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Summary: Ronald Kaback's research is focused primarily on the structure and mechanism of energy-transducing membrane transport proteins.

Lactose permease (LacY) is a paradigm for membrane transport proteins, which comprise a highly significant percentage of the genomes sequenced. Encoded by the lacY gene, LacY transduces free energy stored in an electrochemical H⁺ gradient into a sugar concentration gradient by catalyzing the coupled stoichiometric translocation of galactosides and H⁺ (lactose/ H⁺ symport). In the absence of substrate, LacY does not translocate H⁺, and a substrate concentration gradient in itself generates a H⁺ electrochemical gradient, indicating that the primary trigger for turnover is binding and dissociation of substrate on opposite sides of the membrane.

Site-directed mutagenesis of wild-type LacY and complete Cys-scanning mutagenesis of a functional mutant devoid of Cys residues has allowed delineation of functionally important amino acids. Furthermore, application of a battery of site-directed methods—thiol crosslinking, second-site suppressor analysis, excimer fluorescence, engineered divalent metal-binding sites, spin-spin or metal-spin interactions, chemical cleavage, and identification of discontinuous monoclonal antibody epitopes—to an extensive library of mutants has led to a static structure at the level of ~4 Å by applying distance constraint-based torsion-angle dynamic structure calculation methods (Figure 1), as well as dynamic information indicating that the permease is a highly flexible molecule. Recently, in collaboration with Jeff Abramson, Bernadette Byrne, and So Iwata (Imperial College, London), we have obtained three-dimensional crystals of a LacY mutant locked in one conformation. The crystals are hexagonal rods that are ~0.05 × 0.05 × 0.2 mm in size. They exhibit a space group of P6₄22 with cell dimensions a = 86, b = 86, and c = 412 Å. The crystals exhibit anisotropic diffraction (3.5/5 Å).

	Figure 1. Structural models of LacY... Figure 2. Proposed binding site in LacY... Scheme 1. QuickTime movie: Wild-type mechanism... Scheme 2. QuickTime movie: Rescue of E325D mutant...

Of the 417 residues in LacY, only 6 side chains are irreplaceable for active transport. Glu126 (helix IV) and Arg144 (helix V) are directly involved in substrate binding and specificity. A detailed view of binding site interactions based on biochemical observations is shown in Figure 2, with lactose as ligand. The galactopyranosyl ring contains all of the determinants for specificity, and the C-4 OH is most important. Galactose is the most specific substrate for LacY but has very low affinity (K_d ~ 30 mM); however, various adducts at the 1 position can increase affinity by more than 3 orders of magnitude. Cys148 (helix V) interacts weakly and hydrophobically with the galactosyl moiety, and Ala122 (helix IV) is in close proximity to the nongalactosyl moiety. Although LacY with W151F or W151Y transports lactose almost as well as wild type, the mutants exhibit 50- and 20-fold decreases in affinity, respectively, indicating Trp151 stacks with the hydrophobic face of the galactopyranosyl ring, placing it at a right angle with helix IV and abutting Cys148 near the 1 position. In this orientation, the C-4 OH can H bond directly with either NH1 or NH2 of Arg144, and the C-6 OH is in close proximity to Glu126. Since the C-3 OH is close to Glu269 (helix VIII) but at an angle, it is reasonable to suggest that a water molecule may mediate this interaction.

Arg302 (helix IX), His322 (helix X), and Glu325 (helix X) are involved in H⁺ translocation and/or coupling between H⁺ and substrate translocation. In addition, Arg302, His322, and Glu325 are in close proximity (Figure 1C) and at about the same depth in the membrane as the major components of the substrate-binding site. A mechanistic model for lactose/H⁺ symport has been postulated that describes the interactions between the irreplaceable side chains during LacY turnover. In the ground state (Scheme 1, first panel), LacY is protonated, the H⁺ is shared between His322 and Glu269, and Glu325 is charge paired with Arg302. Since de-energized right-side-out membrane vesicles containing LacY or purified LacY in detergent bind ligand with high affinity, the protonated, high-affinity state of LacY represents the conformation with the lowest free energy. In this conformation, LacY binds ligand primarily at the interface between helices IV and V at the outer surface of the membrane (Scheme 1). Substrate binding induces a conformational change that leads to transfer of the H⁺ from His322/Glu269 to Glu325 and reorientation of the binding site to the inner surface with release of sugar. Glu325 is deprotonated on the inside due to rejuxtaposition with Arg302 as the conformation relaxes, and the His322/Glu269 complex is reprotonated from the outside surface to reinitiate the cycle.

The model explicitly predicts that LacY must be protonated to bind ligand in the ground state and that His322 and Glu269 form the site of protonation. Evidence for this notion was obtained by Miklós Sahin-Tóth, who studied the relationship between substrate affinity and the protonation state of the residues directly involved in H⁺ translocation and/or coupling between sugar and H⁺ translocation. Specifically, he examined the effect of pH on ligand binding in single-Cys148 LacY without or with conservative or neutral replacements for Glu325, Arg302, His322, or Glu269, utilizing a unique binding assay in which substrate protection of Cys148 against alkylation by N-ethylmaleimide (NEM) is quantified. By this means, affinities ranging from submicromolar to high millimolar are measured accurately. The affinity of single-Cys148 LacY mutants with Glu325 (Asp or Gln) or Arg302 replacements (Lys or Ala) is markedly decreased at alkaline pH, exhibiting an apparent pK_a of 8–9, while His322 replacements (Ala, Asn, or Gln) or Glu269 replacement with Asp shows essentially no change in binding affinity as a function of pH. Because the apparent pK_a is significantly higher than the pK_a of an unperturbed imidazole, we concluded that the H⁺ is shared between His322 and Glu269, thereby raising the pK_a.

Another important prediction of the model with respect to H⁺ translocation is that upon substrate binding the Glu269/His322 and Arg302/Glu325 pairs are disrupted and the H⁺ is transferred to Glu325. Subsequently, after substrate dissociates, rejuxtaposition between Arg302 and Glu325 is thought to be the primary driving force for deprotonation of the carboxylic acid as the protein relaxes back to the ground-state conformation. Evidence supporting this is provided by demonstrations that LacY mutants with neutral replacements for Glu325 or Ala or Ser in place of Arg302 are specifically defective in all translocation modes that involve net H⁺ movement (active lactose accumulation, as well as influx or efflux down a concentration gradient), but bind ligand and catalyze equilibrium exchange and counterflow as well or better than wild type. Therefore, Glu325 must play a direct role in H⁺ translocation, and its deprotonation is facilitated by interaction with Arg302 (Scheme 1).

The conservative mutation E325D causes a 10-fold reduction in the V_max for active lactose transport and markedly decreases lactose-induced H⁺ influx with no effect on exchange or counterflow, neither of which involve H⁺ symport. Thus, shortening the side chain may weaken the interaction of the carboxyl group at position 325 with the guanidino group of Arg302. Therefore, Adam Weinglass and Irina Smirnova employed Gly-scanning mutagenesis of helices IX and X and the intervening loop systematically with mutant E325D in an effort to rescue function by introducing flexibility between the two helices. Five Gly-replacement mutants in the E325D background have been identified that exhibit significantly higher transport activity. In particular, mutant V316G/E325D catalyzes active transport, efflux, and lactose-induced H⁺ influx with kinetic properties approaching those of wild-type LacY. Therefore, introduction of flexibility or a change in helix packing at the interface between helices IX and X improves rejuxtapositioning between Arg302 and Asp325 during turnover, thereby allowing more effective deprotonation of the permease on the inner surface of the membrane (Scheme 2).

This work was partially supported by National Institutes of Health.

¹Site-directed mutants are designated by the one-letter amino acid code followed by a number indicating the position of the residue in wild-type lactose permease and a second letter denoting the amino acid replacement at this position.

Last updated June 10, 2004