Crystal Structure of Activated CheY

COMPARISON WITH OTHER ACTIVATED RECEIVER DOMAINS*

Seok-Yong LeeDagger §, Ho. S. ChoDagger , Jeffrey G. PeltonDagger , Dalai Yan, Edward A. BerryDagger , and David E. WemmerDagger §||**

From the Dagger  Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 and the § Graduate Group in Biophysics, the  Department of Plant and Microbial Biology, and the || Department of Chemistry, University of California, Berkeley, California 94720

Received for publication, February 2, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

The crystal structure of BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-activated CheY, with manganese in the magnesium binding site, was determined at 2.4-Å resolution. BeF<UP><SUB>3</SUB><SUP>−</SUP></UP> 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 BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>, 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 BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY are also observed in P-Spo0Ar. Thus, BeF<UP><SUB>3</SUB><SUP>−</SUP></UP> 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 beta -strand beta 4 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 beta 4/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 beta 4/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 BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY, P-Spo0Ar, P-FixJr, and phosphono-CheY is a salt bridge between Lys109 Nzeta 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-PO<UP><SUB>3</SUB><SUP>−</SUP></UP> salt bridge is directly involved in the activation of receiver domains generally.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Two-component signal transduction systems control a variety of cellular processes, including chemotaxis and expression of some genes in bacteria and lower eukaryotes (1-4). 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 (5). The structures of several receiver domains have been solved (4). They all have a similar (beta /alpha )5 fold with an active site comprised of five highly conserved residues, including three aspartates, a lysine, and either threonine or serine (6).

CheY, the response regulator of bacterial chemotaxis, has served as the model for understanding phosphorylation-induced activation of response regulators (7, 8). 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. 6). 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 (9) and, along with Asp12 and Asp13, is required for magnesium binding (10-12). While Thr87 and Lys109 were implicated in post-phosphorylation events (10, 13-15), 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 (16). 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 (17, 18), 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 BeF<UP><SUB>3</SUB><SUP>−</SUP></UP> forms persistent complexes with receiver domains, mimicking the phosphorylation-activated states (19). For example, similar to phosphorylated CheY (P-CheY), BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY shows enhanced binding to the N-terminal 16 residues of its target, FliM, enhanced affinity for CheZ, and decreased affinity for CheA (19, 20). Our recent NMR structure of BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY (20) 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 BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-Asp57, and the side chain of Tyr106 is restrained in a buried conformation. Unfortunately, the positions of the BeF<UP><SUB>3</SUB><SUP>−</SUP></UP> moiety, magnesium cation, and the side chain conformation of Lys109 could not be defined by the NMR data.

Recently, the crystal structures of the phosphorylated forms of two other response regulators, Spo0Ar (21) and FixJr (22), 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 (23). 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 BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY.

We have recently determined the crystal structures of BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY and BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-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 (24). Herein we report the crystal structure of BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY complexed with the divalent cation manganese solved at 2.4-Å resolution. A detailed comparison of the active sites of BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY and P-Spo0Ar (21) clearly shows that BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-aspartate activates receiver domains by reproducing the geometry and electrostatic potential of a phospho-aspartate. Indeed, all of the active site interactions in BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY are identical to those in P-Spo0Ar, indicating that the structural changes induced by BeF<UP><SUB>3</SUB><SUP>−</SUP></UP> activation of response regulators are the same as those induced by phosphorylation. We also show, through a comparison of backbone coordinates of BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-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 (22).

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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Escherichia coli-- CheY was overexpressed and purified as described previously (20, 25) and was prepared as a solution containing 2 mM CheY, 8 mM BeCl2, 50 mM NaF, and 4 mM MnCl2 at pH of 8.4. Crystals of the complex were obtained at room temperature using the hanging-drop vapor diffusion method using a well solution containing 1.8 M ammonium sulfate, 5-10% glycerol, and 100 mM Tris (pH 8.4). The crystallization droplets contained the CheY solution mixed with an equal volume of the well solution. Crystals appeared after 1 day and grew to ~0.7 × 0.4 × 0.4 mm after 3 days. The concentration of glycerol in the well solution was increased (5% at each step) every 2 days to a final concentration of 25 volume %. The glycerol was added as a cryoprotectant to allow freezing for data acquisition. The protein crystallized in space group P212121 with unit cell dimensions a = 53.5 Å, b = 53.8 Å, and c = 161.3 Å with two molecules in the asymmetric unit.

The diffraction data were collected at 100 K on the Mar345 detector at the Stanford Synchrotron Radiation Laboratory using beamline 7-1 (wavelength of 1.08 Å). A crystal detector distance of 180 mm was used to collect data to 2.37-Å resolution. The data set was integrated and scaled to 2.37-Å resolution, using DENZO and SCALEPACK (26).

Cystal Structure of BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY-- Initial phases were determined by molecular replacement (MR) using AMoRe (27). The structure of BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY from a BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY-N16-FliM complex (Protein Data Bank accession code 1F4V) was used as the search model with Tyr106 replaced by alanine. Two solutions were easily found with a correlation coefficient of 67% and an R-factor of 34% (20-3.5 Å). The MR1 model was refined by several rounds of simulated annealing and B-group refinements using CNS (28) to an R-factor and Rfree of 29.5% and 31.4%, respectively. Structure factors (Fc and Phi c) were calculated from this partially refined MR model using SFALL of the CCP4 package (29). These phases were improved by 20 rounds of 2-fold noncrystallographic symmetry averaging and solvent flattening at 2.4 Å using the RAVE package (30), with a mask made from the MR solution and operators from the MR solution refined by the IMP program using the 2Fo - Fc MR map. In each round the map was calculated using coefficients 2Fo - Fc, with Fc and Phi c calculated from the density-modified map of the previous cycle.

An unbiased (2Fo - Fc) map calculated with phases and Fc from the final symmetry-averaged, solvent-flattened map was displayed using the graphics program O (31) and used as a guide in modeling Tyr106, positioning Mn2+ ions, and manually modifying the model in places where it did not fit the electron density. Refinement was performed using CNS (28). Anisotropic B-factor and bulk solvent corrections as well as the cross-validation method (32) were applied throughout the refinement. 15.0 to 2.37 Å data were included in the refinement with tight noncrystallographic symmetry restraints (300 kcal mol-1 Å2). Water picking was performed after the R-factor/Rfree dropped to 24%/27% using CNS.

The electron density for beryllofluoride on Asp57 was clearly seen (10 sigma ) in the resulting CNS Fo - Fc SIGMAA weighted map. This moiety was modeled on both protomers and refinement continued giving a final R-factor and Rfree of 21.0/24.0%. Geometric parameters for the structure were monitored using PROCHECK (33) and WHAT_CHECK (34).

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-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 (12, 35). Given that Mn2+ has the same coordination geometry as Mg2+ and supports phospho-transfer from CheA-P to CheY (11), we expect that it does not perturb the active structure significantly.

BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-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 (22), with helix H4 of one molecule packing against the H4-beta 5-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 BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY remains monomeric even at 3 mM protein concentrations. Refinement statistics are summarized in Table I.


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Fig. 1.   Ribbon diagram of the two BeF<UP><SUB><B>3</B></SUB><SUP><B>−</B></SUP></UP>-activated CheY molecules in the asymmetric unit. The active sites are directed toward the reader. Side chains are shown for BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-Asp57, Thr87, and Tyr106.

                              
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Table I
Data collection and refinement statistics

The overall crystal structure of BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY retains the (beta /alpha )5 fold of receiver domains (6) (Fig. 1) and is very similar to the NMR structure of BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY (20) as well as the crystal structure of inactive Mg2+-bound CheY (36). Superposition of Calpha coordinates (residues 6-125) of the BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY x-ray tructure with the mean BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-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 Calpha coordinates between BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-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 beta 4/H4 loop, the beta 5/H5 loop, and the N terminus of helix H5. The significance of the changes in the beta 4/H4 loop are particularly hard to interpret, because it adopts different conformations in the various crystal structures of inactive CheY (6). Indeed, dynamics studies of Mg2+-bound CheY showed that this region is flexible in solution (37), and a superposition of the x-ray structure of BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY with the NMR structures of Mg2+-bound CheY shows that the beta 4/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 beta 4/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 beta 5/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 (37).


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Fig. 2.   Activation-induced Calpha coordinate changes (active-inactive) for CheY (a), FixJr (b), and phosphono-CheY (c). For CheY, a delta -distance plot comparing crystal structures of Mg2+ and BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY showed that residues 5-55 and 65-84 were the least affected by activation. These residues were used to superimpose the Mg2+ and BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY structures from which changes in Calpha 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 Calpha 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.

Active Site of BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY-- From the NMR structure of BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-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 BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-Asp57. As a consequence of, or in conjunction with, formation of this hydrogen bond, beta -strand beta 4 (along with Thr87) is displaced, and the aromatic ring of Tyr106 becomes buried in a hydrophobic pocket between helix H4 and beta 5. However, the NMR data did not define the positions of either the BeF<UP><SUB>3</SUB><SUP>−</SUP></UP> moiety or the divalent cation. In addition, the NMR data for Lys109, a residue known to be critical for switching to the active conformation (10), were insufficient to define the position of the side chain in the active site accurately. The BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-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 BeF<UP><SUB>3</SUB><SUP>−</SUP></UP> moiety that is bonded to Asp57 Odelta (Odelta -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-BeF<UP><SUB>3</SUB><SUP>−</SUP></UP> in the crystal structure and is coordinated by Asp13 Odelta , Asp57 Odelta , backbone carbonyl oxygen of Asn59, a fluorine atom, and two water molecules. Finally, the side chain of Lys109 forms a salt bridge with BeF<UP><SUB>3</SUB><SUP>−</SUP></UP> and Asp12 Odelta .


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Fig. 3.   Stereo view of the active site of BeF<UP><SUB><B>3</B></SUB><SUP><B>−</B></SUP></UP>-CheY. Carbon, nitrogen, oxygen beryllofluoride, and manganese atoms are colored gray, dark blue, red, yellow, and green, respectively. a, omit map contoured at 3.0 sigma  covering Asp12, Asp13, BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-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 BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-activated CheY active site. Dashed lines and numbers denote active site interactions defined in Table II. c, stereo view of active site residues for BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY(Mn2+) (blue), phosphorylated FixJr(no metal) (lime), and phosphorylated Spo0Ar(Ca2+) (copper). Mn2+ and Ca2+ are shown as red and green balls, respectively. Residue numbers are based on E. coli CheY. For clarity, phosphono-CheY was not included.

Active Site Comparisons with P-Spo0Ar, P-FixJr, and Phosphono-CheY-- Comparison of the BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-activated CheY active site with those of phosphorylated receiver domains determined to high resolution, including P-Spo0Ar (21), P-FixJr (22), and phosphono-CheY (38), provides a structural basis by which BeF<UP><SUB>3</SUB><SUP>−</SUP></UP> mimics the phosphoryl group (Fig. 3). Of these, BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY is best compared with P-Spo0Ar because both contain a divalent cation, hexavalent Mn2+, and heptavalent Ca2+, respectively, in the active site. Similar to the phospho-aspartate in P-Spo0Ar, BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-aspartate acts as a ligand to the divalent metal atom, forms a salt bridge with Lys109 Nzeta , and hydrogen bonds with Thr87 Ogamma and the backbone amides of Trp58, Asn59, and Ala88. The measured distances for the common interactions in the structural models of BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY and P-Spo0Ar are within coordinate uncertainties (~0.3 Å) (Table II). 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.

                              
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Table II
Active sites distances (Å)
M2+ denotes divalent cation; N/A denotes not applicable. Residue numbers correspond to those of E. coli CheY.

Although lacking a divalent cation, the distances for the analogous interactions in P-FixJr are also similar to those seen for BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY and P-Spo0Ar (Table II). The only exception is the large distance (4.1 Å) between Lys109 Nzeta and Asp12 Odelta 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 (23). 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 Nzeta 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 beta 4/H4 and beta 5/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 (38), whereas BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>- and phosphorylation-activated CheY show a 25-fold increase in affinity (19, 39). Considering that the Sgamma -Cdelta bond in phosphono-cysteine is only 0.5 Å longer than the Cgamma -Odelta bond in phospho-aspartate, it is surprising that the phosphonate analog does not better activate CheY. Since the salt bridge formed by Lys109 Nzeta and an active site partner (BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>, PO<UP><SUB>3</SUB><SUP>−</SUP></UP>, phosphonate) is the only common interaction in P-Spo0Ar, P-FixJr, BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-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 beta 5/H5 loop (40, 41).

Activation-induced Conformational Changes-- CheY and FixJr are the only receiver domains that have been solved with sufficient resolution in both active (22) and inactive (36, 42) states to allow a detailed comparison of activation-induced structural changes. The largest activation-induced Calpha coordinate changes for both proteins occur in loop regions, particularly the beta 4/H4 loop. In addition, the beta 5/H5 loop shows significant displacement in CheY, potentially due to the Lys109 Nzeta -BeF<UP><SUB>3</SUB><SUP>−</SUP></UP> salt bridge, but the analogous conformational change is not seen in FixJr. As stated previously, for inactive CheY the beta 4/H4 loop is conformationally flexible according to solution NMR studies (37). 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 beta 4/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 beta 4/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 beta -strands and alpha -helices are slightly displaced, the actual residues that define these elements of secondary structure remain unchanged in both proteins. In both BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-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.


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Fig. 4.   Stereo view of Calpha overlays of inactive and active response regulators. Inactive CheY(Mg2+) (green) and BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-activated CheY(Mn2+) (blue), inactive FixJr(Mn2+) (burgundy) and phospho-activated FixJr(no metal) (yellow), and phospho-activated Spo0Ar(Ca2+) (orange). The superposition was generated by first superimposing residues in the five beta -strands of the three active structures and then superimposing residues 5-55 and 65-84 for inactive CheY(Mg2+) and residues 1-8, 16-52, and 106-122 for inactive FixJr(Mn2+) onto their respective active structures. a, molecules are oriented with the helix H4, beta 5, helix H5 face directed toward the reader. b, inactive and active CheY and fixJr are shown oriented with the active site directed toward the reader. Compared with a the structures are rotated 45° along the z axis and -90° along the x axis. c, only the three active structures are shown with the same orientation as in b. Note helix H2 is not labeled.

The structures of Spo0Ar and NtrCr have also been determined in both the active (21, 43) and inactive states (44, 45). It was difficult to analyze the activation-induced structural changes for Spo0Ar, because the inactive form crystallized as a domain-swapped dimer, the biological relevance of which is unclear. In contrast to CheY and FixJr, the low resolution NMR structures of active and inactive NtrCr show major structural differences, especially for residues that define helix H4. A higher resolution structure of BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-activated NtrCr will more clearly define these changes.

Conclusions-- The comparable interactions in the active sites of BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY and P-Spo0Ar indicates that BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-aspartate is almost a perfect structural mimic of phospho-aspartate. In conjunction with our previous biochemical data that show functional activation of receiver domains with BeF<UP><SUB>3</SUB><SUP>−</SUP></UP> (19, 20), it appears that beryllium fluoride is a convenient tool that can be applied to biochemical as well as structural studies of a host of response regulators.

Given the high sequence conservation, it is perhaps not surprising that the structures of P-Spo0Ar, P-FixJr, and BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>-CheY all show similar interactions in the active site. Based on a comparison of these structures we can begin to define a general mechanism of activation. As predicted by previous biochemical and genetic studies on CheY, the hydroxyl group of Thr87 (Thr84 Spo0Ar, Thr82 FixJr) (13) and the side chain of Lys109 (Lys106 Spo0Ar, Lys104 FixJr) (10) form what appears to be critical active site interactions with the phosphoryl group (or BeF<UP><SUB>3</SUB><SUP>−</SUP></UP>). As a consequence of, or in conjunction with, these interactions, Tyr106 (Phe103 Spo0Ar, Phe101 FixJr) adopts a buried conformation. Just how general these three events are in the transition from an inactive to an active conformation and how they affect the overall structures of receiver domains generally remains to be determined.

Comparison of just crystal structures suggests that there is a coupling between phosphorylation of CheY and FixJ with structural changes, especially in the beta 4/H4 loop. The extent to which phosphorylation induces an actual conformational change versus a stabilization of the active state from a pre-existing equilibrium between the active and inactive conformations in solution is not clear. Positive evidence for the idea of stabilization comes from NMR studies of inactive, constitutively active mutant forms (46), and phosphorylated NtrC (43), which indicate that phosphorylation stabilizes the active conformation (47). Additional NMR and x-ray studies of active and inactive forms of response regulators will help to clarify this issue and will help define how phosphorylation-induced conformational changes ultimately regulate the diverse processes controlled by two-component signal transduction.

    ACKNOWLEDGEMENTS

We thank the staff at Stanford Synchrotron Radiation Laboratory and, in particular, Drs. T. Earnest and D. Shin for providing invaluable advice on crystallographic data collection and processing. We also thank Prof. T. Yeates at UCLA for help with analysis of crystal twinning. We thank L. Huang for help with crystallization procedures and Dr. D. King for expert help with mass spectral analysis. We also thank R. Bourret for a critical reading of the manuscript.

    FOOTNOTES

* This work was supported by the Office of Energy Research, Office of Health and Environmental Research, Health Effects Research Division of the United States Department of Energy under contract number DE-AC03-76SF00098 (to D. E. W.) and through instrumentation Grant DE FG05-86ER75281 from the United States Department of Energy and National Science Foundation Grants DMB 86-09035 and BBS 87-20134 (to D. E. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1FQW) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

** To whom correspondence should be addressed. Tel.: 510-486-4318; Fax: 510-486-6059; E-mail: dewemmer@lbl.gov.

Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M101002200

    ABBREVIATIONS

The abbreviation used is: MR, molecular replacement.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

1. Nixon, B. T., Ronson, C. W., and Ausubel, F. M. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7850-7854[Abstract]
2. Ota, I. M., and Varshavsky, A. (1993) Science 262, 566-569[Medline] [Order article via Infotrieve]
3. Parkinson, J. S., and Kofoid, E. C. (1992) Annu. Rev. Genet. 26, 71-112[CrossRef][Medline] [Order article via Infotrieve]
4. Stock, A. M., Robinson, V. L., and Goudreau, P. N. (2000) Annu. Rev. Biochem. 69, 183-215[CrossRef][Medline] [Order article via Infotrieve]
5. Grebe, T. W., and Stock, J. B. (1999) Adv. Microb. Physiol. 41, 139-227[Medline] [Order article via Infotrieve]
6. Djordjevic, S., and Stock, A. M. (1998) J. Struct. Biol. 124, 189-200[CrossRef][Medline] [Order article via Infotrieve]
7. Stock, J. B., Stock, A. M., and Mottonen, J. M. (1990) Nature 344, 395-400[CrossRef][Medline] [Order article via Infotrieve]
8. Volz, K. (1993) Biochemistry 32, 11741-11753[Medline] [Order article via Infotrieve]
9. Sanders, D., Gillece-Castro, B. L., Stock, A. M., Burlingame, A. L., and Koshland, D. E., Jr. (1989) J. Biol. Chem. 264, 21770-21778[Abstract/Free Full Text]
10. Lukat, G. S., Lee, B. H., Mottonen, J. M., Stock, A. M., and Stock, J. B. (1991) J. Biol. Chem. 266, 8348-8354[Abstract/Free Full Text]
11. Lukat, G. S., Stock, A. M., and Stock, J. B. (1990) Biochemistry 29, 5436-5442[Medline] [Order article via Infotrieve]
12. Needham, J. V., Chen, T. Y., and Falke, J. J. (1993) Biochemistry 32, 3363-3367[Medline] [Order article via Infotrieve]
13. Appleby, J. L., and Bourret, R. B. (1998) J. Bacteriol. 180, 3563-3569[Abstract/Free Full Text]
14. Volz, K., and Matsumura, P. (1991) J. Biol. Chem. 266, 15511-15519[Abstract/Free Full Text]
15. Zhu, X., Rebello, J., Matsumura, P., and Volz, K. (1997) J. Biol. Chem. 272, 5000-5006[Abstract/Free Full Text]
16. Zhu, X. Y., Amsler, C. D., Volz, K., and Matsumura, P. (1996) J. Bacteriol. 178, 4208-4215[Abstract]
17. Feher, V. A., and Cavanagh, J. (1999) Nature 400, 289-293[CrossRef][Medline] [Order article via Infotrieve]
18. Weinstein, M., Lois, A. F., Monson, E. K., Ditta, G. S., and Helinski, D. R. (1992) Mol. Microbiol. 6, 2041-2049[Medline] [Order article via Infotrieve]
19. Yan, D., Cho, H. S., Hastings, C. A., Igo, M. M., Lee, S.-Y., Pelton, J. G., Stewart, V., Wemmer, D. E., and Kustu, S. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 14789-14794[Abstract/Free Full Text]
20. Cho, H. S., Lee, S.-Y., Yan, D., Pan, X., Parkinson, J. S., Kustu, S., Wemmer, D. E., and Pelton, J. G. (2000) J. Mol. Biol. 297, 543-551[CrossRef][Medline] [Order article via Infotrieve]
21. Lewis, R. J., Brannigan, J. A., Muchova, K., Barak, I., and Wilkinson, A. J. (1999) J. Mol. Biol. 294, 9-15[CrossRef][Medline] [Order article via Infotrieve]
22. Birck, C., Mourey, L., Gouet, P., Fabry, B., Schumacher, J., Rousseau, P., Kahn, D., and Samama, J.-P. (1999) Structure (Lond.) 7, 1505-1515[CrossRef][Medline] [Order article via Infotrieve]
23. Welch, M., Oosawa, K., Aizawa, S.-I., and Eisenbach, M. (1994) Biochemistry 33, 10470-10476[Medline] [Order article via Infotrieve]
24. Lee, S.-Y., Cho, H. S., Pelton, J. G., Yan, D., Henderson, R. K., King, D. S., Huang, L., Kustu, S., Berry, E. A., and Wemmer, D. E. (2001) Nature Struct. Biol. 8, 52-56[CrossRef][Medline] [Order article via Infotrieve]
25. Bruix, M., Pascual, J., Santoro, J., Prieto, J., Serrano, L., and Rico, M. (1993) Eur. J. Biochem. 215, 573-585[Abstract]
26. Otwinowski, Z. O., and Minor, W. (1997) Methods Enzymol. 276, 307-326
27. Navaza, J. (1994) Acta Crystallogr. Sect. A Cryst. Struct. Commun. 50, 157-163[CrossRef]
28. Brünger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D Biol. Crystallogr. 54, 905-921[CrossRef][Medline] [Order article via Infotrieve]
29. Bailey, S. (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760-763[CrossRef][Medline] [Order article via Infotrieve]
30. Kleywegt, G. J., and Jones, T. A. (1999) Acta Crystallogr. D Biol. Crystallogr. 55, 941-944[CrossRef][Medline] [Order article via Infotrieve]
31. Jones, T. A., Zou, J.-Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. Sect. A 47, 110-119[CrossRef][Medline] [Order article via Infotrieve]
32. Brünger, A. T. (1992) Nature 355, 472-475[CrossRef]
33. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr. 26, 283-291[CrossRef]
34. Hooft, R. W. W., Vriend, G., Sander, C., and Abola, E. E. (1996) Nature 381, 272[Medline] [Order article via Infotrieve]
35. Kar, L., Matsumura, P., and Johnson, M. E. (1992) Biochem. J. 287, 521-531[Medline] [Order article via Infotrieve]
36. Stock, A. M., Martinex-Hackert, E., Rasmussen, B. F., West, A. H., Stock, J. B., Ringe, D., and Petsko, G. A. (1993) Biochemistry 32, 13375-13380[Medline] [Order article via Infotrieve]
37. Moy, F. J., Lowry, D. F., Matsumura, P., Dahlquist, F. W., Krywko, J. E., and Domaille, P. J. (1994) Biochemistry 33, 10731-10742[Medline] [Order article via Infotrieve]
38. Halkides, C. J., McEvoy, M. M., Casper, E., Matsumura, P., Volz, K., and Dahlquist, F. W. (2000) Biochemistry 39, 5280-5286[CrossRef][Medline] [Order article via Infotrieve]
39. McEvoy, M. M., Bren, A., Eisenbach, M., and Dahlquist, F. W. (1999) J. Mol. Biol. 289, 1423-1433[CrossRef][Medline] [Order article via Infotrieve]
40. Bellsolell, L., Cronet, P., Majolero, M., Serrano, L., and Coll, M. (1996) J. Mol. Biol. 257, 116-128[CrossRef][Medline] [Order article via Infotrieve]
41. Sola, M., Lopez-Hernandez, E., Cronet, P., Lacroix, E., Serrano, L., Coll, M., and Parraga, A. (2000) J. Mol. Biol. 303, 213-225[CrossRef][Medline] [Order article via Infotrieve]
42. Gouet, P., Fabry, B., Guillet, V., Birck, C., Mourey, L., Kahn, D., and Samama, J.-P. (1999) Structure (Lond.) 7, 1517-1526[CrossRef][Medline] [Order article via Infotrieve]
43. Kern, D., Volkman, B. F., Luginbühl, P., Nohaile, M. J., Kustu, S., and Wemmer, D. E. (1999) Nature 402, 894-898[CrossRef][Medline] [Order article via Infotrieve]
44. Lewis, R. J., Muchova, K., Brannigan, J. A., Barak, I., Leonard, G., and Wilkinson, A. J. (2000) J. Mol. Biol. 297, 757-770[CrossRef][Medline] [Order article via Infotrieve]
45. Volkman, B. F., Nohaile, M. J., Amy, N. K., Kustu, S., and Wemmer, D. E. (1995) Biochemistry 34, 1413-1424[Medline] [Order article via Infotrieve]
46. Nohaile, M. J., Kern, D., Wemmer, D. E., Stedman, K., and Kustu, S. (1997) J. Mol. Biol. 273, 299-316[CrossRef][Medline] [Order article via Infotrieve]
47. Volkman, B. F., Wemmer, D. E., and Kern, D. (2001) Science 291, 2429-2433[Abstract/Free Full Text]


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