Loop X/XI, the largest cytoplasmic loop in the membrane-bound melibiose carrier of Escherichia coli, is a functional re-entrant loop.
The melibiose carrier of Escherichia coli is a membrane-bound sugar-cation cotransporter consisting of 12 transmembrane helices connected by cytoplasmic and periplasmic loops, with both N- and C-terminus on the cytoplasmic side. Using a functional cysteine-less carrier, cysteine was substituted individually for residues 347-378 that comprise the largest cytoplasmic loop X/XI. The majority of the cysteine mutants have good protein expression levels. The cysteine mutants were studied for their transport activities, and the inhibitory effects of two sulfhydryl reagents, PCMBS (7-A long) and BM (29-A long). Cysteine substitution resulted in substantial loss of transport in 12 mutants. While PCMBS caused significant inhibition in only two mutants, T373C and V376C, from the periplasmic side (in a substrate-protective manner), more extensive inhibition pattern was observed from the cytoplasmic side, in seven mutants: V353C, Y358C, V371C, Q372C, T373C, V376C and G378C, suggesting that these residues are along the sugar pathway in the aqueous channel, close to the cytoplasmic side. Furthermore, the inhibitory effect of BM on the inside-out vesicles of the above mutants was clearly less than that of PCMBS, suggesting channel space limitation to large molecules, consistent with those residues being inside the channel. Three second-site revertants (A350C/F268L, A350C/I22S, and A350C/I22N) were selected. They may suggest proximities between loop X/XI and helices I and VIII, in agreement with a re-entrant loop structure. Self thiol cross-linkings of the cysteine mutants on loop X/XI failed to form dimers, suggesting that most of the loop is not surface-exposed from cytoplasmic side. Together, these results strongly indicated a functional re-entrant loop mechanistically important in Na+-coupled transporters.
Ding
Ping Z
PZ
Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA. pingding9308@hotmail.com
D002731:4-Chloromercuribenzenesulfonate; D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008553:Melibiose / Q000378:metabolism; D008969:Molecular Sequence Data; D019897:Periplasm / Q000378:metabolism; D010957:Plasmids; D013439:Sulfhydryl Reagents; D027981:Symporters / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D013439:Sulfhydryl Reagents; D027981:Symporters; D002731:4-Chloromercuribenzenesulfonate; C021548:melibiose permease; D008553:Melibiose; D003545:Cysteine