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