In the last decade, the understanding of the molecular mechanisms of membrane transporters has increased dramatically, mainly through experimentally determined conformer structures corresponding to different functional states of membrane transporters. However, these are still insufficient to accurately describe the mechanistic properties of these proteins. Membrane co-transporter proteins undergo major structural rearrangements, generally related to binding events, which are essential for their biological function. By combining structural knowledge with chemical, biophysical and computational approaches, a clearer understanding of the different modes of transport of small molecules and related species is beginning to emerge. In this case, the computational approach represents an important tool that allows the study of the functioning of these systems at the molecular level and the coupling of the biophysical properties of the systems to experimentally measurable quantities.
In addition to their main role as specific carriers for small molecules and ions, membrane carriers can also behave as water channels. However, often in different systems, neither the position of the main water passage pathway in the protein nor their functional mechanism of action is precisely known. In general, the whole process of substrate transport is ensured by "alternating access" transitions between two main conformational states: the outward-facing (OF) and the inward-facing (IF) states, in which the substrate accessibility is shifted from one side of the membrane to the other.
In the context of our study, we focused on the water transport mechanisms in human sodium-glucose co-transporter 1 (SGLT1), the study of which has so far shown numerous conflicting hypotheses of action, which in our opinion have not been sufficiently supported by a detailed biophysical study of the mechanism. The aim of this study is to elucidate the water transport in this transporter as a general mechanism that could be transferred to other membrane transporters as well as to other protein and non-protein systems. By studying the internal protein dynamics using atomistic molecular dynamics methods, the development of methods to trace diffusion- and osmotically-dependent water transport and specific implementations of the principle component analysis (PCA) method, we have elucidated the mechanism of water transport in the carrier protein under investigation. The membrane-embedded SGLT1 protein was investigated in different conformational states, including the states in the presence of ions and substrates, and in the form of inhibitor-protein complexes. We have precisely decomposed the transporter's domain motion modes into individual components that describe the characteristic rocking-bundle motion mode described in the literature and analysed the influence of the presence or absence of ligands or inhibitors on the flexibility properties of each mode as well as of the overall domain motion. We evaluated the effect of the use of inhibitors on the biophysical properties of the protein and found that, in the case of aqueous transport, they express their effect allosterically at a site that is not in direct contact with the binding site for the sugar moiety of the inhibitor binding site. We suggest that the design of new inhibitors should take into account a requirement for the stiffening of protein structure and dynamics upon binding with inhibitor.
Molecular dynamics simulations have detected the transition of the natural sugar ligand to a potential new binding pocket in SGLT1. We have evaluated the biophysical properties of the system insofar as the sugar ligand is bound in either the primary or the alternative binding site. Given an indication of the possibility of ligand binding in the alternative binding site, we performed a virtual search of the compound library and used the developed methods to predict the potential effect of the selected hit.
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