Research Associate Professor
726 Mary Ellen Jones Bldg, CB #7290
We use a combination of structural, biochemical, and biophysical approaches to probe phosphorylation and dephosphorylation events that occur in bacterial signal transduction. Two-component regulatory systems are a widespread means of signal transduction in bacteria and play critical roles in diverse cellular processes varying from morphological development to pathogenesis. Central to these systems is the 'response regulator' protein that essentially functions as a molecular switch: the response regulator is 'turned on' by covalent phosphorylation of an aspartyl residue and subsequently 'turned off' by hydrolysis of the phosphoryl group.
A primary focus has been investigation of phosphatases that catalyze the dephosphorylation of response regulators. Our co-crystal structure of the chemotaxis phosphatase CheZ complexed with CheY (its response regulator substrate) gave us tremendous insight into the catalytic mechanism and served as a framework for detailed biochemical and biophysical studies to elucidate CheZ enzyme kinetics and regulation. We have extended this work to explore the CheC/CheX/FliY family of phosphatases. Remarkably, we found that this family uses an identical catalytic strategy as the CheZ family, despite possessing a distinct three-dimensional fold. Both phosphatase families bind their response regulator substrates such that a strictly conserved amide residue (a glutamine in CheZ and an asparagine in CheC/CheX/FliY) is positioned to interact with a water molecule that attacks the phosphoryl group in a substitution reaction.
We are currently exploring whether the strategy used by the CheZ and CheC/CheX/FliY families can be generalized to other response regulator phosphatase families. Notably, many of the sensor kinases that transfer a phosphoryl group to response regulators also possess phosphatase activity towards their partner response regulator but little is known about the mechanism by which this occurs. We have also initiated a collaboration with Matt Wolfgang's lab to study phosphotransfer reactions within a two-component system involved in the regulation of both cAMP levels and twitching motility in Pseudomonas aeruginosa.
Freeman AM, Mole BM, Silversmith RE, Bourret RB (2011). Action at a Distance: Amino Acid Substitutions That Affect Binding of the Phosphorylated CheY Response Regulator and Catalysis of Dephosphorylation Can Be Far from the CheZ Phosphatase Active Site. J Bacteriol. 193(18):4709-18.
Bourret RB, Thomas SA, Page SC, Creager-Allen RL, Moore AM, Silversmith RE (2010). Measurement of response regulator autodephosphorylation rates spanning six orders of magnitude. Methods Enzymol. 471:89-114.
Silversmith, R.E. (2010) Auxiliary phosphatases in two-component signal transduction. Curr. Opin. Microbiol. 13, 177-183.
Bourret, R.B. & Silversmith, R.E. (2010) Two-component signal transduction. Curr. Opin. Microbiol. 13, 113-115.
Pazy, Y., Motaleb, M.A., Guarinari, M., Charon, N.W., Zhao, R., & Silversmith, R.E. (2010) Identical phosphatase mechanisms achieved through distinct modes of binding phosphoprotein substrate. Proc. Natl. Acad. Sci. U.S.A. 107, 1924-1929.
Pazy, Y., Wollish A.C., Thomas, S.A., Miller, P.J, Collins, E.J., Bourret, R.B., & Silversmith, R.E. (2009) Matching biochemical reaction kinetics to the timescales of life: Structural determinants that influence the autodephosphorylation rate of response regulator proteins. J. Mol. Biol. 392, 1205-1220.
Silversmith, R.E., Levin, M.D., Schilling, E., & Bourret, R.B. (2008) Kinetic characterization of catalysis by the chemotaxis phosphatase CheZ: modulation of activity by the CheYp substrate. J. Biol. Chem. 283, 756-765.
Silversmith, R. E. (2005) High mobility of carboxyl-terminal region of bacterial chemotaxis phosphatase CheZ is diminished upon binding divalent cation or CheY-P substrate. Biochemistry 44, 7768-76.