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.
Sobczak
Iwona
I
Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands.
Lolkema
Juke S
JS
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D002846:Chromatography, Affinity; D005033:Ethylmaleimide / Q000737:chemistry; D007711:Klebsiella pneumoniae / Q000737:chemistry*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017510:Protein Folding; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D002352:Carrier Proteins; C086246:CitS protein, bacteria; D005033:Ethylmaleimide