Role of bundle helices in a regulatory crosstalk in the trimeric betaine transporter BetP.
The Na(+)-coupled betaine symporter BetP regulates transport activity in response to hyperosmotic stress only in its trimeric state, suggesting a regulatory crosstalk between individual protomers. BetP shares the overall fold of two inverted structurally related five-transmembrane (TM) helix repeats with the sequence-unrelated Na(+)-coupled symporters LeuT, vSGLT, and Mhp1, which are neither trimeric nor regulated in transport activity. Conformational changes characteristic for this transporter fold involve the two first helices of each repeat, which form a four-TM-helix bundle. Here, we identify two ionic networks in BetP located on both sides of the membrane that might be responsible for BetP's unique regulatory behavior by restricting the conformational flexibility of the four-TM-helix bundle. The cytoplasmic ionic interaction network links both first helices of each repeat in one protomer to the osmosensing C-terminal domain of the adjacent protomer. Moreover, the periplasmic ionic interaction network conformationally locks the four-TM-helix bundle between the same neighbor protomers. By a combination of site-directed mutagenesis, cross-linking, and betaine uptake measurements, we demonstrate how conformational changes in individual bundle helices are transduced to the entire bundle by specific inter-helical interactions. We suggest that one purpose of bundle networking is to assist crosstalk between protomers during transport regulation by specifically modulating the transition from outward-facing to inward-facing state.
Department of Structural Biology, Max-Planck Institute of Biophysics, Max-von-Laue Strasse 3, 60438 Frankfurt am Main, Germany.
Rebecca M
RM
Gärtner
Camilo
C
Perez
Caroline
C
Koshy
Christine
C
Ziegler
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001622:Betaine / Q000737:chemistry*; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D048230:Corynebacterium glutamicum / Q000378:metabolism*; D018360:Crystallography, X-Ray / Q000379:methods; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D027981:Symporters
D001426:Bacterial Proteins; C101652:BetP protein, Corynebacterium glutamicum; D002352:Carrier Proteins; D027981:Symporters; D001622:Betaine