Structural and Biochemical Characterization of the Vitamin B₁₂ ABC Transporter, BtuCD-F

ABC transporters;BtuCD;BtuF;Vitamin B12

Borths, Elizabeth Loraine ; Rees, Douglas C.

University:California Institute of Technology

Department:Chemistry and Chemical Engineering

关键词: ABC transporters;    BtuCD;    BtuF;    Vitamin B12;   

Others  :  https://thesis.library.caltech.edu/9/7/Prologue.pdf

美国|英语

来源: Caltech THESIS

【 摘 要 】

BtuCD-F is a binding protein-dependent ABC transporter system that uses the power of ATP hydrolysis to pump vitamin B₁₂ into the cytoplasm of E. coli.The crystal structure of BtuF, the protein that binds vitamin B₁₂ and delivers it to the transporter, BtuCD, has been solved by x-ray crystallography.BtuF is a bi-lobed protein and B₁₂ is bound in a deep cleft formed at the interface between the two lobes.A stable complex between BtuF and BtuCD is demonstrated to form in vitro and was modeled using the individual crystal structures.Two conserved surface glutamates from BtuF may interact with conserved arginine residues on the periplasmic surface of the BtuCD transporter, playing a role in docking and the transmission of conformational changes.

BtuCD has also been reconstituted in vitro into proteoliposomes.In the presence of ATP, BtuCD proteoliposomes can mediate uptake of vitamin B₁₂ in a BtuF dependent fashion.In the absence of ATP, B₁₂ appears to become sequestered between BtuF and BtuCD.The ATPase activity of BtuCD was examined in proteoliposomes as well as in detergent solution.BtuCD has a significant basal rate of hydrolysis under all conditions tested, and B₁₂-bound and apo-BtuF can stimulate that rate.Interestingly, the rate of ATP hydrolysis, as well as the effect of BtuF, vitamin B₁₂ and sodium ortho-vanadate on that rate, is different in each detergent and lipid environment.These results indicate that ABC transporters are highly sensitive to their environment and underline the importance of detergent or lipid choice in functional reconstitution and membrane protein crystallization experiments.Our results lead us to propose a revised model of the ABC transport cycle.