Structural and biochemical characterization of the vitamin B12 ABC transporter, BtuCD-F

by Borths, Elizabeth Loraine

Abstract (Summary)
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.
Bibliographical Information:

Advisor:Douglas C. Rees; Pamela Jane Bjorkman; David Chan; Scott E. Fraser

School:California Institute of Technology

School Location:USA - California

Source Type:Master's Thesis



Date of Publication:10/27/2004

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