POSTERS - BLAST X - University of Utah

BLAST X Mon. Morning Session
A STRUCTURAL INVESTIGATION OF RESPONSE REGULATOR
AUTODEPHOSPHORYLATION
Yael Pazy 1 , Amy C. Wollish 1 , Stephanie A. Thomas 1 , Peter J. Miller 2 , Edward J. Collins 1,2 ,
Robert B. Bourret 1 , and Ruth E. Silversmith 1
Departments of Microbiology & Immunology 1 and Biochemistry & Biophysics 2 , University of
North Carolina, Chapel Hill, NC 27599
Assorted two-component regulatory systems are used to control a wide variety of
biological processes, which occur over a broad range of time scales. To appropriately
synchronize the adaptive responses implemented by response regulators with the
environmental stimuli detected by sensor kinases, the kinetics of biochemical signaling
reactions must be at least as fast as the biological process that they regulate. The fraction of
response regulators in the phosphorylated state is determined by the net result of
phosphorylation and dephosphorylation. Autodephosphorylation rates reported for various
wildtype response regulators span a range of at least 40,000x, consistent with timescales
ranging from about one second to one day. Furthermore, the range of rates observed suggests
that autodephosphorylation rates are an important contributor to setting the overall timescales of
two-component signaling systems.
We previously found that changing the amino acids at the variable active site positions
corresponding to residues 59 and 89 of Escherichia coli CheY could alter response regulator
autodephosphorylation rates about 100x (1). Thus, the particular amino acids found at positions
'59' and '89' in response regulators can account for two orders of magnitude in
autodephosphorylation rate, but other factors to account for an additional two to three orders of
magnitude in reaction rate must also exist and remain to be identified. To begin to characterize
the structural basis of response regulator autodephosphorylation rate, we determined highresolution
X-ray crystal structures for five CheY mutants that bear amino acid substitutions at
positions 14, 59, and 89 and consequently exhibit autodephosphorylation rates six to 40 times
slower than wildtype CheY. Each structure was determined in the presence of the phosphoryl
group analog BeF3 - and thus represents the starting point of the autodephosphorylation
reaction. Comparison of mutant and wildtype CheY structures, which are matched at all but
three residues and yet support different reaction rates, can potentially provide insight into the
mechanistic basis by which positions '59' and '89' influence autodephosphorylation rate.
Similarly, comparison of each mutant CheY structure with the structure of a wildtype response
regulator that is matched at eight (three variable and five conserved) active site residues and
yet catalyzes autodephosphorylation at rates 10-80x slower than the CheY mutants can
potentially suggest candidates for additional factors that might contribute to
autodephosphorylation rate.
Response regulator autodephosphorylation likely involves an inline attack on the
phosphoryl group by a nucleophilic water molecule. The structural comparisons outlined above
clearly suggest that autodephosphorylation rate is influenced by the extent to which amino acid
sidechains at positions '59' or '89' sterically occlude access to the phosphoryl group. Additional
factors potentially affecting autodephosphorylation rate will also be discussed.
Reference
1. Thomas, S.A., Brewster, J.A., & Bourret, R.B. (2008) Two nonconserved active site residues
affect response regulator phosphoryl group stability. Molecular Microbiology 69, 453-465.
2