Loop VIII/IX of the Na+-citrate transporter CitS of Klebsiella pneumoniae folds into an amphipathic surface helix

The sodium ion-dependent citrate transporter CitS of Klebsiella pneumoniae is a member of, the 2-hydroxycarboxylate transporter (2HCT) family whose members transport divalent citrate in symport with two sodium ions. Profiles of the hydrophobic moment suggested the presence of an amphipathic helical structure in the cytoplasmic loop between transmembrane segments (TMSs) VIII and IX (the AH loop) in all members of the family. Cysteine-scanning mutagenesis was used to study the secondary structure of the AH loop. We have mutated 20 successive residues into cysteine residues, characterized each of the mutants for its transport activity, and determined the accessibility of the residues. Three of the mutants, G324C, F331C, and F332C, had very low citrate transport activity, and two others, I321C and S333C, exhibited significantly decreased activity after treatment of right-side-out membranes with membrane permeable thiol reagent N-ethylmaleimide (NEM), but not with membrane impermeable 4-acetamido-4 '-maleimidylstilbene-2,2 '-disulfonic acid (AmdiS) and [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET). No protection against NEM was observed with citrate or sodium ions. Labeling of the cysteine residues in the 20 mutants with the fluorescent probe fluorescein 5-maleimide, in membrane vesicles with an inverted orientation, resulted in a clear periodicity in the accessibility of the residues. Residues expected to be at the hydrophobic face of the putative alpha-helix were not accessible for the label, whereas those at the hydrophilic face were easily accessed and labeled. Pretreatment of whole cells and inside-out membranes expressing the mutants with the membrane impermeable reagent AmdiS confirmed the cytoplasmic localization of the AH region. It is concluded that the loop between TMSs VIII and IX folds into an amphipathic surface helix

Accessibility of cysteine residues in a cytoplasmic loop of CitS of Klebsiella pneumoniae is controlled by the catalytic state of the transporter

The citrate transporter CAS of Klebsiella pneumoniae is a secondary transporter that transports citrate in symport with two sodium ions and one proton. Treatment of CAS with the alkylating, agent N-ethylmaleimide resulted in a complete loss of transport activity. Treatment of mutant proteins in which the five endogenous cysteine residues were mutated into serines in different combinations revealed that two cysteine residues located in the C-terminal cytoplasmic loop, Cys-398 and Cys-414, were responsible for the inactivation. Labeling with the membrane impermeable methanethiosulfonate derivatives MTSET and MTSES in right-side-out membrane vesicles showed that the cytoplasmic loop was accessible from the periplasmic side of the membrane. The membrane impermeable but more bulky maleimide AmdiS did not inactivate the transporter in right-side-out membrane vesicles. Inactivation by N-ethylmaleimide, MTSES, and MTSET was prevented by the presence of the co-ion Na+. Protection was obtained upon binding 2 Na+, which equals the transport stoichiometry. In the absence of Na+, the substrate citrate had no effect on the inactivation by permeable or impermeable thiol reagents. In contrast, when subsaturating concentrations of Na+ were present, citrate significantly reduced inactivation suggesting ordered binding of the substrate and co-ion; citrate is bound after Na+. In the presence of the proton motive force, the reactivity of the Cys residues was increased significantly for the membrane permeable N-ethylmaleimide, while no difference was observed for the membrane impermeable thiol reagents. The results are discussed in the context of a model for the opening and closing of the translocation pore during turnover of the transporter

Secondary transporters of the 2HCT family contain two homologous domains with inverted membrane topology and trans re-entrant loops

The 2-hydroxycarboxylate transporter (2HCT) family of secondary transporters belongs to a much larger structural class of secondary transporters termed ST3 which contains about 2000 transporters in 32 families. The transporters of the 2HCT family are among the best studied in the class. Here we detect weak sequence similarity between the N- and C-terminal halves of the proteins using a sensitive method which uses a database containing the N- and C-terminal halves of all the sequences in ST3 and involves BLAST searches of each sequence in the database against the whole database. Unrelated families of secondary transporters of the same length and composition were used as controls. The sequence similarity involved major parts of the N- and C-terminal halves and not just a small stretch. The membrane topology of the homologous N- and C-terminal domains was deduced from the experimentally determined topology of the members of the 2HCT family. The domains consist of five transmembrane segments each and have opposite orientations in the membrane. The N terminus of the N-terminal domain is extracellular, while the N terminus of the C-terminal domain is cytoplasmic. The loops between the fourth and fifth transmembrane segment in each domain are well conserved throughout the class and contain a high fraction of residues with small side chains, Gly, Ala and Ser. Experimental work on the citrate transporter CitS in the 2HCT family indicates that the loops are re-entrant or pore loops. The re-entrant loops in the N- and C-terminal domains enter the membrane from opposite sides (trans-re-entrant loops). The combination of inverted membrane topology and trans-re-entrant loops represents a new fold for secondary transporters and resembles the structure of aquaporins and models proposed for Na+/Ca2+ exchangers