Substrate-induced changes in the structural properties of LacY

Abstract

The lactose permease (LacY) of Escherichia coli, a paradigm for the major facilitator superfamily, catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H + across the cytoplasmic membrane. To catalyze transport, LacY undergoes large conformational changes that allow alternating access of sugarand H + -binding sites to either side of the membrane. Despite strong evidence for an alternating access mechanism, it remains unclear how H + -and sugar-binding trigger the cascade of interactions leading to alternating conformational states. Here we used dynamic single-molecule force spectroscopy to investigate how substrate binding induces this phenomenon. Galactoside binding strongly modifies kinetic, energetic, and mechanical properties of the N-terminal 6-helix bundle of LacY, whereas the C-terminal 6-helix bundle remains largely unaffected. Within the N-terminal 6-helix bundle, the properties of helix V, which contains residues critical for sugar binding, change most radically. Particularly, secondary structures forming the N-terminal domain exhibit mechanically brittle properties in the unbound state, but highly flexible conformations in the substrate-bound state with significantly increased lifetimes and energetic stability. Thus, sugar binding tunes the properties of the N-terminal domain to initiate galactoside/H + symport. In contrast to wild-type LacY, the properties of the conformationally restricted mutant Cys154➝Gly do not change upon sugar binding. It is also observed that the single mutation of Cys154➝Gly alters intramolecular interactions so that individual transmembrane helices manifest different properties. The results support a working model of LacY in which substrate binding induces alternating conformational states and provides insight into their specific kinetic, energetic, and mechanical properties.atomic force microscopy | membrane | transport protein | membrane protein structure | membrane protein folding | membrane transport T he lactose permease of Escherichia coli (LacY) of the major facilitator superfamily (MFS) (1, 2) catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H + (sugar/H + symport) (3-6). Uphill (i.e., active) symport of galactoside against a concentration gradient is achieved by transduction of free energy released from the downhill movement of H + with the electrochemical H + gradient (Δμ H + ; interior negative and/or alkaline). Conversely, because coupling between sugar and H + is obligatory, downhill galactoside transport from a high to a low sugar concentration is coupled to uphill H + transport with the generation of Δμ H +, the polarity of which depends upon the direction of the sugar concentration gradient (7-10).LacY monomers reconstituted into proteoliposomes are functional (11, 12), and X-ray crystal structures reveal 12, mostly irregular, transmembrane α-helices organized into two pseudosymmetrical 6-helix bundles surrounding a large interior hydrophilic cavity open to the cytoplasm (13-16). At the apex of the h...

Symmetry and Structure in the POT Family of Proton Coupled Peptide Transporters

“…Our experiments, models, and molecular dynamics simulations suggest that in the POT family, the first six helices are less dynamic than the last six, consistent with a functional separation between the N-and C-terminal bundles. A similar asymmetry was also observed in LacY [44], suggesting this separation may be a feature of proton coupled MFS members. We also observe a breakdown in the simple two salt bridge mechanism with respect to PepT So2 and NRT1.1, which clearly don't require a second salt bridge to stabilise the close state of the extracellular gate.…”

Function, Structure, and Evolution of the Major Facilitator Superfamily: The LacY Manifesto

“…Accordingly, increased substrate-protein interaction in the substrateoccluding state provides the binding energy to compensate Results obtained employing dynamic single-molecule force spectroscopy support and expand this hypothesis by providing quantitative insights into kinetic, energetic, and mechanical properties of the sugar-free and sugar-bound states of LacY [116]. Sugar liganding induces a dramatic change in structural properties of the N-terminal 6-helix bundle leading to a global increase in conformational flexibility.…”

“…C154G LacY mutant exhibits fewer liganddependent conformational changes than observed with WT LacY and catalyzes little or no transport but binds ligand with high affinity [72,115,116]. The X-ray structure demonstrates that helix V crosses helix I in the approximate middle of the membrane in such a manner that Cys154 lies close to Gly24 (helix I).…”

“…In view of these considerations and the direct observation that Δ̃H+ has no effect whatsoever on equilibrium exchange or counterflow, the conclusion emerges that galactoside binding and dissociationnot Δ̃H+ -are the driving force for the alternating access mechanism (Figures 8(a) and 8(b), Steps 6 and 7). With galactoside dissociation (Figure 8(a), conformer HE + S), the transporter returns to a more static conformation [116]. The initiation of another round of transport (Figure 8 Lactose/H + symport in the active transport, (Figure 1(b); against the lactose concentration gradient-uphill) or efflux or influx, (Figures 3(a) and 3(b); thermodynamically favored movement with lactose concentration gradient-downhill) energetic modes are precisely the same reaction (Figure 8(a)).…”

“…It appears that coupling between sugar and H + translocation is solely energetic; the transporter (e.g., LacY) transduces the potential energy stored in the concentration gradient of one substrate (e.g., H + ) to facilitate the accumulation of another substrate (e.g., galactoside) against its concentration gradient. Accordingly, the hypothetical energy landscapes for the transport cycle in which the occluded intermediates occupy higher energy levels relative to a comparatively facile energy profile between the open conformers are the conformationally dynamic species [116]. The higher energy occluded intermediates tend to relax to lower-energy open conformations (Figure 8(b)).…”