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

Abstract

SummaryA 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 a -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.

Study of Amide-proton Exchange of Escherichia coliMelibiose Permease by Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy

“…It should finally be mentioned that alignment of the amino acid sequence of homologous transporters composing the galactosides-pentoses-hexuronides family (52,53) supports the existence of conserved motifs in almost every loop 4 -5 ( 132 (P/ G)X 4 DXRER 141 ) and in cytoplasmic loop 10 -11 ( 351 DX 2 (E/ D)YGX 6 R 364 ). These data further emphasize the potential functional/structural importance of the loops with significant ␤-sheet content for sugar transport.…”

A melibiose transporter and an operon containing its gene in Enterobacter cloacae

“…However, the level of sequence similarity between MelY of E. cloacae and the MelB transporters of these bacteria was not high. We found some similarity between a region (Phe26 to Ala338) of MelY and a region (Phe19 to Thr337) of E. coli MelB (revised numbering) (16,18). There were 15% identity and 58% similarity in these regions of E. cloacae MelY and E. coli MelB.…”

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

“…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). MelB models strongly suggest that it is a 12 transmembrane (TM) helices transporter (18)(19)(20)(21)(22)(23).…”