An investigation of cysteine mutants on the cytoplasmic loop X/XI in the melibiose transporter of Escherichia coli by using thiol reagents: implication of structural conservation of charged residues.
The melibiose transporter (Mel B) of Escherichia coli is a cation-coupled (H(+), Li(+), and Na(+)) membrane protein (MW 50 kDa) consisting of 12 transmembrane helices that are connected by periplasmic and cytoplasmic loops, with both the C- and N-ends located on the cytoplasmic side of the membrane. Previous investigations on the largest cytoplasmic loop X/XI indicated that it is a functional re-entrant loop. In this communication, the cysteine mutants on loop X/XI were studied with charged thiol reagents MTSES, MTSET, and IAA for both the inhibition patterns and charge replacement/function rescue of inactive mutants in which the original charged residues were replaced by neutral cysteines. Strong inhibitions were observed in T373C and V376C by both MTSES and MTSET, consistent with previous results of PCMBS inhibition. The thiol reagents failed to recover the activities of inactive mutants D351C, D354C, and R363C and to inhibit active mutants E357C, K359C, and E365C to any significant extent, suggesting a structural conservation at D351, D354, and R363 and tolerance of structural variations at E357, K359, and E365. The results are consistent with previous observation of structural conservation of functionally charged residues in the transmembrane domains and extend to a loop the contention that in the melibiose transporter functionally important charged residues are structurally conserved.
Ding
Ping Z
PZ
Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA. ping_ding@hms.harvard.edu
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002478:Cells, Cultured; D003545:Cysteine / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D019807:Iodoacetic Acid / Q000494:pharmacology; D008553:Melibiose / Q000378:metabolism; D008698:Mesylates / Q000494:pharmacology; D009154:Mutation; D017433:Protein Structure, Secondary; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D027981:Symporters / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics
D000596:Amino Acids; D029968:Escherichia coli Proteins; D008698:Mesylates; D013439:Sulfhydryl Reagents; D027981:Symporters; C109153:(2-sulfonatoethyl)methanethiosulfonate; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D002731:4-Chloromercuribenzenesulfonate; C021548:melibiose permease; D008553:Melibiose; D003545:Cysteine; D019807:Iodoacetic Acid