Analysis of Receiver Domain Dephosphorylation KineticsPublic Deposited
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MLAPage, Stephani. Analysis of Receiver Domain Dephosphorylation Kinetics. 2016. https://doi.org/10.17615/f250-rp89
APAPage, S. (2016). Analysis of Receiver Domain Dephosphorylation Kinetics. https://doi.org/10.17615/f250-rp89
ChicagoPage, Stephani. 2016. Analysis of Receiver Domain Dephosphorylation Kinetics. https://doi.org/10.17615/f250-rp89
- Last Modified
- March 21, 2019
- Affiliation: School of Medicine, Department of Biochemistry and Biophysics
- Plants and microorganisms use two-component signal transduction systems (TCSs) to mediate responses to stimuli. Canonical TCSs consist of a sensory component, the sensor kinase (SK), and a response-mediating component. The phosphorylation state of the SK is regulated by the presence of stimuli. The response-mediating component, the response regulator (RR) accepts the phosphoryl group from the SK. Receiver domains are the conserved domain of RRs, and contain the site of phosphorylation, a conserved Asp. Receiver domain dephosphorylation results in signal termination, and can occur through phosphotransfer to water or histidine. The kinetics of self-catalyzed Asp → water phosphotransfer (autodephosphorylation) of RRs appear to be tuned to the timescales of the biological processes in which RRs participate, and reported rate constants span six orders of magnitude. More complex variations of TCSs, called phosphorelays, contain histidine-containing phosphotransfer domain (Hpt), which contain the phosphoaccepting histidine in Asp → His phosphotransfer. First, we propose a strategy for assessment of functional variation within protein families. We used bioinformatics to guide experimental analysis of non-conserved receiver domain active site residues D+2 and T+2 (two amino acids C-terminal to conserved Asp phosphorylation site and Thr/Ser, respectively). Using bioinformatics, we were able to determine a representative and feasible set of D+2/T+2 pairs to assess for kinetic effects on receiver domain autodephosphorylation. We also report results of additional bioinformatics analysis of receiver domain sequences assigned to RR subfamilies (determined by the presence of specific output domains). Second, this work represents the first reported demonstration of a small molecule analog for Hpts in TCSs. Imidazole is the functional group of His. Here, we characterize the imidazole-mediated dephosphoryation reaction of receiver domains. Additionally, we use imidazole to probe non-conserved active site residues as potential determinants of TCS Asp → His phosphotransfer reactions. Further, we show the structure of a receiver domain in the presence of a phosphomimic and imidazole. This work presents novel approaches to answering fundamental questions regarding receiver domain dephosphorylation reactions in two-component systems, and proposes avenues to further probe phosphotransfer reactions in TCSs and functional variation in protein families in general.
- Date of publication
- May 2016
- Resource type
- Rights statement
- In Copyright
- Cotter, Peggy
- Collins, Edward
- Campbell, Sharon
- Lee, Andrew
- Bourret, Robert R.
- Doctor of Philosophy
- Degree granting institution
- University of North Carolina at Chapel Hill Graduate School
- Graduation year
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