Metadynamics Simulations Reveal a Na+ Independent Exiting Path of Galactose for the Inward-Facing Conformation of vSGLT

Sodium-Galactose Transporter (SGLT) is a secondary active symporter which accumulates sugars into cells by using the electrochemical gradient of Na+ across the membrane. Previous computational studies provided insights into the release process of the two ligands (galactose and sodium ion) into the cytoplasm from the inward-facing conformation of Vibrio parahaemolyticus sodium/galactose transporter (vSGLT). Several aspects of the transport mechanism of this symporter remain to be clarified: (i) a detailed kinetic and thermodynamic characterization of the exit path of the two ligands is still lacking; (ii) contradictory conclusions have been drawn concerning the gating role of Y263; (iii) the role of Na+ in modulating the release path of galactose is not clear. In this work, we use bias-exchange metadynamics simulations to characterize the free energy profile of the galactose and Na+ release processes toward the intracellular side. Surprisingly, we find that the exit of Na+ and galactose is non-concerted as the cooperativity between the two ligands is associated to a transition that is not rate limiting. The dissociation barriers are of the order of 11-12 kcal/mol for both the ion and the substrate, in line with kinetic information concerning this type of transporters. On the basis of these results we propose a branched six-state alternating access mechanism, which may be shared also by other members of the LeuT-fold transporters

Atomistic Study of Structural and Functional Properties of Membrane Proteins

Living cells exploit membrane proteins to carry out crucial functions like transport of nutrients, signal transduction, energy conversion, etc. Recently, the remarkable and continuous improvement of computational algorithms and power allowed simulating and investigating relevant aspects of the mechanisms of this important class of proteins. In this thesis we focused on the study of two membrane proteins: a transporter and an ion channel. Firstly, we investigated the bacterial homologue of Sodium Galactose Transporter (SGLT), which plays an important role in the accumulation of sugars (i.e. glucose or galactose) inside cells, assuring a correct intestinal absorption and renal re-absorption. Using enhanced sampling techniques, we focused in understanding selected aspects of its transport mechanism. First, we identified a stable Na+ ion binding site, which was not solved crystallographically. Second, based on the results of the first study, we investigated the mechanism of the binding/release of both ligands to/from the protein in the inward-facing conformation and their interplay during this process. Finally, we also worked on another membrane protein: the Cyclic Nucleotide-Gated (CNG) channel. Using a chimera, the NaK2CNG mimic, we investigated the structural basis of the linkage among gating and permeation and of the voltage dependence shown by this channel. Large-scale molecular dynamics (MD) simulations, together with electrophysiology and X-ray crystallography, have been used to study the permeation mechanism of this mimic as a model system of CNG in presence of different alkalications

Stochastic steps in secondary active sugar transport.

Secondary active transporters, such as those that adopt the leucine-transporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function. Here, we report a series of molecular simulations carried out on the sugar transporter vSGLT that lend insight into how substrate and ions are released from the inward-facing state of the transporter. Our simulations reveal that the order of release is stochastic. Functional experiments were designed to test this prediction on the human homolog, hSGLT1, and we also found that cytoplasmic release is not ordered, but we confirmed that substrate and ion binding from the extracellular space is ordered. Our findings unify conflicting published results concerning cytoplasmic release of ions and substrate and hint at the possibility that other transporters in the superfamily may lack coordination between ions and substrate in the inward-facing state

The molecular mechanism of secondary sodium symporters elucidated through the lens of the computational microscope

Transport of molecules across cellular membranes is a key biological process for normal cell function. As such, secondary active transporters exploit electrochemical ion gradients to carry out fundamental processes, i.e. nutrients uptake, ion regulation, neurotransmission, and substrate extrusion. Despite their modest sequence similarity, several Na+ symporters share the same fold of LeuT (leucine transporter), a prokaryotic member of the neurotransmitter-sodium symporter family, pinpointing to a common structural/functional mechanism of transport. This is associated with specific conformational transitions occurring along a so-called alternating access mechanism. Thanks to recent advances in computer simulation techniques and the ever-increasing computational power that has become available in the last decade, molecular dynamics (MD) simulations have been largely employed to provide atomistic insights into mechanistic, kinetic, and thermodynamic aspects of this family of transporters. Here we report a detailed overview of selected Na+-symporters belonging to the LeuT-fold superfamily for which different aspects of the transport mechanism have been addressed using both experimental and computational studies. The aim of this review is to describe current state-of-the-art knowledge on the mechanism of these transporters showing how molecular simulations have contributed to elucidate mechanistic aspects and can provide nowadays a spatial and temporal resolution, allowing the interpretation of experimental findings, complementing biophysical methods, and filling the gaps in fragmentary experimental information