Determinants of sugar-induced influx in the mammalian fructose transporter GLUT5

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    The current manuscript investigates the energy landscape of the mammalian sugar porter GLUT5 using enhanced molecular dynamics simulations and biochemical assays. The approach generates important insights into the mechanism of GLUT5 conformational change, and into mechanistic diversity among the GLUT sugar porters more generally. The overall strategy is solid, but without an additional error analysis, the computational components remain incomplete. These findings will be of interest to the transporter and membrane biology communities.

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Abstract

In mammals, glucose transporters (GLUT) control organism-wide blood-glucose homeostasis. In human, this is accomplished by 14 different GLUT isoforms, that transport glucose and other monosaccharides with varying substrate preferences and kinetics. Nevertheless, there is little difference between the sugar-coordinating residues in the GLUT proteins and even the malarial Plasmodium falciparum transporter Pf HT1, which is uniquely able to transport a wide range of different sugars. Pf HT1 was captured in an intermediate ‘occluded’ state, revealing how the extracellular gating helix TM7b has moved to break and occlude the sugar-binding site. Sequence difference and kinetics indicated that the TM7b gating helix dynamics and interactions likely evolved to enable substrate promiscuity in Pf HT1, rather than the sugar-binding site itself. It was unclear, however, if the TM7b structural transitions observed in Pf HT1 would be similar in the other GLUT proteins. Here, using enhanced sampling molecular dynamics simulations, we show that the fructose transporter GLUT5 spontaneously transitions through an occluded state that closely resembles Pf HT1. The coordination of D -fructose lowers the energetic barriers between the outward- and inward-facing states, and the observed binding mode for D -fructose is consistent with biochemical analysis. Rather than a substrate-binding site that achieves strict specificity by having a high affinity for the substrate, we conclude GLUT proteins have allosterically coupled sugar binding with an extracellular gate that forms the high-affinity transition-state instead. This substrate-coupling pathway presumably enables the catalysis of fast sugar flux at physiological relevant blood-glucose concentrations.

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  1. Author Response

    Reviewer #3 (Public Review):

    The authors investigated the mechanism of transport of the GLUT5 sugar porter using enhanced sampling molecular dynamics simulations and biochemical analysis. The results suggest a possible general mechanism by which binding to a transported substrate stabilizes an occluded intermediate conformation between outward and inward-facing states of the alternating access conformational change of the protein, thereby enabling transport.

    The authors also identified key elements of this transition, associated with residues involved in sugar binding, and through elegant biochemical experiments demonstrated how mutations of the latter affect the protein function, including mutations of gating residues that can recover the function of inactive mutants.

    The general computational methodology used by …

  2. eLife assessment

    The current manuscript investigates the energy landscape of the mammalian sugar porter GLUT5 using enhanced molecular dynamics simulations and biochemical assays. The approach generates important insights into the mechanism of GLUT5 conformational change, and into mechanistic diversity among the GLUT sugar porters more generally. The overall strategy is solid, but without an additional error analysis, the computational components remain incomplete. These findings will be of interest to the transporter and membrane biology communities.

  3. Reviewer #1 (Public Review):

    The current paper tackles a central conundrum in transporter mechanism: how substrate recognition and conformational change are coupled to achieve substrate selectivity. The focus of this manuscript is the GLUT family of sugar importers, specifically GLUT5, a fructose importer. Using information from multiple GLUT structures in different conformational states, together with enhanced molecular dynamic simulations, the authors reconstruct a free energy landscape for the outward-open to inward-open GLUT5 conformational transition in the presence and absence of fructose. The authors are thorough in their approach, considering alternative approaches (for example, including vs. excluding a distantly related GLUT transporter).

    These experiments provide insight into the energy barriers, fructose coordination in the …

  4. Reviewer #2 (Public Review):

    In this essential study for the field, McComas et al. use a combination of MD simulations and experiments to construct a unifying transport cycle for a single GLUT protein, GLUT5. The authors demonstrate that GLUT5 likely moves through a transient, intermediate-occluded state like that observed in PfHT1. They also demonstrate that substrate-binding, the specificity of which is regulated by allosteric coupling of the substrate binding site to the extracellular gate, lowers the energetic barriers for the transition from outward- to inward-facing states. The manuscript is clearly and logically written, the data is presented clearly, and the conclusions are sound.

  5. Reviewer #3 (Public Review):

    The authors investigated the mechanism of transport of the GLUT5 sugar porter using enhanced sampling molecular dynamics simulations and biochemical analysis.
    The results suggest a possible general mechanism by which binding to a transported substrate stabilizes an occluded intermediate conformation between outward and inward-facing states of the alternating access conformational change of the protein, thereby enabling transport.

    The authors also identified key elements of this transition, associated with residues involved in sugar binding, and through elegant biochemical experiments demonstrated how mutations of the latter affect the protein function, including mutations of gating residues that can recover the function of inactive mutants.
    The general computational methodology used by authors is appropriate …