Biochemical and biophysical characterization of N-terminal domain of KdpD, a potassium biosensor

Mani Singh, Purdue University

Abstract

In many bacterial two-component signal transduction systems, membrane-bound sensors transmit environmental signals to response regulators that launch cellular responses. This study focuses on KdpD, a sensor kinase capable of autophosphorylation, followed by activation of the transcriptional factor, KdpE, by transphosphorylation. The signaling is terminated by dephosphorylation promoted by KdpD. In KdpD, a central unit containing four transmembrane segments is flanked at the N- and C-termini by 400 residue regions that are exposed to the cytoplasm. The C-terminal region contains a classical histidine kinase domain, whereas the atypical amino-terminal region contains two domains, namely `NTP' (N-terminal P-loop motif) and `Usp' (universal stress protein), whose precise roles are unclear. Previous studies suggest that KdpD integrates multiple signals including drop in concentration of extracellular K+, changes in turgor resulting from ionic hyperosmolarity, and modification of membrane phospholipid composition. However, the precise signal(s) and parts of the protein responsible for signal perception are unknown. Our hypothesis is that during potassium limitation, NTP domain senses the modified membrane composition by directly interacting with the anionic phospholipids and regulates the autophosphorylation-dephosphorylation equilibrium of KdpD in an ATP dependent manner. The NTP domain is the most conserved region of the protein and contains the classic Walker A and Walker B nucleotide binding motifs. Using purified NTP domain, we show that the protein interacts with the negatively charged lipids and undergoes detergent and lipid dependent oligomerization in addition to secondary structural changes. Furthermore, the strength of ATP binding is dependent on the presence of surfactant and lipid mixtures and most interestingly, the NTP domain is capable of hydrolyzing ATP. Although, no significant change in ATP hydrolytic activity of NTP domain occurs in the presence of anionic lipids, deletion of this NTP domain shows significant reduction in kdpFABC expression under K+ limiting conditions. These studies suggest that the NTP domain interaction with membranes rich in negatively charged lipids and NTP domain's ability to hydrolyze ATP are two independent events. In summary, our data indicates a complex regulatory role of NTP domain of KdpD in sensing and for regulating the kdpFABC expression.

Degree

Ph.D.

Advisors

Stauffacher, Purdue University.

Subject Area

Biochemistry|Biophysics

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