RESUMO
Cation-coupled active transport is an essential cellular process found ubiquitously in all living organisms. Here, we present two novel ligand-free X-ray structures of the lactose permease (LacY) of Escherichia coli determined at acidic and neutral pH, and propose a model for the mechanism of coupling between lactose and H+ translocation. No sugar-binding site is observed in the absence of ligand, and deprotonation of the key residue Glu269 is associated with ligand binding. Thus, substrate induces formation of the sugar-binding site, as well as the initial step in H+ transduction.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Lactose/metabolismo , Proteínas de Transporte de Monossacarídeos/química , Simportadores/química , Sítios de Ligação , Concentração de Íons de Hidrogênio , Transporte de Íons , Ligantes , Conformação Proteica , Especificidade por Substrato , Difração de Raios XRESUMO
Crystallization is a major bottleneck to obtaining x-ray structures of membrane proteins. By applying an established crystallization protocol for the lactose permease (LacY) of Escherichia coli, a systematic study of the effect of phospholipids (PL) on crystallization of LacY was undertaken. We observe three different crystal forms that diffract to increasingly better resolution in a manner that correlates with the concentration of copurified PL. Consistently, progressive addition of E. coli PL to delipidated LacY leads to different crystal forms. Tetragonal crystals are obtained with improved diffraction quality for a stable mutant by carefully adjusting PL content. Furthermore, crystals of good quality from wild-type LacY, a particularly difficult protein, were also obtained by using same approach. Thus, it is likely that manipulation of PL is a good strategy for predominantly hydrophobic membrane proteins like LacY.
Assuntos
Cristalização/métodos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/metabolismo , Fosfolipídeos/química , Simportadores/química , Simportadores/metabolismo , Cromatografia em Gel , Dimerização , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Modelos Moleculares , Proteínas de Transporte de Monossacarídeos/genética , Estrutura Quaternária de Proteína , Simportadores/genéticaRESUMO
Biochemical, luminescence and mass spectroscopy approaches indicate that Trp-151 (helix V) plays an important role in hydrophobic stacking with the galactopyranosyl ring of substrate and that Glu-269 (helix VIII) is essential for substrate affinity and specificity. The x-ray structure of the lactose permease (LacY) with bound substrate is consistent with these conclusions and suggests that a possible H-bond between Glu-269 and Trp-151 may play a critical role in the architecture of the binding site. We have now probed this relationship by exploiting the intrinsic luminescence of a single Trp-151 LacY with various replacements for Glu-269. Mutations at position 269 dramatically alter the environment of Trp-151 in a manner that correlates with binding affinity of LacY substrates. Furthermore, chemical modification of Trp-151 with N-bromosuccinimide indicates that Glu-269 forms an H-bond with the indole N. It is concluded that 1) an H-bond between the indole N and Glu-269 optimizes the formation of the substrate binding site in the inward facing conformation of LacY, and 2) the disposition of the residues implicated in sugar binding in different conformers suggests that sugar binding by LacY involves induced fit.
