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

Abstract

In mammals, glucose transporters (GLUT) control organism-wide blood glucose homeostasis. In human, this is accomplished by fourteen 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 PfHT1, which is uniquely able to transport a wide range of different sugars. PfHT1 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 PfHT1, rather than the sugar-binding site itself. It was unclear, however, if the TM7b structural transitions observed in PfHT1 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 PfHT1. Furthermore, we observe that inclusion of fructose lowers the energetic barriers between the outward and inward-facing states, and that its binding is coupled to TM7b gating by a strictly-conserved asparagine residue and a GLUT5-specific tyrosine-histidine pairing. 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.

Reconstructing the transport cycle in the sugar porter superfamily using coevolution-powered machine learning

“…Notably, the relaxed GLUT5 occluded state does not fall on the largest energetic barrier corresponding to the transition state (Figure 5). However, in the presence of a substrate sugar, GLUT5 passes through a transition state that closely matches the occluded state of Pf HT1 40 . Our analysis indicates that coevolution-driven MD simulations are detecting an energetic minimum for an occluded state prior to the transition state which has not been experimentally observed (Figure S3).…”

“…Here, we decided to generate a free energy landscape for the fructose transporter GLUT5 using family-wide information in the form of coevolving contacts. We chose GLUT5 because it is the only transporter with experimental structures in both fully outward- and inward-facing conformations 21 and because our recently determined free energy landscape using a more traditional enhanced sampling method can serve as a comparison 40 .…”

AtSWEET13 transporter discriminates sugars by selective facial and positional substrate recognition

“…These residues instead predominantly line the AtSWEET13 periphery, echoing the importance of distant residues for evolved function in other proteins, including membrane transporters. [32][33][34][35] Still, AtSWEET13 maintains high fluctuations within these regions for both GLC and SUC transport reactions, suggesting that movements from these residues are essential for general alternate access regardless of substrate.…”

Tackling hysteresis in conformational sampling — how to be forgetful with MEMENTO

“…In transition path sampling, 20,21 an ensemble of transition paths can be constructed from one rare event trajectory, and the string method 22,23 has been applied with success in transitions as complex as membrane transporter conformational changes. 24,25 Since tMD paths like the P38α runs we presented above can be highly metastableto the extent that they appear converged in REUS even with hundreds of nanoseconds of simulation time per window -we decided not to attempt refining paths with MD. Instead, we focus on alternative path-generation algorithms.…”

“…In transition path sampling, 20,21 an ensemble of transition paths can be constructed from one rare event trajectory, and the string method 22,23 has been applied with success in transitions as complex as membrane transporter conformational changes. 24,25…”