(Received for publication, May 17, 1995; and in revised form, July 10, 1995)
From the
CheY is the response regulator of Escherichiacoli chemotaxis and is one of the best studied response regulators of the two-component signaling system. CheY can receive phosphate from the histidine kinase, CheA. Phospho-CheY interacts with the motor-switch complex to induce clockwise flagellar rotation, thus causing the cell to tumble. We used an enzyme-linked immunosorbent assay to study the direct interaction between the kinase, CheA, and the regulator, CheY. The products of random, suppressor, and site-specific cheY mutants were assayed for their ability to bind CheA. Nine mutants showed altered binding. We sequenced and mapped these point mutations on the crystal structure of CheY, and a high degree of spatial clustering was revealed, indicating that this region of CheY is involved in CheA binding. Interestingly, five of these altered binding mutants were previously defined as being involved in motor-switch binding interactions. This suggested a possible overlap between the motor-switch binding and CheA binding surfaces of CheY. Using CheY (Trp-58) fluorescence quenching, we determined the equilibrium dissociation constants of CheA(124-257) binding for these CheY mutants. The results from the fluorescence quenching are in close agreement with our initial enzyme-linked immunosorbent assay results. Therefore, we propose that the CheA and the motor binding surfaces on CheY partially overlap and that this overlap allows CheY to interact with either the CheA or the flagellar motor, depending on its signaling (phosphorylation) state.
Bacterial response to hostile environmental conditions is regulated by a complex network of interacting proteins, with the most predominant interactions being generated by members of two-component systems(1) . In Escherichia coli chemotaxis, the interaction between the receptor-coupled histidine autokinase, CheA, and the response regulator, CheY, controls the bacterial response to chemical environmental changes(1, 2, 3, 4) . In response to changes in the receptor's occupancy and adaptation, CheA autophosphorylates (5, 6) and subsequently transfers its phosphate to either CheY or CheB(7, 8, 15) . This phosphotransfer results from CheY's intrinsic autophosphorylation activity and is not due to catalysis by CheA(9, 10) . Studies indicate that phospho-CheY is the activated form that binds to a motor-switch complex, causing a clockwise flagellar rotation and a net change in the bacterial swimming direction(11, 12, 13) .
Allosteric changes within a large complex containing a receptor
dimer, a CheA dimer, and two copies of a small coupling protein, CheW,
regulate CheA autophosphorylation and subsequent phosphotransfer to
CheY(11, 14, 22, 23) . The
autophosphorylation site, His-48, lies on the N terminus of CheA, and
the CheY binding determinants lie between residues 124 and 257,
commonly known as the P2 domain(17) . However, the CheA binding
site on CheY is not defined. Structural studies indicate that CheY is a
single domain protein that folds into a (/
)
topology, with five
-strands forming the hydrophobic core,
surrounded by five
-helices(18, 19) . Three
aspartate residues, Asp-12, -13, and -57 form the molecule's
active site, with Asp-57 being the site where CheY receives the
phospho-group from CheA His-48(20, 21) .
Phosphorylated CheY loses its affinity for CheA (23) and shows
high binding affinity for FliM, one of the motor-switch
components(13) .
It has been estimated that E. coli contains about 50 homologous two-component systems, which govern
various cellular responses to stress(1, 3) . Since the
active site of response regulators is highly conserved(1) ,
CheA must be able to differentiate CheY from other potential response
regulators. Very recently, an NMR study on the interaction between an
N-terminal fragment of CheA(1-233) and CheY has defined the CheA(1-233)
binding site on CheY by measuring the chemical shift changes in CheY
upon CheA(1-233) addition(26) . Residues identified here lie
distinct from the active site on -4,
-4,
-5, and
-5
of CheY.
In the present study, we have employed a CheY-CheA binding
assay, based on the ELISA, ()to characterize, in
vitro, the interaction between full-length CheA and CheY and to
screen CheY mutants for possible defects in CheA binding. The mutants
that altered the CheA interaction had amino acid positions clustered on
a face of the CheY protein, and this three-dimensional clustering
suggested that this region of CheY is involved in CheA binding.
Figure 1: Schematic diagram of the ELISA-based CheY-CheA binding assay. Each well was coated with serially diluted anti-CheY antibody. The sequence in which other proteins were added is shown in the leftwell (from bottom to top). Alk. Phos., alkaline phosphatase; Ab, antibody; MAb, monoclonal antibody. The optical density was read at 405 nm in an ELISA reader.
The assay was
performed using cell lysates overexpressing CheY and CheA/CheW. To
measure background activity, lysates with either no CheY or a CheY
deletion (18-28) were used. As seen in Fig. 2, the
wells containing wild-type CheY produced stronger signals than the
negative controls, with the signal to noise ratio being at least 2 to
1. The signal obtained from the negative controls was due to a
cross-reactivity of the anti-CheY antibody to CheA, which could not be
completely eliminated. The mouse monoclonal antibody to CheA and the
class-specific anti-mouse antibody did not cross-react with CheY (data
not shown).
Figure 2:
Binding of CheA to immobilized CheY.
Binding of CheA/CheW complex to immobilized CheY was determined by the
ELISA-based CheY-CheA binding assay for wild-type CheY (circles) and CheY (18-28, squares) by
measuring the absorbance at 405 nm plotted as a function of the
anti-CheY antibody serial dilution. The background was determined by
using CheA/CheW without CheY (triangles). All the Che proteins
were obtained from overexpressing cell
lysates.
Figure 3:
CheY mutants with altered CheA binding.
Nine CheY mutants (bars), which showed significant differences
in CheA binding (at least ±25% of wild-type binding, out of 73
screened) as determined by ELISA. Results are mean ± S.D. of
three experiments, normalized to binding of wild-type CheY to CheA. The
location (shown by the numbers) of mutation and the amino acid
change (shown by singlelettersymbols, wild
type first) in the mutants were determined by DNA sequencing. The CheY
mutants (also the wild type indicated by WT) are arranged in
increasing order of K values as
determined by fluorescence quenching (line).
Figure 4:
Stereographs showing the locations of some cheY mutations on the CheY (an 5-
5 protein)
structure (18) . Highlighted residues are as follows: redatoms represent residues where mutation affects CheA
binding, and greenatoms represent residues where
mutation does not affect CheA binding. The
-carbon backbone of
CheY is in white. Whitestippling depicts
solvent-accessible surfaces of the residues that were implicated in
motor-switch binding(25) . The top and bottompictures are two different views, 90° apart from each
other.
The substitutions that affected binding also make sense chemically in the context of an altered protein-interaction surface. For example, the two glutamate-to-lysine substitutions (E93K, E117K) involve charge changes that could affect the electrostatic interactions between the interacting proteins. The other substitutions involve side group volume changes (A90V, Y106W, V108M) as well as the hydrophobicity changes (T87I, T112I). Among these residues, only Thr-87 and Phe-111 have their side chains directed in toward CheY's hydrophobic core, and in these cases, only the backbone portion of these residues seems to contribute to the proposed CheA binding surface, so the influence of the side chain may be more indirect.
The goal of this study was to define the CheA binding face of CheY. We screened cheY mutants for altered CheA interaction, using a modified version of the ELISA(32) , which is a specific, economic, and sensitive assay. A technical problem with this method was a reduced signal to noise ratio caused by a high background. This was determined to be due to the cross-reactivity of the polyclonal anti-CheY antibody to CheA. Affinity purification of the CheY antibody on a CheY column reduced but did not eliminate this cross-reactivity. However, none of the other components nor antibodies demonstrated significant background.
Use of this assay enabled us to identify nine point mutants, D13K, T87I, A90V, E93K, Y106W, V108M, F111V, T112I, and E117K, which showed altered CheA binding. The most striking result is their location on the CheY molecule and the fact that they clearly cluster on the face of CheY. This clustering becomes functionally more significant, since many of these residues (90, 108, 111, 112, and 117) were previously identified by genetic suppression analysis to be involved in motor-switch interaction(25) . The motor-switch suppressor E27K was the only putative position that interacts with the motor switch that did not alter CheA binding. Another study, using NMR spectroscopy to examine the interaction of CheA1-233 with CheY(26) , came to the same conclusion that an overlap exists between the motor switch and the P2 binding surfaces on CheY. Interestingly, another study indicated that a number of residues, showing chemical shift changes upon phosphorylation, lie in this region(24) . Combining these results with the observations that phospho-CheY does not stay in complex with CheA (23) but binds to FliM with a higher affinity than apo-CheY(13) , we propose that 1) the overlap region in the unphosphorylated state contributes to the CheA binding surface, 2) phosphorylation alters the topology of this overlap region, and 3) the phosphorylation-induced changes may be responsible for both CheA releasing CheY and CheY's increased affinity for the flagellar motor. This kind of overlap enables CheY to interact with several proteins with the specificity for each interaction being governed by its signaling state.
Although the
region where our mutants clustered is similar to the region identified
by the P2-CheY NMR study(26) , some residues were unique to
each study. In our study, the mutagenesis may not have been saturated,
despite the recurrence of T112I, and since we screened for total
non-chemotaxis, it is possible that we might have missed some mutants
that had reduced binding and partial chemotaxis function. In the NMR
study, chemical shift changes in the backbone amide residues were
measured, and only those chemical shift changes greater than 60 Hz were
considered significant. It is possible that some residues, indicated in
this study, may affect the binding through their solvent-accessible
side chains but do not result in Hz greater than 60. The side
chain of Phe-111 is buried inside CheY's hydrophobic core (18) and may seem to be an exception, but NMR data show that
Phe-111 shifts upon adding CheA(1-233), although the observed chemical
shift change is less than 60 Hz. Since CheA has been shown to bind to
CheY as a dimer(23) , some of the residues we identified, which
are not indicated by NMR studies, may be critical for CheA dimer
binding.
The D13K mutant is a dominant tumbly mutation, also found to be defective in CheA binding. One possible explanation is that this mutant has acquired a conformation resembling phospho-CheY, and hence it is defective in CheA binding. Welch et al.(13) found that it binds to FliM with higher affinity than the apo-CheY, and this is consistent with the possibility of a conformational similarity between this mutant and the phospho-CheY.
Our results identify mutations that can alter the CheY-CheA binding,
and it is possible that some of our mutations may have introduced a
structural change that sterically disrupted binding rather than remove
an interaction contributing to the binding. Also, a mutation can alter
CheA binding, either by specifically changing the interaction surface
or by nonspecifically causing a change in folding. Since all of the
mutants we characterized were overproduced in the stable form and can
be phosphorylated by CheA (except D13K (data not shown)), the
probability of any major folding defects occurring is reduced. Also,
the crystal structures of T87I and Y106W ()do not show
altered folding(33) . The most striking evidence is derived
from the clustering of these mutations on one face of CheY molecule. We
also mapped the positions of five cheY mutants not displaying altered
CheA binding, and these mutations are not found on our proposed CheA
binding face (greenatoms, Fig. 4).
It is clear that the CheA binding face of CheY that our study identified lies distinct from the active site of the molecule. Recognition by CheA, away from the highly conserved active site of CheY (3) , may be a way of acquiring specificity for this interaction. On the other hand, evidence for an overlap between CheA binding face and motor-switch binding face makes the structural aspects of CheY more interesting. While more work will be needed for the absolute determination of the region of overlap between the two faces, it will be interesting to know the region of CheY involved in the binding of CheZ, the only protein besides CheA and motor-switch proteins, that is known to interact with CheY(34) . This will provide a better understanding of the structural aspects of CheY's activity and the regulation of chemotaxis.