Melibiose Permease of Escherichia coli: Substrate-Induced Conformational Changes Monitored by Tryptophan Fluorescence Spectroscopy

Abstract

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.

Structural insights into the activation mechanism of melibiose permease by sodium binding

“…As a result of the substrate coupling, Trp fluorescence intensity increases upon binding of Na þ to MelB WT or C-less in the presence of melibiose, reflecting conformational changes responsible for the Na þ -induced increase in the affinity for the melibiose (13). None of the mutants studied here showed any significant intensity variation upon incubation with melibiose or with Na þ (in the presence of melibiose) (Fig.…”

“…S1). Evidence that conformational changes occur at different stages of the MelB transport cycle has been obtained using biochemical and electrophysiological methods, intrinsic and fluorescence energy transfer spectroscopy, and substrate-induced infrared difference (IR diff ) spectroscopy by attenuated total reflection (ATR) (13,15,(31)(32)(33)(34).In the past, several mutagenesis studies have shown that four Asp residues (Asp19, Asp55, Asp59, Asp124), located in the putative N-terminal six-helices bundle of MelB, are crucial for Na þ -dependent affinity increase and transport of melibiose, justifying their assignment as Na þ ligands (35-38). Nevertheless, direct evidence showing that mutation of these residues directly interferes with Na þ binding is still lacking and the assigned role of these residues remains still tentative.…”

“…S1). Evidence that conformational changes occur at different stages of the MelB transport cycle has been obtained using biochemical and electrophysiological methods, intrinsic and fluorescence energy transfer spectroscopy, and substrate-induced infrared difference (IR diff ) spectroscopy by attenuated total reflection (ATR) (13,15,(31)(32)(33)(34).…”

“…MelB efficiently couples the uphill transport of α-or β-galactosides to the favorable entry of Na þ , (10,11). In the past, this property has been extensively exploited to investigate the molecular and structural basis of the ion-MelB interaction and implications in the coupling properties inherent to the symport mechanism (9,(12)(13)(14)(15)(16). This strategy led to show, for example, that the three cations compete for the same binding site, that the sugar/ion stoichiometry is 1∕1 and that either cosubstrate enhances the affinity for its companion substrate as a manifestation of their coupling (9,17).…”

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

“…Spectroscopic and/or crystallographic methods are presently used to determine the structure of these transporters and also to obtain additional insight into the structure-function relationships. Illustration of this future line of research is given by a recent fluorescence spectroscopy study of purified MelB EC , which has shown that sugar and cation binding is co-operative and induces conformational change(s) in the transporter (Mus-Veteau et al, 1995).…”