Protein-Protein Interactions And The Energy Coupling Mechanism In TonB-Dependent Transport

Abstract

BtuB is a TonB-dependent outer membrane transporter of vitamin B12 in E. coli. In this work, we investigated the interaction between BtuB and the inner membrane protein TonB using site-directed spin labeling (SDSL). In CHAPS/POPC mixed micelles, the Ton box of BtuB undergoes an order-to-disorder transition upon addition of vitamin B12 which appears to be identical to that seen in POPC bilayers. Under these conditions, addition of a C-terminal fragment of TonB broadens the EPR lineshapes, indicating that there is an ordering of the Ton box and an interaction between the transporter Ton box and this C-terminal fragment. Residues N-terminal to the Ton box do not appear to interact with TonB. These changes appear to be independent of the addition of the substrate, vitamin B12. The EPR data obtained are generally consistent with the crystal structure that has been obtained for this complex (Shultis et al. Science 312, (2006)); however, preliminary distance measurements using DEER indicate that there may be multiple states of TonB when it is bound to BtuB. Spin labels incorporated into TonB also become ordered upon interaction with BtuB, and the EPR lineshapes indicate that there is a decrease in backbone dynamics of TonB upon association with BtuB. An EPR based equilibrium binding assay was carried out to determine the affinity between this C-terminal TonB fragment and BtuB, and was performed using either labels on BtuB or labels on TonB. Both labels indicate that there is an affinity of approximately 50 μM between BtuB and TonB, which is, unexpectedly, independent of substrate addition.This work was supported by GM035215.