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

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

A candidate ion-retaining state in the inward-facing conformation of sodium/galactose symporter: Clues from atomistic simulations

The recent Vibrio parahaemolyticus sodium/galactose (vSGLT) symporter crystal structure captures the protein in an inward-facing substrate-bound conformation, with the sodium ion placed, by structural alignment, in a site equivalent to the Na2 site of the leucine transporter (LeuT). A recent study, based on molecular dynamics simulations, showed that the sodium ion spontaneously leaves its initial position diffusing outside vSGLT, toward the intracellular space. This suggested that the crystal structure corresponds to an ion-releasing state of the transporter. Here, using metadynamics, we identified a more stable Na+ binding site corresponding to a putative ion-retaining state of the transporter. In addition, our simulations, consistently with mutagenesis studies, highlight the importance of D189 that, without being one of the NA(+)-coordinating residues, regulates its binding/release

Exit pathway of galactose.

<p>On the left is shown the free energy profile (kcal/mol) along the path collective variable representing Gal exit. On the right it is reported the position of the Gal in the different minima along exit pathway on the top of the protein. This latter is depicted in pink cartoons, the substrate in licorice, while the Na⁺ is depicted as a yellow sphere. The curved path is connected by dark dashed lines.</p

The cooperativity in the release mechanism.

<p>(A) Free energy surface (kcal/mol) depicted with respect to the distance between Na⁺ and its binding site (Å), and the galactose exit path. Note that the deepest minima for both exit pathways are the same, i.e. . (B) Close view of the initial part of the FES. (C) The projection of the free energy (kcal/mol) along the path collective variable representing Gal exit. Black and green lines correspond to the simulations carried out in presence or in absence of Na⁺. On the two sides of the image, the most relevant structures corresponding to minima and transitions states (TS) are depicted.</p

Exit pathway of sodium ion.

<p>Free energy profile (kcal/mol) projected along the distance ion - center of mass of its binding site (Å) is displayed. On the right the position of the different states along the exit path of Na⁺ are shown on the top of the protein.</p

Six-state kinetic model for Na⁺-galactose cotransporter updated to a branched six-state model.

<p>In the absence of ligands, the transporter can be in two states: outward or inward-facing conformation (C1 and C6, respectively). After the binding of Na⁺ to the outward conformation (C2Na), the substrate enters the protein and finds its site (C3NaS). This step is then followed by the crucial event that sees the transporter switching to the inward-facing conformation (C4NaS). In case the transport follows the dashed line, namely passing from C2Na to C5Na, a uniport of Na⁺ ion happens. From the ligands-loaded inward conformation (C4NaS) the protein can lose at first the substrate (C5Na), as suggested in Ref. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004017#pcbi.1004017-Wright1" target="_blank">[11]</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004017#pcbi.1004017-Eskandari1" target="_blank">[48]</a>, or, based on our results (blue region), in an independent way characterized by similar barriers, the ion (C5S).</p

A Candidate Ion-Retaining State in the Inward-Facing Conformation of Sodium/Galactose Symporter: Clues from Atomistic Simulations

The recent <i>Vibrio parahaemolyticus</i> sodium/galactose (vSGLT) symporter crystal structure captures the protein in an inward-facing substrate-bound conformation, with the sodium ion placed, by structural alignment, in a site equivalent to the Na2 site of the leucine transporter (LeuT). A recent study, based on molecular dynamics simulations, showed that the sodium ion spontaneously leaves its initial position diffusing outside vSGLT, toward the intracellular space. This suggested that the crystal structure corresponds to an ion-releasing state of the transporter. Here, using metadynamics, we identified a more stable Na⁺ binding site corresponding to a putative ion-retaining state of the transporter. In addition, our simulations, consistently with mutagenesis studies, highlight the importance of D189 that, without being one of the Na⁺-coordinating residues, regulates its binding/release