title: Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP.
abstract: Sodium-coupled substrate transport plays a central role in many biological processes. However, despite knowledge of the structures of several sodium-coupled transporters, the location of the sodium-binding site(s) often remains unclear. Several of these structures have the five transmembrane-helix inverted-topology repeat, LeuT-like (FIRL) fold, whose pseudosymmetry has been proposed to facilitate the alternating-access mechanism required for transport. Here, we provide biophysical, biochemical, and computational evidence for the location of the two cation-binding sites in the sodium-coupled betaine symporter BetP. A recent X-ray structure of BetP in a sodium-bound closed state revealed that one of these sites, equivalent to the Na2 site in related transporters, is located between transmembrane helices 1 and 8 of the FIRL-fold; here, we confirm the location of this site by other means. Based on the pseudosymmetry of this fold, we hypothesized that the second site is located between the equivalent helices 6 and 3. Molecular dynamics simulations of the closed-state structure suggest this second sodium site involves two threonine sidechains and a backbone carbonyl from helix 3, a phenylalanine from helix 6, and a water molecule. Mutating the residues proposed to form the two binding sites increased the apparent K(m) and K(d) for sodium, as measured by betaine uptake, tryptophan fluorescence, and (22)Na(+) binding, and also diminished the transient currents measured in proteoliposomes using solid supported membrane-based electrophysiology. Taken together, these results provide strong evidence for the identity of the residues forming the sodium-binding sites in BetP.
authors.affiliation: Computational Structural Biology Group, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany.
authors.forename: Kamil
authors.initials: K
authors.lastname: Khafizov
authors.forename: Camilo
authors.initials: C
authors.lastname: Perez
authors.forename: Caroline
authors.initials: C
authors.lastname: Koshy
authors.forename: Matthias
authors.initials: M
authors.lastname: Quick
authors.forename: Klaus
authors.initials: K
authors.lastname: Fendler
authors.forename: Christine
authors.initials: C
authors.lastname: Ziegler
authors.forename: Lucy R
authors.initials: LR
authors.lastname: Forrest
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D050485:GABA Plasma Membrane Transport Proteins; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; C072207:betaine plasma membrane transport proteins; D012964:Sodium