Close approximation of putative alpha-helices II, IV, VII, X, and XI in the translocation pathway of the lactose transport protein of Streptococcus thermophilus

The lactose transport protein (LacS) of Streptococcus thermophilus belongs to a family of transporters in which putative alpha-helices II and TV have been implicated in cation binding and the coupled transport of the substrat e and the cation, Here, the analysis of site-directed mutants shows that a positive and negative charge at positions 64 and 71 in helix II are essenti al for transport, but not for lactose binding. The conservation of charge/s ide-chain properties is less critical for Glu-67 and Ile-70 in helix II, an d Asp-133 and Lys-139 in helix TV, but these residues are important for the coupled transport of lactose together with a proton. The analysis of secon d-site suppressor mutants indicates an ion pair exists between helices II a nd IV, and thus a close approximation of these helices can be made. The sec ond-site suppressor analysis also suggests ion pairing between helix II and the intracellular loops 6-7 and 10-11, Because the C-terminal region of th e transmembrane domain, especially helix XI and loop 10-11, is important fo r substrate binding in this family of proteins, we propose that sugar and p roton binding and translocation are performed by the joint action of these regions in the protein. Indeed, substrate protection of maleimide labeling of single cysteine mutants confirms that cy-helices II and TV are directly interacting or at least conformationally involved in sugar binding and/or t ranslocation, On the basis of new and published data, we reason that the he lices II, IV, VII, X, and XI and the intracellular loops 6-7 and 10-11 are in close proximity and form the binding sites and/or the translocation path way in the transporters of the galactosides-pentosides-hexuronides family.