RT Journal Article SR Electronic T1 Osmotic stress response in BetP: How lipids and K+ team up to overcome downregulation JF bioRxiv FD Cold Spring Harbor Laboratory SP 2022.06.02.493408 DO 10.1101/2022.06.02.493408 A1 Veronika Heinz A1 Günnur Güler A1 Vanessa Leone A1 M. Gregor Madej A1 Stanislav Maksimov A1 Rebecca M. Gärtner A1 Olga Rudi A1 Farzad Hamdi A1 Panagiotis L. Kastritis A1 Werner Mäntele A1 Reinhard Krämer A1 Lucy R. Forrest A1 Camilo Perez A1 Christine Ziegler YR 2022 UL http://biorxiv.org/content/early/2022/06/03/2022.06.02.493408.abstract AB The trimeric betaine symporter BetP senses an osmotic upshift via its osmosensory C-terminal domain and responds rapidly with a steep increase in transport rate. Full activation requires both an elevated internal K+ concentration and a yet unknown membrane stimulus. Moreover, the molecular mechanisms of stress sensing and upregulation remain unclear. Here, we show that K+ binding to BetP in vivo is highly cooperative. Using X-ray crystallography, we identify four putative K+ interaction sites at the C-terminal domains of BetP. Single particle CryoEM on BetP reconstituted in amphipols (AMP-BetP), in the absence of K+, revealed that the three C-terminal domains are oriented symmetrically near the membrane surface, representing a new downregulated state. The presence of K+ resulted in asymmetric partial unfolding of the C-terminal domains, which was assigned as an intermediate between the downregulated state and the conformation observed in crystal structures. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy in 2D crystals of BetP reveal glutamate/aspartate and tyrosine responses to K+, in agreement with the identified K+ interaction sites, as well as specific unfolding events in the C-terminal domain upon activation. A rearrangement of the relative protomer orientations confers upregulation through key structural elements involved in the alternating access of BetP affecting sodium and betaine binding affinities. Although K+-specific regulation is unique to BetP we discuss unfolding/refolding of sensory domains as a unifying element in hyperosmotic stress response of osmoregulated transporters.Competing Interest StatementThe authors have declared no competing interest.