed to be the physiological signal for tumble generation(17) .

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(18) and Salmonella typhimurium(19) , as well as two unphosphorylated, Mg-bound forms(19, 20) . CheY is a single domain protein that folds into a (/) topology with five parallel -strands forming the hydrophobic core, surrounded by five -helices. Three aspartate residues, Asp, Asp, and Asp, are highly conserved within the superfamily of bacterial response regulators, and form the phosphorylation site near the carboxyl termini of the strands. The side chain of another conserved residue Lys reaches into the active site and forms a strong hydrogen bond with the Asp side chain in the Mg-free, inactive form(18) . The active site binds monophosphates (21) and a Mg ion, required for catalysis(20, 22, 23) . Two other conserved sites near the active site, Thr and Ala, located at the COOH terminus of the -4 strand, lead into the 90's loop connecting -4 to -4. The conserved aromatic position on -5, 106, is a tyrosine residue which assumes two widely different rotameric environments in the 1.7-Å structure of wild-type, Mg-free E. coli CheY. The functional roles of most of these conserved residues are known(17, 24, 25) , but there is as yet no definitive assignment of biological importance for residues Thr, Ala, and Tyr 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 -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.


MATERIALS AND METHODS

Strains and Plasmids

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(26) . 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.



Mutant Phenotype Characterization

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.(11) Reactions were carried out in a phosphorylation buffer (50 mM Tris, pH 7.9, 5 mM MgCl, 50 mM KCl, and 0.5 mM [-P]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-PAGE()sample buffer, and the reaction products were separated on SDS-PAGE (15% polyacrylamide) and autoradiographed.

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 [P]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 -particle scanner (Ambis Inc.) to quantitate the P-label attached to CheY.

Purification and Crystallization

T87I mutant CheY was purified to homogeneity by dye-ligand chromatography and gel filtration chromatography, as described for the wild-type protein(26) . Protein concentration was estimated by a Coomassie dye binding assay kit (Pierce), with bovine serum albumin as the standard(27) . 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(18) ). The crystals used for data collection grew to dimensions of 0.45 0.10 0.08 mm over a period of months.

Crystallographic Data Collection and Processing

The crystals of the T87I mutant were found to be of the monoclinic space group P2. Unit cell dimensions of a = 53.59 Å, b = 71.81 Å, c = 35.83 Å, and = 109.07° were determined from a least-squares fit of 23 intense reflections measured on an Enraf Nonius CAD4 diffractometer with 2 settings in the range of 13.98° 2 27.36°. Assumption of 2 molecules/asymmetric unit yields a reasonable Matthews' coefficient (28) of 2.39 Å/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 K 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 2 for a theoretical upper resolution limit of 2.04 Å. Data merging and reduction were done using the XENGEN program package(29) . 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.



Phasing by Molecular Replacement

Phasing of the structure was initiated by the results of molecular replacement calculations (30) using the software package MERLOT(31) . 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 2 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%.

Structural Refinement

The molecular replacement solutions were used as the starting model for further refinement of atomic positions using the restrained least-squares method(32) , 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 0.5F. The average temperature factor for the two molecules at this stage was 21.9 Å. 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 2 were used in the final refinement.

Calculation of electron density maps and other data processing were carried out with the XTAL software package (33) and several locally developed programs. Least-squares refinement was done with the software packages PROTIN and PROFFT(32, 34) . Visualization of electron density maps and model rebuilding were done using the graphics package FRODO (35) on an Evans and Sutherland PS300 system. Inspection of results was done with the program insightII on a Silicon Graphics Personal Iris.


RESULTS AND DISCUSSION

T87I's Functional Defects

Plasmid pRL22Z was mutagenized in vitro with hydroxylamine, and the mutants were screened for Pen and Che (non-chemotactic) phenotypes in a cheY 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 cheY 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.


Figure 1: CheA phosphoryl group transfer reactions to wild-type and T87I mutant CheY. The experiment was performed as described under ``Materials and Methods.'' Each reaction contained 0.5 mM [-P]ATP, incubated for 10 min with the following reactants: lane 1, 15 pmol of CheA alone; lane 2, 15 pmol of CheA and 57 pmol of wild-type CheY; lane 3, 15 pmol of CheA and 57 pmol of T87I mutant CheY.



Since the results described in Fig. 1suggested 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).


Figure 2: Stability of phosphorylated wild-type and T87I mutant CheY. The experiments were performed as described under ``Materials and Methods.'' denotes wild-type; denotes T87I mutant. The first-order rate constant for wild-type CheY is 3.04 10 s, corresponding to a t of 22.8 s; that for T87I mutant CheY is 4.77 10 s, for a t of 145 s.



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(5) . Such a high degree of conservation within the superfamily implies an important functional role for Thr. The possibility of Thr being an alternate site of phosphorylation has been previously suggested(18) , as well as a role in facilitating hydrolysis of the acylphosphate on Asp(13) . Our data demonstrate that Thr 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 Thr 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(25) . 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 Thr position of OmpR(37) , corresponding to Thr of CheY. Since the T83A mutation of OmpR can intragenically suppress the effect of non-phosphorylatable D55Q mutant in OmpR,()the Thr 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 Thr in CheY.

General Description of Structural Results

The final electron density maps from the 2F - F and F - F 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 C: 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 Å, 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 C-C 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, Tyr, occurs at a conserved site in the two-component response regulator superfamily(5) . 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 Tyr side chain is discussed below.


Figure 3: Average temperature factor versus residue number for T87I. A, molecule A; B, molecule B. Average temperature factors for main chain and side chain groups were calculated from arithmetic averages of their respective atoms. Average main chain temperature factors for wild-type CheY are plotted as continuous lines for comparison. A schematic of the secondary structure elements is included.



The overall structure of the central -sheet and the topology of the surrounding -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, C, C, and O) of the -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 carbons of the least-squares superimposed mutant and wild-type structures are summarized in Table 3. A Luzzati analysis (38) suggests errors on the order of 0.20- 0.40 Å for structures refined to this resolution and R factor(39) . The root mean square differences in corresponding -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(40) . 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 NH-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 NH-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.



Interpretations of positional shifts of macromolecules from different crystal forms (in this case, P222 for the wild-type and P2 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 distance plots of Fig. 5, where comparisons of the interatomic distances of equivalent C atoms were done in pairwise fashion for all three molecules (T87I, T87I, and wild-type). In the distance plots, positive contours correspond to regions of contraction of the second molecule compared to the first, while negative values indicate molecular expansion. Note that since distance plots do not depend on least-squares superpositions, they are not influenced by subjective assignments of structural similarity or dissimilarity.


Figure 5: -Distance plots between C atoms of T87I and wild-type CheY molecules. A, difference plot of [T87I- T87I]. B, difference plot of [wild-type - T87I]; C, difference plot of [wild-type - T87I]. Contour levels are at 0.4 Å intervals. Positive displacements are contoured with solid lines and negative displacements with dashed lines. Every 10th residue is marked on the diagonal by a cross.



The internal comparison of molecules A and B (Fig. 5A) identifies three features that arise from packing effects in this structure. First, the differences due to packing appear as low, broad peaks. Second, the majority of the peaks correspond to regions of the molecule far removed from the mutation site. Third, all the differences between molecules A and B can be verified as participating in intermolecular contacts. In this packing case, T87I molecule B is slightly contracted compared to molecule A, involving the relative positions of the COOH terminus of -2, the loop between -3 and -4, and the NH terminus of -4, primarily affecting residues 44-50, 76-80, and 91-98. In contrast, the distance plots between the wild-type and each of the mutant molecules show sharp displacement peaks up to 2.0 Å involving the 90's loop connecting the top of -4 to the NH terminus of -4. Helix -4 is repositioned on average 1 Å closer to the -5/-5 terminal portion in the T87I mutant CheY molecules. These structural changes are illustrated in Fig. 4.


Figure 4: Stereo diagram of T87I mutant CheY molecules A and B least-squares superimposed on wild-type CheY, highlighting the mutation site. Superpositions were based on residues 3-86 only. Wild-type CheY is shown in green, and the T87I molecules are in white. The hydrogen-bonding network for wild-type CheY residues Asp, Lys, Thr, Tyr, and the solvent molecules 3, 37, and 46 is shown in red. Details are given under ``Results and Discussion.''



Comparison of Active Sites

An inspection of the phosphorylation site of the mutant and wild-type proteins reveals no meaningful changes. The relative positions of Asp, Asp, and Lys, the 3 key residues implicated in the activation mechanism of the CheY molecule, are not significantly altered in this mutant (Fig. 4). The root mean square differences in positions of all atoms of these residues in the least-squares superimposed structures are 0.168 and 0.176 Å for T87I molecules A and B when compared to wild-type. The close interaction between N of Lys and O of Asp (the site of phosphorylation) in the wild-type protein suggested that a disruption of this interaction occurs upon phosphorylation(18) . In both molecules of the T87I mutant this strong interaction is retained. The distance between N of Lys and O of Asp are 2.53 and 2.80 Å for mutant molecules A and B, respectively. A lack of distortion in the active site is consistent with the phosphorylatability of the T87I mutant CheY. The only side chain close to the phosphorylation site with an altered position in the mutant is Phe. In both molecule A and molecule B, Phe occupies a rotameric position trans to its amide nitrogen, whereas it is g in the high resolution wild-type structure. This alternate position is due to crystal packing; it has been observed in other crystal forms of the wild-type,()and bears no functional significance.

The affinity of the T87I mutant for Mg ion compares well with that of wild-type CheY; the T87I mutant binds Mg with a dissociation constant of 0.6 mM at pH 7.0 and 20 °C(23) , similar to wild-type. The well-ordered solvent molecules A13 and B3 are in the active sites' Mg binding positions (solvent molecules are numbered according to increasing temperature factors). The locations of these solvent molecules are identical to the proposed cation binding site in wild-type E. coli CheY(18) , and to the location of the Mg ion in the Mg-CheY structures(19, 20) .

T87I's Effect on Residue Tyr

Besides threonine at position 87 and the aspartate residues in the active site, there are 3 other well conserved residues in the CheY superfamily, corresponding to Ala, Lys, and Tyr in the CheY molecule. The relative positions of Ala and Lys are retained in the T87I mutant structure; the root mean square differences in the position of all atoms of Ala and Lys are less than 0.10 Å when compared to wild-type CheY. Only Tyr had undergone a conformational rearrangement.

The T87I mutant's effect on the phenolic group of Tyr is essentially a restriction to one rotational conformation. In the wild-type structure, the only interpretable electron density for the Tyr side chain was found in two widely differing positions, so the side chain was accordingly built as a two-state rotamer, with values of 88 and 180°(18) . These two positions have very different environments: the inside, a relatively non-polar environment sealing off the interior of the protein core, and the outside, a highly solvent accessible external position (Fig. 4). No functional significance was attributed to this unusual residue at that time. In the T87I mutant structure, this side chain is unambiguously identified with the Tyr phenolic ring in the outer position. This is a direct result of the hydrophobicity and bulk of the isoleucine side chain at position 87 in the mutant, which sterically occludes the internal position of the Tyr side chain.

Conformational heterogeneity in amino acid side chains has been studied in a number of refined crystal structures and is considered as evidence of ``molecular breathing'' within the crystalline lattice(41) . Discrete rotational conformers are usually limited to solvent-accessible charged or polar residues. Such positional rearrangements of tyrosine side chains are known to be important in other systems, such as antigen-antibody recognition(42) . Two additional cases of rotameric rearrangements have had functional significance attributed to such subtle structural changes. One example is the binding of guanosine and other substrate analogues to the guanosine binding site of RNase T, which restricts the rotation of a valine side chain located 12 Å away from the substrate binding site(43) . Variations in the rotational freedoms of side chains have also been observed in the hydrophobic core mutants of T4 lysozyme (44) and barnase (45) from a number of mutant crystal structures. For CheY, earlier NMR spectroscopic results suggested a conformational alteration of Tyr due to the effect of the T87I mutation(23) . That result is consistent with the crystal structure of the mutant. In addition, proton resonances in the Tyr side chain of the wild-type protein were found to be affected by the approach of phosphate containing compounds to the metal-bound active site(21) . These results from solution studies supports the notion that the Tyr side chain may undergo a conformational change during the activation of CheY in vivo.

Interestingly, a mutation at the site corresponding to 106 in a CheY homologue, Y102C in OmpR, causes constitutive activity(46) . Based on this present work, other CheY mutants at position 106 have recently been isolated that give rise to both smooth-swimming and tumbly phenotypes.()Thus Tyr may play a role in CheY's signaling functions, although it is unclear whether it is the restriction of dynamic movement or the absolute positional relocation of the side chain that contributes to the molecular determinant of T87I's phenotype.

Distortion of CheY's Putative Signaling Surface

As shown in Fig. 4and the -distance plots of Fig. 5, the largest backbone conformational changes for the T87I mutant CheY are in the 90's loop, connecting -4 to -4. This exposed loop forms a prominent ridge in the topography of the CheY molecule, intervening between (and contiguous with) the solvent accessible surfaces of the active site and Lys regions. This same surface was proposed in previous work to be part of the ``signaling surface'' of the CheY molecule, based on mapping of extragenic suppressors of non-chemotactic flagellar switch mutants in S. typhimurium(47) and E. coli CheY(48) . Since those suppressor mutants can suppress clockwise bias in the flagellar motor(47) , and most of the suppressor mutant forms are phosphorylatable(48) , it is likely that this putative signaling surface partakes in the activation mechanism of the CheY molecule subsequent to phosphorylation. In addition, this same surface was found to be significantly modified upon Mg binding to CheY(20) .

The root mean square shift in the backbone atoms in the type VIII turn (residues 87-90) preceeding the 90's loop is 0.45 and 0.48 Å for molecule A and B, respectively, when compared to wild-type. In addition to displacements in the position of backbone atoms, this part of the molecule also shows conformational changes and solvent rearrangement as discussed below. Two of the highly conserved residues, Thr and Ala, occupy the first two positions of the type VIII turn. A functional significance for this loop is suggested by the number of mutants that have been isolated with diverse phenotypes in a wide variety of two-component systems (Table 4). Hydrophobicity and charge distribution in this region may be important structural determinants with functional consequences. For example, an A88S mutant of CheY apparently confers genetic phenotypes similar to that of T87I (Table 4).



The isoleucine side chain of the T87I mutant CheY obviously does not participate in hydrogen bonding interactions, whereas the O of Thr in the wild-type structure has a hydrogen bond to the amide nitrogen of Glu, and also bonds to 2 solvent molecules (37 and 46) that participate in the hydrogen bonding network of the active site: the former is bound to N of Asn and to the hydroxyl group of Tyr (in the inside rotameric position), and the latter is only one solvent molecule removed from Asp itself (Fig. 4). These interactions are eliminated in the T87I mutant. In addition, the carbonyl oxygen of Glu forms a new hydrogen bond with the amide nitrogen of Lys, which is the basis for the change in backbone conformation of the 90's loop. These observed structural rearrangements, in combination with the occurrence of non-signaling phenotypes of the various mutants of two component response regulators (Table 4), implicate an important role of this region in the signaling functions of CheY.

Preliminary results for a threonine to serine mutation at position 87 of CheY support our interpretation of the structure and function of T87I.()Behaviorally, the T87S mutant is partially functional, its phosphorylation stability is intermediate between T87I and wild-type CheY, and it does not have the structural perturbations seen here in the T87I mutant. These results will appear in a separate publication.

Clearly our data do not provide a complete structural understanding of the activation of CheY, since we do not know the structural effects of phosphorylation in either the wild-type or the mutant. F NMR studies suggested possible structural modifications subsequent to phosphorylation of the protein, such as separation of the -strands in the central -sheet(49) ; but these studies did not monitor mobilities in the Thr region of the CheY molecule due to the lack of F probes in its vicinity. Notwithstanding the limitations of our crystallographic studies, the fact remains that T87I is a mutant that is phosphorylatable but non-chemotactic. Lack of distortion in the active site of the mutant in comparison to the wild-type is consistent with the defect in signaling activity being downstream from the event of phosphorylation.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants AI18985 (to P. M.) and GM47522 (to K. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (pdb1vlz.ent and r1vlz.ss) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

§
To whom correspondence should be addressed. Tel.: 312-996-2314; Fax: 312-996-6415.

The abbreviation used is: PAGE, polyacrylamide gel electrophoresis.

Asp is the site of phosphorylation in OmpR, although a D11A mutation also lost the ability to be phosphorylated (36).

K. Volz, unpublished observations.

X. Y. Zhu, K. Volz, and P. Matsumura, unpublished observations.

X. Y. Zhu, J. Rebello, P. Matsumura, and K. Volz, unpublished observations.


ACKNOWLEDGEMENTS

The biochemical characterization of the T87I mutant was initiated as a collaborative project with J. F. Hess and M. I. Simon at the California Institute of Technology. We thank E. Westbrook and M. Westbrook for their help and generosity in data collection at Argonne National Laboratory, and C. Amsler for critical reading of the manuscript.


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