Alternating Access and a Pore-Loop Structure in the Na+-Citrate Transporter CitS of Klebsiella pneumoniae

Abstract

CitS of Klebsiella pneumoniae is a secondary transporter that transports citrate in symport with 2 Na ؉ ions. Reaction of Cys-398 and Cys-414, which are located in a cytoplasmic loop of the protein that is believed to be involved in catalysis, with thiol reagents resulted in significant inhibition of uptake activity. The reactivity of the two residues was determined in single Cys mutants in different catalytic states of the transporter and from both sides of the membrane. The single Cys mutants were shown to have the same transport stoichiometry as wild type CitS, but the C398S mutation was responsible for a 10-fold loss of affinity for Na ؉ . Both cysteine residues were accessible from the periplasmic as well as from the cytoplasmic side of the membrane by the membrane-impermeable thiol reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MT-SET) suggesting that the residues are part of the translocation site. Binding of citrate to the outward facing binding site of the transporter resulted in partial protection against inactivation by N-ethylmaleimide, whereas binding to the inward facing binding site resulted in essentially complete protection. A 10-fold higher concentration of citrate was required at the cytoplasmic rather than at the periplasmic side of the membrane to promote protection. Only marginal effects of citrate binding were seen on reactivity with MTSET. Binding of Na ؉ at the periplasmic side of the transporter protected both Cys-398 and Cys-414 against reaction with the thiol reagents, whereas binding at the cytoplasmic side was less effective and discriminated between Cys-398 and Cys-414. A model is presented in which part of the cytoplasmic loop containing Cys-398 and Cys-414 folds back into the translocation pore as a pore-loop structure. The loop protrudes into the pore beyond the citrate-binding site that is situated at the membranecytoplasm interface.The secondary citrate transporter CitS of Klebsiella pneumoniae is a member of the bacterial 2-hydroxycarboxylate transporter (2HCT) 1 family. Members of the 2HCT family transport substrates that contain the 2-hydroxycarboxylate motif, as in citrate, malate, lactate, etc. (1). The family contains H ϩ and Na ϩ symporters that couple the translocation of the substrate to the co-ion as well as precursor/product exchangers that couple the uptake of the substrate to the excretion of a metabolic end product (2, 3). CitS is a Na ϩ symporter. It obligatorily couples the translocation of the divalent citrate anion (Hcit 2Ϫ ) to the translocation of two sodium ions and one proton (4, 5). The CitS protein is an integral membrane protein containing 446 amino acid residues (47.5 kDa). The protein is believed to consist of a bundle of 11 hydrophobic transmembrane segments (TMSs) with the N and C terminus located in the cytoplasm and periplasm, respectively ( Fig. 1) (6 -9). An additional hydrophobic segment, predicted to be transmembrane as well, was shown to reside in the periplasm between TMSs V and VI. The segment, termed Vb, and a stretch ...

The 2-Hydroxycarboxylate Transporter Family: Physiology, Structure, and Mechanism

“…8) was obtained by labeling of endogenous or engineered cysteine residues in the loops. Two native cysteine residues located in the Xa region were shown to be inaccessible from the periplasm with the membrane-impermeative reagent AmdiS (4-acetamido-4Ј-maleimidylstilbene-2,2Ј-disulfonate) but were readily accessible from the cytoplasm (135). The same results were obtained for a series of engineered cysteine residues in loop AH (see below) (136).…”

“…The coupling stoichiometry of two Na ϩ ions was observed at the pH range of pH 6 to 8, suggesting that H ϩ does not compete with Na ϩ for the two binding sites (76). A strict coupling between citrate and Na ϩ ions is also supported by point mutations that lower the affinity for Na ϩ by an order of magnitude but leave the stoichiometry unaltered (135). The differing opinions on the coupling stoichiometry of CitS extend to the kinetic mechanism of the transporter (see "Kinetics" in "MECHANISMS" below) (109).…”

“…More recently, small-scale purification methods were developed for members of the 2HCT family strictly for analytical purposes (64,135,136). The purification method is based on Ni 2ϩ -nitrilotriacetic acid affinity chromatography and yields enough protein for visualization by SDS-PAGE.…”

“…The purification method is based on Ni 2ϩ -nitrilotriacetic acid affinity chromatography and yields enough protein for visualization by SDS-PAGE. This method was used in labeling studies of single-Cys mutants of CimH of B. subtilis (63) and CitS of K. pneumoniae (135,136).…”

Substrate Binding Tunes Conformational Flexibility and Kinetic Stability of an Amino Acid Antiporter

“…Transporters alternate between two conformations to expose their binding sites to the cytoplasmic and extracellular side (15)(16)(17)(18)(19)(20)(21)(22). However, prior to conformational changes substrates have to be recognized and bound.…”