The role of trimerization in the osmoregulated betaine transporter BetP

Abstract

The osmoregulated betaine transporter BetP is a stable trimer. Structural studies have shown that individual protomers can adopt distinct transport conformations, implying a functional role for the trimeric state in transport, although the role of trimerization in regulation is not yet understood. We designed putative monomeric mutants by molecular-dynamics simulations and in silico alanine-scanning mutagenesis. Several mutants including BetP-W101A/T351A were monomeric in detergent as well as in the membrane, as shown by blue native gel electrophoresis, crosslinking and electron microscopy. This monomeric form retains the ability to accumulate betaine, but is no longer regulated by hyperosmotic shock.

Role of Bundle Helices in a Regulatory Crosstalk in the Trimeric Betaine Transporter BetP

“…We investigated the impact of each interaction on transport and regulation by site-directed mutagenesis, cross-linking, and betaine uptake measurements and show striking similarities to ionic networks in LeuT. Consistent to very recent data on the role of the trimeric state, 12 we give evidence that also the interaction network on the periplasmic side between adjacent protomers is involved in transport regulation.…”

Ectoine and Hydroxyectoine as Protectants against Osmotic and Cold Stress: Uptake through the SigB-Controlled Betaine-Choline- Carnitine Transporter-Type Carrier EctT from Virgibacillus pantothenticus

“…The transport activity of BetP is activated in less than a second in response to osmotic stress (32). BetP activation is elicited by an osmotic stressderived cellular signal (possibly increases in the intracellular K ϩ pool) that modulates the intersubunit interaction of the extended carboxy terminus of BetP within the BetP trimer assembly (39,42). The EctT transporter from V. pantothenticus lacks such an extended carboxy terminus, and our finding that the transport activity of EctT is not strongly activated by an osmotic stimulus (Fig.…”

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

“…MD simulations were carried out on (a) a BetP trimer from PDB ID 2WIT prepared and carried out as described previously (37), with two sodium ions added in each protomer at previously proposed locations pNa1 and pNa2 (Fig. S2) (4); and (b) on a BetP monomer from PDB ID 4AIN chain B, which is in a closed conformation (ref.…”

“…S2 and Materials and Methods) are suitable for binding sodium, we first performed MD simulations of structures of BetP trimers from Ressl et al (4), embedded into hydrated palmitoyl oleoyl phosphatidylglycerol (POPG) bilayers. The structures of the protomers within the trimers were stable over the course of the simulations, as measured by the rmsd of the Cα atoms in the TM helices from the crystal structure, which were in the range 1.2-1.5 Å (± 0.1 Å), averaged over each simulation (37). It should be noted that although the structure used for these simulations appears to be sterically occluded with respect to the substrate betaine, it nev- ertheless contains a narrow pathway that is accessible to water molecules from the cytoplasm (4).…”

“…The functional transporting unit of BetP is the protomer, whereas the trimer is a prerequisite for osmoregulation of transport activity (37). Each protomer contains a separate substrate pathway and is expected to bind one betaine molecule along with two sodium ions, reflecting the stoichiometry of substrate transport (4,38,39).…”