Background and Aims: Solute carrier (SLC) proteins are secondary, tertiary, and facilitative transporters making up a significant portion of cellular membrane proteins responsible for transmembrane transport of solutes [1]. Due to their pivotal roles in controlling cellular homeostasis, SLCs have been linked to a large variety of diseases and are increasingly seen as having a yet unexploited therapeutic potential [2]. Despite their membrane localization, little is known about how lipid bilayer components interact with SLCs. Nevertheless, sporadic reports suggest that lipids are able to modulate the function of SLC transporters through direct interaction, either acting as inhibitory agents or as stabilizers that can potentiate transporter function. Reports of the development of bioactive lipid inhibitors can potentially open new therapeutic options [3]. However, a systematic study of lipid-binding sites in SLCs is still lacking. Here, we aim to discover novel SLC-lipid interactions using a combination of large-scale in silico tools and in vitro validation methods.

Method: We employ in silico coarse-grain (CG) molecular dynamics (MD) simulations to screen a large number of transporters for specific lipid-binding sites. Human protein structures based on AlphaFold predictions are immersed in three different model membranes: (1) an asymmetric native-like lipid mixture representing the 63 major constituents of mammalian plasma membranes; generic palmitoyl oleoyl phosphatidylcholine (POPC) bilayers containing either of the signaling lipids (2) sphingosine-1-phosphate (S1P) or (3) N-arachidonoylglycine (NAGly). After extensive MD simulations of 10+ μs involving multiple replicas using the MARTINI CG forcefield [4], statistics on interactions between protein amino acid residues and various lipids, as well as on the spatial localization of lipid species are collected.

Results: Our methods have been applied to all proteins in the human SLC6 (neurotransmitter and amino acid transporter) and SLC39 (zinc/iron/manganese transporter) families, encompassing 33 proteins in total. Analyzing the binding patterns for cholesterol, a lipid species important for membrane protein stability, we have been able to reproduce cholesterol-binding sites previously observed for SLC6A3 and SLC6A4 transporters [5]. SLC38 transporters also show a conserved pattern of cholesterol binding, indicating that cholesterol might play an important role in the stability of these proteins. In addition, novel phosphatidyl inositol 4,5-phosphate (PIP2) binding sites, as well as non-conserved interaction patterns for NAGly and S1P have also been identified in several proteins.

Conclusion: Our systematic approach has uncovered previously unknown, potential transporter-lipid interactions that can be important in either regulating transporter stability and function, or in the involvement in lipid signaling networks. Currently, follow-up studies are planned using all-atom MD simulations to describe the identified transporter-lipid interactions in more detail. Experimental validation using functional assays or mass spectrometric analysis can be used to support the proposed interactions. Our results are planned to be expanded to other SLC families and made publicly available for the research community, and can potentially aid the development of specific bioactive lipid inhibitors in the future.