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  • Title: From membrane to molecule to the third amino acid from the left with a membrane transport protein.
    Author: Kaback HR, Wu J.
    Journal: Q Rev Biophys; 1997 Nov; 30(4):333-64. PubMed ID: 9634651.
    Abstract:
    The lac permease of E. coli is a paradigm for secondary active transporter proteins that transduce the free energy stored in electrochemical ion gradients into work in the form of a concentration gradient. This hydrophobic, polytopic, cytoplasmic membrane protein catalyses the coupled, stoichiometric translocation of beta-galactosides and H+, and it has been solubilized, purified, reconstituted into artificial phospholipid vesicles and shown to be solely responsible responsible for beta-galactoside transport as a monomer. The lacY gene which encodes the permease has been cloned and sequenced, and all available evidence indicates that the protein has 12 transmembrane domains in alpha-helical configuration that traverse the membrane in zigzag fashion connected by hydrophilic loops with the N and C termini on the cytoplasmic face of the membrane. Extensive use of site-directed and Cys-scanning mutagenesis indicates that very few residues in the permease are directly involved in the transport mechanism, but the permease appears to be a highly flexible protein that undergoes widespread conformational changes during turnover. Based on a variety of site-directed approaches which include second-site suppressor analysis and site-directed mutagenesis, excimer fluorescence, engineered divalent metal binding sites, chemical cleavage, EPR, thiol crosslinking and identification of discontinuous mAb epitopes, a helix packing model has been formulated.A mechanism for the coupled translocate ion of substrate and H+ by the lac permease of E. coli is proposed. Four residues are irreplaceable with respect to coupling, and the residues are paired in the tertiary structure--Arg-302 (helix IX) with Glu-325 (helix 10) and His-322 (helix 10) with Glu-269 (helix VIII). In an adjacent region of the molecule at the interface between helices VIII and V is the substrate translocation pathway in which Glu-126 and Arg-144 appear to play key roles. Because of this arrangement, interfacial changes between helices VIII and V are transmitted to the interface between helices IX and X and vice versa. Upon ligand binding, a structural change at the interface between helices V and VIII disrupts the interaction between Glu-269 and His-322, Glu-269 displaces Glu-325 from Ag-302 and Glu-325 is protonated.Simultaneously, protonated Glu-325 becomes inaccessible to water which drastically increases its pKa. In this configuration, the permease undergoes a freely reversible conformational change that corresponds to translocation of the ternary complex. In order to return to ground state after release of substrate, the Arg-302-Glu-325 interaction must be reestablished which necessitates loss of H+ from Glu-325. The H+ is released into a water-filled crevice between helices IX and X which becomes transiently accessible to both sides of the membrane due to a change in helix tilt, where it is acted upon equally by either the membrane potential or the pH gradient across the membrane. Remarkably few amino-acid residues appear to be critically involved in the transport mechanism of lac permease, suggesting that relatively simple chemistry drives the mechanism. On the other hand, widespread, cooperative conformational changes appear to be involved in turnover. As a whole the data suggest that the 12 helices which comprise the permease are loosely packed with a considerable amount of water in the interstices and that surface contours are important for sliding or tilting motions that occur during turnover. This surmise coupled with the indication that few residues are essential to the mechanism is encouraging in that it suggest that the possibility that a relatively low resolution structure (i.e. helix packing) plus localization of the critical residues and the translocation pathway can provide important insights into the mechanism. (ABSTRACT TRUNCATED)
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