|M.Sc Student||Sabri Alabad Siwar|
|Subject||Substrate Recognition and ATPase Activity of the E. Coli|
Cysteine/Cystine ABC Transporter YecSC-FliY
|Department||Department of Medicine||Supervisor||? Oded Lewinson|
|Full Thesis text|
All cells depend on high-affinity transport proteins for the acquisition of essential nutrients. Among these transporters are the ATP binding cassette transporters (ABC transporters) which comprise the largest family of membrane proteins of any proteome. This family of transporters use the energy derived from ATP hydrolysis to move a wide range of biomolecules against their concentration gradients. ABC transporters exist as either exporters or importers, with the exporters present in all three kingdoms of life, while importers present solely in bacteria, where they are the major determinant for the high affinity acquisition of the main elements of life (nitrogen, carbon, sulfur, and phosphor).
Among these elements, sulfur is indispensable for many biological processes such as amino acids biogenesis, formation of iron-sulfur clusters, redox homeostasis, transition metal coordination and the biosynthesis of the sulfur-containing amino acids L-methionine and L-cysteine and taurine .Therefore, bacteria had evolved elaborate systems to acquire sulfur from sulfur-containing organic compounds.
In this work, I studied the FliY-YecSC ABC import system for cystine and cysteine, both of which are important bacterial sulfur sources. For this, I over-expressed and purified the components of the FliY-YecSC system and studied their function in vitro using a combination of biochemical and biophysical approaches.
I find that FliY, the substrate binding protein (SBP) binds L-cysteine, D-cysteine, L-cystine (but not D-cystine) with micro-molar affinity. However, the L- and D-enantiomers induce different conformational changes, and only the L-enantiomers interact with FliY productively.
YecSC (the transporter) has low basal ATPase activity, which is stimulated by apo-FliY, more strongly by D-cysteine-bound FliY, and maximally by L-cysteine- or L-cystine- bound FliY. However, at high concentrations of FliY maximal ATPase rates are reached, independent of the presence of substrate. The results suggest that FliY exists in a conformational equilibrium between an open-unliganded form, which does not bind to the transporter, and closed-unliganded and -liganded forms that bind to the transporter with different affinities yet equally stimulate its ATPase activity. These findings are different from what had been observed for similar ABC transporters, and further demonstrate the extent of mechanistic diversity in this large and important family of proteins.