Secondary structure components and properties of the melibiose permease from Escherichia coli: a fourier transform infrared spectroscopy analysis.

The structure of the melibiose permease from Escherichia coli has been investigated by Fourier transform infrared spectroscopy, using the purified transporter either in the solubilized state or reconstituted in E. coli lipids. In both instances, the spectra suggest that the permease secondary structure is dominated by alpha-helical components (up to 50%) and contains beta-structure (20%) and additional components assigned to turns, 3(10) helix, and nonordered structures (30%). Two distinct and strong absorption bands are recorded at 1660 and 1653 cm(-1), i.e., in the usual range of absorption of helices of membrane proteins. Moreover, conditions that preserve the transporter functionality (reconstitution in liposomes or solubilization with dodecyl maltoside) make possible the detection of two separate alpha-helical bands of comparable intensity. In contrast, a single intense band, centered at approximately 1656 cm(-1), is recorded from the inactive permease in Triton X-100, or a merged and broader signal is recorded after the solubilized protein is heated in dodecyl maltoside. It is suggested that in the functional permease, distinct signals at 1660 and 1653 cm(-1) arise from two different populations of alpha-helical domains. Furthermore, the sodium- and/or melibiose-induced changes in amide I line shape, and in particular, in the relative amplitudes of the 1660 and 1653 cm(-1) bands, indicate that the secondary structure is modified during the early step of sugar transport. Finally, the observation that approximately 80% of the backbone amide protons can be exchanged suggests high conformational flexibility and/or a large accessibility of the membrane domains to the aqueous solvent.

Evidence for a role of helix IV in connecting cation- and sugar-binding sites of Escherichia coli melibiose permease.

To improve the structural organization model of melibiose permease, we assessed the individual contributions of the N-terminal tryptophans to the transporter fluorescence variations induced by the binding of cations and beta-configured sugars, by replacement of the six N-terminal tryptophans by phenylalanines and the study of the signal changes. Only two mutations, W116F located in helix IV and W128F located in the cytoplasmic loop 4-5, impair permease activity. The intrinsic fluorescence spectroscopy analysis of the other mutants suggests that W54, located in helix II, W116, and W128 are mostly responsible for the cation-induced fluorescence variations. These tryptophans, W116 and W128, would also be responsible for the beta-galactoside-induced fluorescence changes observed in the N-terminal domain of the transporter. The implication of W116 and W128 in both the cation- and beta-galactoside-induced fluorescence variations led us to investigate in detail the effects of their mutations on the functional properties of the permease. The results obtained suggest that the domains harboring the two tryptophans, or the residues themselves, play a critical role in the mechanism of Na(+)/sugar symport. Taken together, the results presented in this paper and previous results are consistent with a fundamental role of helix IV in connecting cation- and sugar-binding sites of the melibiose permease.

Structural studies of the melibiose permease of Escherichia coli by fluorescence resonance energy transfer. I. Evidence for ion-induced conformational change.

Further insight into the cosubstrate-induced structural change of the melibiose permease (MelB) of Escherichia coli has been sought by investigating the binding and spectroscopic properties of the fluorescent sugar 2'-(N-5-dimethylaminonaphthalene-1-sulfonyl)aminoethyl 1-thio-beta-D-galactopyranoside (Dns2-S-Gal) and related analogs (Dns3-S-Gal or Dns6-S-Gal with a propyl or hexyl instead of an ethyl linker, respectively) interacting with MelB in membrane vesicles or in proteoliposomes. The three analogs efficiently inhibit melibiose transport and bind to MelB in a sodium-dependent fashion. Their dissociation constants (Kd) are in the micromolar range in the presence of NaCl and an order of magnitude higher in its absence. In the presence of NaCl and Dns2-S-Gal, sample excitation at 335 or 297 nm gives rise to a fluorescent signal at around 465 nm, whereas Dns3-S-Gal or Dns6-S-Gal emits a fluorescence light at 490 or 506 nm, respectively. Detailed study of the Dns2-S-Gal signal elicited by a 297 nm illumination indicates that a tryptophan-mediated fluorescence resonance energy transfer phenomenon is involved in the response. All fluorescence signals below 500 nm are prevented by addition of melibiose in excess, and the kinetic constants describing their dependence on the probe or NaCl concentrations closely correlate with the probe binding constants. Finally, the Dns2-S-Gal signal recorded in sodium-free medium is red shifted by up to 25 nm from that recorded in the presence of NaCl. Taken together, these results suggest (i) that the fluorescence signals below 500 nm arise from Dns-S-Gal molecules bound to MelB, (ii) the presence of a highly hydrophobic environment close to or at the sugar-binding site, the polarity of which increases on moving away from the sugar-binding site, and (iii) that the interaction of sodium ions with MelB enhances the hydrophobicity of this environment. These results are consistent with the induction of a cooperative change of the structure of the sugar-binding site or of its immediate vicinity by the ions.

Structural studies of the melibiose permease of Escherichia coli by fluorescence resonance energy transfer. II. Identification of the tryptophan residues acting as energy donors.

In the accompanying paper, we demonstrated the presence of a fluorescence resonance energy transfer (FRET) between the tryptophans of the melibiose permease (MelB) of Escherichia coli and a fluorescent sugar, 2'-(N-5-dimethylaminonaphthalene-1-sulfonyl)aminoethyl-1-thio-beta-D- galactopyranoside (Dns2-S-Gal) bound at the sugar-binding site (Maehrel, C., Cordat, E., Mus-Veteau, I., and Leblanc, G. (1998) J. Biol. Chem. 273, 33192-33197). To identify the tryptophans that transfer their energy to the fluorescent sugar, we analyzed the FRET properties of MelB mutants carrying the replacement of each of the eight MelB tryptophans by a phenylalanine. The data indicate that Trp64, localized in loop 2-3 from the N-terminal domain, and Trp299, localized in helix IX in the C-terminal domain, are responsible for up to 80% of the FRET signal. Moreover, by assuming that only Trp299 transfers energy to Dns2-S-Gal in mutant W64F, whereas only Trp64 transfers energy to Dns2-S-Gal in mutant W299F, we calculated that Trp299 and Trp64 are about 14 and 20 A away from the probe, respectively. In addition, we observed that mutating Trp342, localized in helix X of the C-terminal domain, produces a significant increase of the polarity of the fluorescent sugar environment, suggesting its proximity to the sugar-binding site. Taken together, these data provide additional support for the suggestion that (i) the sugar-binding site is localized in the C-terminal part of the transporter, probably close to membrane segments IX and X, and (ii) the N-terminal domain, and particularly cytoplasmic loop 2-3, is also close to the sugar-binding site.

Melibiose permease of Escherichia coli: structural organization of cosubstrate binding sites as deduced from tryptophan fluorescence analyses.

Binding of the coupling ion (Na+ or Li+) and sugars to the purified melibiose permease of Escherichia coli, reconstituted in proteoliposomes, produces selective and cooperative changes of the transporter tryptophan fluorescence. To assess the individual contribution of N- or C-terminal domains of the permease to these substrate-induced fluorescence variations, we replaced the two tryptophans located in its C-terminal half (W299 and W342) by a phenylalanine and compared the signal change in mutants and wild-type permease. None of the mutations significantly impairs transport activity. Persistence of the ion-induced signal quenching in a permease carrying only the six other tryptophans of the N-terminal domain is consistent with a previous suggestion that this domain accommodates the ion-binding site. On the other hand, the sugar-induced fluorescence increase varies from mutant to mutant in a sugar-specific fashion. While alpha-galactosides increase essentially the fluorescence of W299 and W342, beta-galactosides enhance the signal of W299 and of one (or more) of the N-terminal tryptophans but quench that of W342. Moreover, addition of sugars producers a 10 nm blue shift of both W299 and W342 emission spectra, suggesting reduced accessibility of these residues to solvent following substrate binding. These data suggest that W299 and W342 are at or close to the sugar binding site and that this latter is lined by the C-terminal helices IX and X. Moreover, as sugars with the beta-configuration also enhance the fluorescence of the N-terminal tryptophans, it is suggested that one (or more) helix of the N-terminal half may be also at or near the sugar binding site. This implies close proximity and/or tight functional linkage between some N-terminal helices and helices IX and X of the C-terminal domain of the transporter.

Cation and sugar selectivity determinants in a novel family of transport proteins.

A new family of homologous membrane proteins that transport galactosides-pentoses-hexuronides (GPH) is described. By analysing the aligned amino acid sequences of the GPH family, and by exploiting their different specificities for cations and sugars, we have designed mutations that yield novel insights into the nature of ligand binding sites in membrane proteins. Mutants have been isolated/constructed in the melibiose transport proteins of Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium, and the lactose transport protein of Streptococcus thermophilus which facilitate uncoupled transport or have an altered cation and/or substrate specificity. Most of the mutations map in the amino-terminal region, in or near amphipathic alpha-helices II and IV, or in interhelix-loop 10-11 of the transport proteins. On the basis of the kinetic properties of these mutants, and the primary and secondary structure analyses presented here, we speculate on the cation binding pocket of this family of transporters. The regulation of the transporters through interaction with, or phosphorylation by, components of the phosphoenolpyruvate:sugar phosphotransferase system is also discussed.

Melibiose permease of Escherichia coli: substrate-induced conformational changes monitored by tryptophan fluorescence spectroscopy.

Tryptophan fluorescence spectroscopy has been used to investigate the effects of sugars and coupling cations (H+, Na+, or Li+) on the conformational properties of purified melibiose permease after reconstitution in liposomes. Melibiose permease emission fluorescence is selectively enhanced by sugars, which serve as substrates for the symport reaction, alpha-galactosides producing larger variations (13-17%) than beta-galactosides (7%). Moreover, the sugar-dependent fluorescence increase is specifically potentiated by NaCl and LiCl (5-7 times), which are well-established activators of sugar binding and transport by the permease. The potentiation effect is greater in the presence of LiCl than NaCl. On their own, sodium and lithium ions produce quenching of the fluorescence signal (2%). Evidence suggesting that sugars and cations compete for their respective binding sites is also given. Both the sugar-induced fluorescence variation and the NaCl(or LiCl)-dependent potentiation effect exhibit saturation kinetics. In each ionic condition, the half-maximal fluorescence change is found at a sugar concentration corresponding to the sugar-binding constant. Also, half-maximal potentiation of the fluorescence change by sodium or lithium occurs at a concentration comparable to the activation constant of sugar binding by each ion. The sugar- and ion-dependent fluorescence variations still take place after selective inactivation of the permease substrate translocation capacity by N-ethylmaleimide. Taken together, the data suggest that the changes in permease fluorescence reflect conformational changes occurring upon the formation of ternary sugar/cation/permease complexes.

Involvement of histidine residues in the catalytic mechanism of hydrogenases.

In spite of their structural and amino acid sequence differences, Fe-only and Ni-containing hydrogenases achieved the same catalytic reactions. A chemical modification of histidine residues using a highly specific reagent (pentaammineruthenium II) has been carried out on Desulfovibrio vulgaris Hildenborough Fe-hydrogenase and Desulfovibrio desulfuricans Norway Ni-Fe-Se-hydrogenase. The preliminary results obtained suggest the existence of a general mechanism involving histidine residues in the two groups of hydrogenases. These residues may be part of the histidine-containing motive shown to be present in both Fe- and Ni-Fe-hydrogenase sequences by Hydrophobic Cluster Analysis. This analysis also allows us to suggest a functional role for the small subunit of Desulfovibrio vulgaris Hildenborough Fe-hydrogenase.

Site-directed mutagenesis of tetraheme cytochrome c3. Modification of oxidoreduction potentials after heme axial ligand replacement.

The nature of the axial ligands of a heme group is an important factor in maintaining the oxidation-reduction potential of a c-type cytochrome. Cytochrome c3 from Desulfovibrio vulgaris Hildenborough contains four bis-histidinyl coordinated hemes with low oxidation-reduction potentials. Site-directed mutagenesis was used to generate a mutant in which histidine 70, the sixth axial ligand of heme 4, has been replaced by a methionine. The mutant protein was expressed in Desulfovibrio desulfuricans G200 at a level similar to the wild type cytochrome. A model for the three-dimensional structure of D. vulgaris Hildenborough cytochrome c3 was generated on the basis of the crystal structure of D. vulgaris Miyazaki cytochrome c3 in order to investigate the effects of the H70M mutation. The model, together with NMR data, suggested that methionine 70 has effectively replaced histidine 70 as the sixth axial ligand of heme 4 without significant alteration of the structure. A large increase of at least 200 mV of one of the four oxidation-reduction potentials was observed by electrochemistry and is interpreted in terms of structure/potential relationships.