Diversity in kinetics correlated with structure in nano body-stabilized LacY.

Funder: research foundation-flandersThe structure of lactose permease, stabilized in a periplasmic open conformation by two Gly to Trp replacements (LacYww) and complexed with a nanobody directed against this conformation, provides the highest resolution structure of the symporter. The nanobody binds in a different manner than two other nanobodies made against the same mutant, which also bind to the same general region on the periplasmic side. This region of the protein may represent an immune hotspot. The CDR3 loop of the nanobody is held by hydrogen bonds in a conformation that partially blocks access to the substrate-binding site. As a result, kon and koff for galactoside binding to either LacY or the double mutant complexed with the nanobody are lower than for the other two LacY/nanobody complexes though the Kd values are similar, reflecting the fact that the nanobodies rigidify structures along the pathway. While the wild-type LacY/nanobody complex clearly stabilizes a similar 'extracellular open' conformation in solution, judged by binding kinetics, the complex with wild-type LacY did not yet crystallize, suggesting the nanobody does not bind strongly enough to shift the equilibrium to stabilize a periplasmic side-open conformation suitable for crystallization. However, the similarity of the galactoside binding kinetics for the nanobody-bound complexes with wild type LacY and with LacYWW indicates that they have similar structures, showing that the reported co-structures reliably show nanobody interactions with LacY

The Alternating Access Transport Mechanism in LacY

Lactose permease of Escherichia coli (LacY) is highly dynamic, and sugar binding causes closing of a large inward-facing cavity with opening of a wide outward-facing hydrophilic cavity. Therefore, lactose/H+ symport via LacY very likely involves a global conformational change that allows alternating access of single sugar- and H+-binding sites to either side of the membrane. Here, in honor of Stephan H. White’s seventieth birthday, we review in camera the various biochemical/biophysical approaches that provide experimental evidence for the alternating access mechanism

Real-time conformational changes in LacY.

Galactoside/H(+) symport across the cytoplasmic membrane of Escherichia coli is catalyzed by lactose permease (LacY), which uses an alternating access mechanism with opening and closing of deep cavities on the periplasmic and cytoplasmic sides. In this study, conformational changes in LacY initiated by galactoside binding were monitored in real time by Trp quenching/unquenching of bimane, a small fluorophore covalently attached to the protein. Rates of change in bimane fluorescence on either side of LacY were measured by stopped flow with LacY in detergent or in proteoliposomes and were compared with rates of galactoside binding. With LacY in proteoliposomes, the periplasmic cavity is tightly sealed and the substrate-binding rate is limited by the rate of opening of this cavity. Rates of opening, measured as unquenching of bimane fluorescence, are 20-30 s(-1), independent of sugar concentration and essentially the same in detergent or in proteoliposomes. On the cytoplasmic side of LacY in proteoliposomes, slow bimane quenching (i.e., closing of the cavity) is observed at a rate that is also independent of sugar concentration and similar to the rate of sugar binding from the periplasmic side. Therefore, opening of the periplasmic cavity not only limits access of sugar to the binding site of LacY but also controls the rate of closing of the cytoplasmic cavity

Protonation and sugar binding to LacY

The effect of bulk-phase pH on the apparent affinity (Kdapp) of purified wild-type lactose permease (LacY) for sugars was studied. Kdapp values were determined by ligand-induced changes in the fluorescence of either of two covalently bound fluorescent reporters positioned away from the sugar-binding site. Kdapp for three different galactopyranosides was determined over a pH range from 5.5 to 11. A remarkably high pKa of ≈10.5 was obtained for all sugars. Kinetic data for thiodigalactoside binding measured from pH 6 to 10 show that decreased affinity for sugar at alkaline pH is due specifically to increased reverse rate. A similar effect was also observed with nitrophenylgalactoside by using a direct binding assay. Because affinity for sugar remains constant from pH 5.5 to pH 9.0, it follows that LacY is fully protonated with respect to sugar binding under physiological conditions of pH. The results are consistent with the conclusion that LacY is protonated before sugar binding during lactose/H+ symport in either direction across the membrane

Probing of the rates of alternating access in LacY with Trp fluorescence

Sugar/H+ symport by lactose permease (LacY) utilizes an alternating access mechanism in which sugar and H+ binding sites in the middle of the molecule are alternatively exposed to either side of the membrane by sequential opening and closing of inward- and outward-facing hydrophilic cavities. Here, we introduce Trp residues on either side of LacY where they are predicted to be in close proximity to side chains of natural Trp quenchers in either the inward- or outward-facing conformers. In the inward-facing conformer, LacY is tightly packed on the periplasmic side, and Trp residues placed at positions 245 (helix VII) or 378 (helix XII) are in close contact with His-35 (helix I) or Lys-42 (helix II), respectively. Sugar binding leads to unquenching of Trp fluorescence in both mutants, a finding clearly consistent with opening of the periplasmic cavity. The pH dependence of Trp-245 unquenching exhibits a pKa of 8, typical for a His side chain interacting with an aromatic group. As estimated from stopped-flow studies, the rate of sugar-induced opening is ≈100 s−1. On the cytoplasmic side, Phe-140 (helix V) and Phe-334 (helix X) are located on opposite sides of a wide-open hydrophilic cavity. In precisely the opposite fashion from the periplasmic side, mutant Phe-140→Trp/Phe-334→His exhibits sugar-induced Trp quenching. Again, quenching is pH dependent (pKa = 8), but remarkably, the rate of sugar-induced quenching is only ≈0.4 s−1. The results provide yet another strong, independent line of evidence for the alternating access mechanism and demonstrate that the methodology described provides a sensitive probe to measure rates of conformational change in membrane transport proteins

An Asymmetric Conformational Change in LacY

Galactoside/H⁺ symport by the lactose permease of <i>Escherichia coli</i> (LacY) involves reciprocal opening and closing of periplasmic and cytoplasmic cavities so that sugar- and H⁺-binding sites become alternatively accessible to either side of the membrane. After reconstitution into proteoliposomes, LacY with the periplasmic cavity sealed by cross-linking paired-Cys residues does not bind sugar from the periplasmic side. However, reduction of the S–S bond restores opening of the periplasmic cavity and galactoside binding. Furthermore, nanobodies that stabilize the double-Cys mutant in a periplasmic-open conformation and allow free access of galactoside to the binding site do so only after reduction of the S–S bond. In contrast, when cross-linked LacY is solubilized in detergent, galactoside binding is observed, indicating that the cytoplasmic cavity is patent. Sugar binding from the cytoplasmic side exhibits nonlinear stopped-flow kinetics, and analysis reveals a two-step process in which a conformational change precedes binding. Because the cytoplasmic cavity is spontaneously closing and opening in the symporter with a sealed periplasmic cavity, it is apparent that an asymmetrical conformational transition controls access of sugar to the binding site

Outward-facing conformers of LacY stabilized by nanobodies.

The lactose permease of Escherichia coli (LacY), a highly dynamic polytopic membrane protein, catalyzes stoichiometric galactoside/H(+) symport by an alternating access mechanism and exhibits multiple conformations, the distribution of which is altered by sugar binding. We have developed single-domain camelid nanobodies (Nbs) against a LacY mutant in an outward (periplasmic)-open conformation to stabilize this state of the WT protein. Twelve purified Nbs inhibit lactose transport in right-side-out membrane vesicles, indicating that the Nbs recognize epitopes on the periplasmic side of LacY. Stopped-flow kinetics of sugar binding by WT LacY in detergent micelles or reconstituted into proteoliposomes reveals dramatic increases in galactoside-binding rates induced by interaction with the Nbs. Thus, WT LacY in complex with the great majority of the Nbs exhibits varied increases in access of sugar to the binding site with an increase in association rate constants (kon) of up to ∼ 50-fold (reaching 10(7) M(-1) ⋅ s(-1)). In contrast, with the double-Trp mutant, which is already open on the periplasmic side, the Nbs have little effect. The findings are clearly consistent with stabilization of WT conformers with an open periplasmic cavity. Remarkably, some Nbs drastically decrease the rate of dissociation of bound sugar leading to increased affinity (greater than 200-fold for lactose)