Intersubunit Cross-talk in the Betaine Permease BetP from Corynebacterium glutamicum

The secondary active glycine-betaine transporter BetP of the gram-positive soil bacterium Corynebacterium glutamicum has become a paradigm for osmo-regulated transport systems. BetP is the only transport protein regulated by osmolality for which the sensing domain is clearly established and for which structural information at the level of atomic resolution is available. Biochemical studies have shown that the transporter is activated by raising potassium concentrations both in vivo and in vitro which are sensed by the C-terminal, hydrophilic domain, located in the cytoplasm. The C-terminal sensor domain was shown to be involved in inter-protomer contacts in the homo-trimeric quarternary structure of the transporter. It was furthermore shown that mutations impairing the trimerisation of the transporter severely affect the regulatory function. Furthermore several structural studies reported conformational asymmetry of single protomers to occur in BetP. These findings suggested the occurrence of a regulatory relevant cross-talk between the protomers of the homo-trimeric BetP. The interaction of the C-terminal domain with the cytoplasmic loop2 of the adjacent protomer was speculated to be crucial for the osmosensing function. To test the relevance of conformational cross-talk between BetP protomers, artificial hetero-trimeric BetP was established in the current work. With this system it was possible to clearly exclude any functionally relevant cross-talk in the activation of BetP. The established hetero-trimers where shown to be a useful tool for the analysis of the molecular dynamics of the transporter via spectroscopic methods. Beside BetP only two different transport proteins have been prooven to be regulated by the external osmolality. Purification and reconstitution of the osmoregulated betaine transporter LcoP from C. glutamicum proved the osmosensing function of this carrier as well. For LcoP the osmosensing domains in this transporter could be identified in the C- and N-terminal hydrophilic domains. Furthermore it could be shown that this protein is a promising candidate for structural analysis. It may therefore be used as test system for conclusions that were drawn for the whole family of Betaine-Carnithine-Choline-Transporters based on data obtained from the the betaine permease BetP