ABCMdb

ABCC8 p.Val343Gly

Predicted by SNAP2:	A: N (82%), C: N (78%), D: N (78%), E: N (87%), F: N (82%), G: N (72%), H: N (72%), I: N (93%), K: N (82%), L: N (87%), M: N (93%), N: N (82%), P: N (61%), Q: N (82%), R: N (78%), S: N (93%), T: N (93%), W: D (66%), Y: N (87%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, W: N, Y: N,

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Publications
[hide] Cytoplasmic loop connecting helices IV and V of th... J Biol Chem. 2003 Jan 17;278(3):1518-24. Epub 2002 Nov 5. Abdel-Dayem M, Basquin C, Pourcher T, Cordat E, Leblanc G
Cytoplasmic loop connecting helices IV and V of the melibiose permease from Escherichia coli is involved in the process of Na+-coupled sugar translocation.
J Biol Chem. 2003 Jan 17;278(3):1518-24. Epub 2002 Nov 5., [PMID:12421811]

Abstract [show]
Previous photolabeling and limited proteolysis studies suggested that one of the four basic residues (Arg-141) of the N-terminal cytoplasmic loop connecting helices IV and V (loop 4-5) of the melibiose permease (MelB) from Escherichia coli has a potential role in its symport function (Ambroise, Y., Leblanc, G., and Rousseau, B. (2000) Biochemistry 39, 1338-1345). A mutagenesis study of Arg-141 and of the other three basic residues of loop 4-5 was undertaken to further examine this hypothesis. Cys replacement analysis indicated that Arg-141 and Arg-149, but not Lys-138 and Arg-139, are essential for MelB transport activity. Replacement of Arg-141 by neutral residues (Cys or Gln) inactivated transport and energy-independent carrier-mediated flows of substrates (counterflow, efflux), whereas it had a limited effect on co-substrate binding. R141C sugar transport was partially rescued on reintroducing a positive charge with a charged and permeant thiol reagent. Whereas R149C was completely inactive, R149K and R149Q remained functional. Strikingly, introduction of an additional mutation in the C-terminal helix X (Gly for Val-343) of R149C restored sugar transport. Impermeant thiol reagents inhibited R149C/V343G transport activity in right-side-out membrane vesicles and prevented sugar binding in a sugar-protected manner. All these data suggest that MelB loop 4-5 is close to the sugar binding site and that the charged residue Arg-141 is involved in the reaction of co-substrate translocation or substrate release in the inner compartment.

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Sentences [show]
No. Sentence Comment
6 Strikingly, introduction of an additional mutation in the C-terminal helix X (Gly for Val-343) of R149C restored sugar transport. Login to comment
7 Impermeant thiol reagents inhibited R149C/V343G transport activity in right-side-out membrane vesicles and prevented sugar binding in a sugar-protected manner. Login to comment
73 Identification of R149C/V343G Second-site Revertants-A strategy developed by Ding and Wilson was used (28). Login to comment
83 The second-site mutation present in helix X (V343G) of the revertant R149C/V343G described below is indicated in bold face. Login to comment
104 TABLE I NPG binding constants of the C-less permease, of the permease carrying different substitutions at position 141 or 149, and of the second-site revertant R149C/V343G All RSO membrane vesicles harboring the C-less or MelB mutants were prepared and concentrated (15-25 mg of protein/ml) in Na- -free, 0.1 M potassium phosphate buffer (pH 7) containing carbonyl cyanide m-chlorophenylhydrazone (5 ␮M) and monensin (0.75 ␮M) as described in the Fig. 3 legend. Login to comment
111 Mutant Location Bmax (nmoles/mg protein) KD KNa ␮M mM C-less 0.6 0.6 0.6 R141C 0.2 2.5 0.1-0.5 R141K Loop 4-5 0.2 1.7 0.3 R141Q 0.3 3.1 0.3 R149C NS NS NS R149K Loop 4-5 0.3 7.7 0.3 R149Q 0.3 7.3 0.4 R149C/V343G Loop 4-5/TM X 0.2 1.7 0.2 V343G TM X 0.3 4.4 1.6 FIG. 3. Login to comment
168 Fig. 6 shows that R149C/V343G RSO membrane vesicles accumulated melibiose up to 10% of the levels observed in C-less MelB vesicles. Login to comment
170 Remarkably, although R149C/V343G had limited capacity to concentrate the substrates, it displayed sugar affinity and Naϩ activation constants very close or identical to those of C-less MelB (Table I). Login to comment
172 One may finally note that the double mutant has a comparatively better affinity for ␣-galactosides (KD) or for Naϩ ions on the basis of the KNa value than any of the individual single R149C or V343G mutants (Table I). Login to comment
173 Finally, the effects of various thiol reagents on the transport and sugar binding properties of R149C/V343G RSO membrane vesicles were analyzed. Login to comment
177 To identify the step of the R149C/V343G transport cycle inhibited by MTSETϩ inhibited, we analyzed the effect of this reagent on the ␣-[3 H]NPG binding to RSO membrane vesicles and looked for an eventual protection by the co-substrates (Fig. 7). Login to comment
182 Inhibition of Na؉ -dependent sugar transport of R149C/ V343G in RSO membrane vesicles by MTSET؉ . Login to comment
188 Selective protection against inactivation by MTSET؉ of ␣-[3 H]NPG binding to R149C/V343G RSO membrane vesicles by the sugar. Login to comment
224 Analysis of the properties of the second-site suppressor mutant R149C/V343G provided further clues to the mechanism by which Arg-149 participates in the process of sugar binding. Login to comment
225 The presence of an additional mutation (V343G) led to partial recovery of transport activity despite the absence of the native arginine at position 149. Login to comment
230 The observed inhibition of both R149C/V343G sugar transport and binding activity by MTSETϩ or PCMBS- provides insight into the relative localization of Arg-149 with respect to the sugar binding site. Login to comment