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 (MTSET) 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 membrane-cytoplasm interface.
Highlights
The secondary citrate transporter CitS of Klebsiella pneumoniae is a member of the bacterial 2-hydroxycarboxylate transporter (2HCT)1 family
The rate of uptake in Right side out (RSO) membranes prepared from E. coli cells expressing CitS and energized by the artificial electron donor system ascorbate/ phenazine methosulfate (PMS) increased sigmoidally with increasing Naϩ concentration
We investigated the accessibility of residues Cys-398 and Cys-414 in region Xa of the Naϩ citrate transporter CitS of K. pneumoniae in different catalytic states of the transporter
Summary
Recent crystal structures of the LacY and GlpT proteins, both secondary transporters from the major facilitator superfamily, support the alternating access model for the translocation mechanism of secondary transporters [12, 13] In this model, the proteins contain single binding sites for substrate and co-ions that alternately are exposed to the two sides of the membrane during the catalytic cycle. In a number of cases, this was observed for residues in loop regions, rather than TMSs, which let to the suggestion of pore-loop structures or re-entrant loops in secondary transportmethylammonium)ethyl] methanethiosulfonate bromide; FM, fluorescein 5-maleimide; MTS, methanethiosulfonate; DTT, dithiothreitol The results are consistent with a pore-loop structure formed by part of region Xa
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