Thiol cross-linking of cytoplasmic loops in the lactose permease of Escherichia coli

The N- and C-terminal halves of lactose permease, each with a single-Cys re sidue in a cytoplasmic loop, were coexpressed, and cross-linking was studie d in the absence or presence of ligand. Out of the 68 paired-Cys mutants in cytoplasmic loops NN and VII/IX or X/XI, three pairs in loop IV/V and X/XI , (i) Arg135 --> Cys/Thr338 --> Cys, (ii) Arg134 --> Cys/Va1343 --> Cys, an d (iii) Arg134 --> Cys/Phe345 --> Cys, form a spontaneous disulfide bond, i ndicating that loops IV/V and X/XI are in close proximity. In addition, spe cific paired-Cys residues in loop IV/V (132-138) and loop VIII/IX (282-290) or loop X/XI (335-345) cross-link with iodine and/or the homobifunctional cross-linking agents N,N'-o-phenylenedimaleimide, N,N'-p-phenylenedimaleimi de, and 1,6-bis(maleimido)hexane, The results demonstrate that loop IV/V is close to both loop VIII/IX and loop X/XI, On the other hand, similar thoug h less extensive cross-linking studies indicate that neither the N terminus nor loop II/III appear Co be close to loops VIII/IX or X/XI. The findings suggest that the longer cytoplasmic loops are highly flexible and interact in a largely random fashion. However, although a Cys residue at position 13 4 in loop IV/V, for example, is able to cross-link with a Cys residue at ea ch position in loop VIII/IX or loop X/XI, Cys residues at other positions i n loop IV/V exhibit markedly different cross-linking; patterns. Therefore, although the domains appear to be very flexible, the interactions are not c ompletely random, suggesting that there are probably at least some structur al constraints that Limit the degree of flexibility. In addition, evidence is presented suggesting that ligand binding induces conformational alterati ons between loop IV/V and loop VII/IX or X/XI.