6108 Marsico Hall
Campus Box 7290
Chapel Hill, NC 27599-7290
Signal transduction and protein phosphorylation. The ability to respond to stimuli is often considered to be a key characteristic of life. Cells can detect new conditions, transduce that information into a usable form, and execute an appropriate response. One common signal transduction strategy in both prokaryotic and eukaryotic organisms is to represent information by the specific and transient placement of phosphoryl groups on proteins. Errors in signal transduction can lead to diseases (e.g. cancer, diabetes), and drugs have been developed to block aberrant signaling processes. Understanding the mechanisms, regulation, and impact of phosphoryl group transfer among proteins is thus of fundamental interest, as well as of practical significance to human health.
Two-component signaling biochemistry and kinetics. Microorganisms are the dominant form of life on Earth by many measures, including genetic diversity, raw numbers, environmental distribution, and evolutionary experience. Thus, it is logical to seek basic signal transduction principles in microbes. Microorganisms from all three phylogenetic domains use two-component regulatory systems to regulate pathogenesis, antibiotic resistance, physiology, development, behavior, etc. Sensor kinases (the first component) detect stimuli and record this information in the form of phosphoryl groups. Response regulators (the second component) catalyze transfer of phosphoryl groups to themselves from sensor kinases (or from small molecules) to turn output function on, and from themselves to water to turn output function off. Kinases and phosphatases accelerate response regulator autocatalytic reactions to achieve physiologically appropriate signaling speeds, but do not alter the intrinsic reaction mechanisms. The kinetics of biochemical signaling reactions are crucial to synchronize responses with stimuli, and can differ substantially for biological processes that operate on different timescales.
An innovative research strategy for the genomics era. Our long-term goal is comprehensive understanding of signal transduction by two-component regulatory systems. Genome sequencing presents both a challenge (a rapidly widening gap between the number of known proteins and what can be studied experimentally) and an opportunity (the availability of diverse and extensive sequence data). Tens of thousands of two-component system proteins have been identified, but amino acid sequences currently reveal little beyond the presence of the conserved domains that define sensor kinases or response regulators. Our strategy is to elucidate general principles rather than clarify specific systems. To learn how to deduce properties of two-component proteins from sequence data alone, we investigate the consequences of sequence differences (rather than similarities) between the conserved domains of sensor kinases or response regulators. For example, we can alter the rate of response regulator phosphorylation and dephosphorylation by at least two orders of magnitude by changing variable residues in the active site. We are currently trying to identify the factors that control phosphodonor binding and response regulator autophosphorylation, response regulator autodephosphorylation, and sensor kinase-mediated dephosphorylation of response regulators. We are also characterizing the molecular mechanisms of these three processes.
Multidisciplinary methods. Our research utilizes an integrated approach that draws on an exceptionally wide range of techniques from biochemistry, bioinformatics, biophysics, genetics, microbiology, molecular biology, and structural biology. It is also noteworthy that laboratory work involving bacteria typically progresses much more rapidly than with eukaryotes.
Potential impact. Antibiotic resistance of bacterial and fungal pathogens is a major and increasing threat to human health. Our study of the binding of small molecules to response regulators may influence design of therapeutic agents to disable critical two-component systems of microbial pathogens. The results of our project could also be used to predict or manipulate the signaling kinetics of two-component systems, or engineer synthetic regulatory circuits with specific timing characteristics. Fundamental principles of signal transduction may also emerge.
Creager-Allen, R., Silversmith, R.E., & Bourret, R.B. (2013) A link between dimerization and autophosphorylation of the response regulator PhoB. J. Biol. Chem. 288, 21755-21769.
Thomas, S.A., Immormino, R.M., Bourret, R.B., & Silversmith, R.E. (2013) Nonconserved active site residues modulate CheY autophosphorylation kinetics and phosphodonor preference. Biochemistry 52, 2262-2273.
Freeman, A.M., Mole, B.M., Silversmith, R.E., & Bourret, R.B. (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, 4709-4718.
Miller, J., Parker, M., Bourret, R.B., & Giddings, M.C. (2010) An agent-based model of signal transduction in bacterial chemotaxis. PLoS One 5, e9454.
Silversmith, R.E. (2010) Auxiliary phosphatases in two-component signal transduction Curr. Opin. Microbiol. 13, 177-183.
Bourret, R.B. (2010) Receiver domain structure and function in response regulator proteins. Curr. Opin. Microbiol. 13, 142-149.
Bourret, R.B., & Silversmith, R.E. (2010) Two-component signal transduction. Curr. Opin. Microbiol. 13, 113-115.
Bourret, R.B., Thomas, S.A., Page, S. C., Creager-Allen, R.L., Moore, A.M., & Silversmith, R.E. (2010) Measurement of response regulator autodephosphorylation rates spanning six orders of magnitude. Meth. Enzymol. 471, 89-114.
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.