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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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 …
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 authors is appropriate for addressing these questions and compared to other techniques has the advantage of bringing forth an unbiased molecular description of the transport process. The results are overall qualitatively in line with the proposed conclusions.
A major weakness of this work is that, in contrast to previous studies with the same type of methodology, the authors do not report error analysis or careful statistical assessment of the computational results. Therefore, it is not clear whether the latter is solid or if they support the proposed conclusions. The computational data might generally benefit from an improved methodological design, such as including more degrees of freedom (or collective variables) in the description of the minimum free energy pathway, e.g. the salt-bridges.
This has now been addressed in the essential revisions above.
Another weakness is that some of the details of the computational analysis are not reported, therefore other investigators would not know how to reproduce the results.
We have extended the methods section to include much more detail about the MSM construction and other computational analysis. Data files needed for reproduction are now found in a public repository with links provided in the Methods section.
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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.
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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 …
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 occluded conformation, and the coupling between substrate binding, the motion of the extracellular gate, and conformational change. Uptake assays are used to test predictions about gating residues and residues predicted to bind fructose in the occluded state. Overall, this is a comprehensive study that provides broad insight into mechanistic diversity among GLUT sugar porters.
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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.
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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 …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 for addressing these questions and compared to other techniques has the advantage of bringing forth an unbiased molecular description of the transport process. The results are overall qualitatively in line with the proposed conclusions.A major weakness of this work is that, in contrast to previous studies with the same type of methodology, the authors do not report error analysis or careful statistical assessment of the computational results. Therefore, it is not clear whether the latter is solid or if they support the proposed conclusions. The computational data might generally benefit from an improved methodological design, such as including more degrees of freedom (or collective variables) in the description of the minimum free energy pathway, e.g. the salt-bridges.
Another weakness is that some of the details of the computational analysis are not reported, therefore other investigators would not know how to reproduce the results.
Once these issues are addressed, this work could potentially provide important insights into the mechanism of transport of sugar porters, which as suggested by other recent studies might also apply to other types of membrane transporters.
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