(Received for publication, January 27, 1995; and in revised form, May 3, 1995)
From the
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
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
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
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
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
[
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.
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 [
Figure 2:
Stability of phosphorylated wild-type and
T87I mutant CheY. The experiments were performed as described under
``Materials and Methods.''
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
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.
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
Figure 5:
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
The
affinity of the T87I mutant for Mg
The T87I mutant's effect on the phenolic group
of Tyr
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
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.
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
Preliminary results for a threonine to
serine mutation at position 87 of CheY support our interpretation of
the structure and function of T87I.
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.
The
atomic coordinates and structure factors (pdb1vlz.ent and r1vlz.ss)
have been deposited in the Protein Data Bank, Brookhaven National
Laboratory, Upton, NY.
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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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.
(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) .
-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.
-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.
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.
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
0.5
F
. 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.
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.
-
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).
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.
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.
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.
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, P22
2
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.
-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.
, 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.
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.
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.
, 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.
(
)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) .
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.
(
)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.
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.
is the site of phosphorylation in OmpR, although a D11A mutation
also lost the ability to be phosphorylated (36).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.