Mutational Analysis of the Proteins BtuB and TonB: Their Roles in Cobalamin Transport

Barekzi, Nazir, Department of Microbiology, University of Virginia
Nakamoto, Bob, Department of Microbiology, University of Virginia
Bouton, Amy, Department of Microbiology, University of Virginia
Fox, Jay, Department of Microbiology, University of Virginia
Cafiso, David, Department of Chemistry, University of Virginia
Goldberg, Joanna, Department of Microbiology, University of Virginia
Smith, Mitch, Department of Microbiology, University of Virginia
Brown, Jay, Department of Microbiology, University of Virginia

The E. coli transporter, BtuB, is an integral membrane protein which translocates vitamin B 12 (CNCbl) and other cobalamin derivatives from the extracellular milieu into the periplasmic compartment by an active transport process. BtuB is part of a membrane system that can be utilized in vitro and in vivo providing a model system for the study of active transport. Substrateinduced structural changes of the wild-type and mutant BtuB proteins relating to the enzyme mechanism have been examined. Radiolabeled CNCbl substrate was used to probe transport in whole cells and purified membranes. The binding and transport of CNCbl was affected by mutations in the Tonbox that resulted in transport-negative mutants. In this study, we show that substitutions in the amino-terminal Tonbox residues (6-DTLVVTA-12) alter CNCbl binding at the extracellular face of BtuB. Cold-chase experiments show that the substitution of five alanines in the Tonbox of a functional mutant (V10C) results in a BtuB mutant named 5AV10C (BtuB- 6A+7A+8A+9A+10C+11A) with a 60-fold slower release rate of bound CNCbl. Interestingly, the 5AV10C mutant with higher affinity for substrate still interacts with TonB. Experiments using N-biotinoyl-N-6-maleimideohexanoyl-hydrazide labeling and probe dynamics by electron paramagnetic resonance analysis of the 5AV10C mutant amino-terminus shows a constitutively accessible Tonbox. These data suggest that the Tonbox of a mutant with higher affinity for CNCbl may be in an extended conformation. Furthermore, in the presence of iii uncouplers of the proton motive force, the constitutively accessible conformation of the Tonbox was evident in the V10C mutant which has wild-type activity. A proposed mechanism for active transport that is consistent with these observations is a BtuB protein recycling model. In this model substrate binding, transport and release from one side of the membrane to the other is dependent on three factors: 1) Specific residues in the Tonbox; 2) a functional proton motive force; and 3) an unaltered communication signal between the Tonbox and substrate binding site. In sum, we have identified critical specificity determinants in the periplasmic face of BtuB that is required for active transport of cobalamin in E. coli.

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PHD (Doctor of Philosophy)
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