Alteration of sugar-induced conformational changes of the melibiose permease by mutating Arg141 in loop 4-5.
The melibiose permease (MelB) from Escherichia coli couples the uptake of melibiose to that of Na+, Li+, or H+. In this work, we applied attenuated total reflection Fourier transform infrared (ATR-FTIR) difference spectroscopy to obtain information about the structural changes involved in substrate interaction with the R141C mutant and with the wild-type MelB reacted with N-ethylmaleimide (NEM). These modified permeases have the ability to bind the substrates but fail to transport them. It is shown that the sugar-induced ATR-FTIR difference spectra of the R141C mutant are different from those corresponding to the Cys-less permease from which it is derived. There are alterations of peaks assigned to turns and beta-structures located most likely in loop 4-5. In addition, and quite notably, a peak at 1659 cm(-1), assigned to changes at the level of one alpha-helix subpopulation, disappears in the melibiose-induced difference spectrum in the presence of Na+, suggesting a reduction of the conformational change capacity of the mutated MelB. These helices may involve structural components that couple the cation- and sugar-binding sites. On the other hand, MelB-NEM difference spectra are proportionally less disrupted than the R141C ones. Hence, the transport cycle of these two permeases, modified at two different loops, is most likely impaired at a different stage. It is proposed that the R141C mutant leads to the generation of a partially defective ternary complex that is unable to catalyze the subsequent conformational change necessary for substrate translocation.
León
Xavier
X
Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, Barcelona, Spain. xavier.leon@uab.cat
Leblanc
Gérard
G
Padrós
Esteve
E
D001120:Arginine / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide; D008553:Melibiose / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D011487:Protein Conformation; D013379:Substrate Specificity; D027981:Symporters / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D027981:Symporters; C021548:melibiose permease; D001120:Arginine; D008553:Melibiose; D005033:Ethylmaleimide