Comprehensive Characterization of LAT1 Cholesterol-Binding Sites

The human L-type amino acid transporter 1 (LAT1; SLC7A5), is an amino acid exchanger protein, primarily found in the blood–brain barrier, placenta, and testis, where it plays a key role in amino acid homeostasis. Cholesterol is an essential lipid that has been highlighted to play a role in regulating the activity of membrane transporters, such as LAT1, yet little is known about the molecular mechanisms driving this phenomenon. Here we perform a comprehensive computational analysis to investigate cholesterol’s role in LAT1 structure and function, focusing on four cholesterol-binding sites (CHOL1-4) identified in a recent LAT1-apo inward-open conformation cryo-EM structure. Through a series of independent molecular dynamics (MD) simulations, molecular docking, MM/GBSA free energy calculations, and other analysis tools, we explored the interactions between LAT1 and cholesterol. Our findings suggest that CHOL3 forms the most stable and favorable interactions with LAT1. Principal component analysis (PCA) and center of mass (COM) distance assessments show that CHOL3 binding stabilizes the inward-open state of LAT1 by preserving the spatial arrangement of the hash and bundle domains. Additionally, we propose an alternative cholesterol-binding site for originally assigned CHOL1. Overall, this study improves the understanding of cholesterol’s modulatory effect on LAT1 and proposes candidate sites for the discovery of future allosteric ligands with rational design

Comprehensive Characterization of LAT1 Cholesterol-Binding Sites

The human L-type amino acid transporter 1 (LAT1; SLC7A5), is an amino acid exchanger protein, primarily found in the blood–brain barrier, placenta, and testis, where it plays a key role in amino acid homeostasis. Cholesterol is an essential lipid that has been highlighted to play a role in regulating the activity of membrane transporters, such as LAT1, yet little is known about the molecular mechanisms driving this phenomenon. Here we perform a comprehensive computational analysis to investigate cholesterol’s role in LAT1 structure and function, focusing on four cholesterol-binding sites (CHOL1-4) identified in a recent LAT1-apo inward-open conformation cryo-EM structure. Through a series of independent molecular dynamics (MD) simulations, molecular docking, MM/GBSA free energy calculations, and other analysis tools, we explored the interactions between LAT1 and cholesterol. Our findings suggest that CHOL3 forms the most stable and favorable interactions with LAT1. Principal component analysis (PCA) and center of mass (COM) distance assessments show that CHOL3 binding stabilizes the inward-open state of LAT1 by preserving the spatial arrangement of the hash and bundle domains. Additionally, we propose an alternative cholesterol-binding site for originally assigned CHOL1. Overall, this study improves the understanding of cholesterol’s modulatory effect on LAT1 and proposes candidate sites for the discovery of future allosteric ligands with rational design

Protein disorder linked to habitat more than to phylogeny.

<p>The fractions of proteins with long disordered regions are predicted by two disorder predictor methods (MD in green bars and IUPred in red bars). Eukaryotes are predicted with substantially more disorder than prokaryotes. Within the kingdoms predictions vary greatly: organisms in similar habitats tend to resemble each other in terms of disorder more than they resemble their closest phylogenetic relatives. (A) Hyperthermophilic archaea (dark red) are more ordered than their phylogenetic neighbors; halophilic archaea are more disordered (green). (B) Halophilic bacteria also appear more disordered than their relatives. (C) The bacterial thermophile (red) also has less disorder than its relatives. Other extreme organisms included: psychrophile (blue), psychrotolerant (light blue), radiation resistant (purple) and alkalophile (pink). We could also find organisms with relative high/low disorder content explainable separately.</p

Environmental Pressure May Change the Composition Protein Disorder in Prokaryotes

<div><p>Many prokaryotic organisms have adapted to incredibly extreme habitats. The genomes of such extremophiles differ from their non-extremophile relatives. For example, some proteins in thermophiles sustain high temperatures by being more compact than homologs in non-extremophiles. Conversely, some proteins have increased volumes to compensate for freezing effects in psychrophiles that survive in the cold. Here, we revealed that some differences in organisms surviving in extreme habitats correlate with a simple single feature, namely the fraction of proteins predicted to have long disordered regions. We predicted disorder with different methods for 46 completely sequenced organisms from diverse habitats and found a correlation between protein disorder and the extremity of the environment. More specifically, the overall percentage of proteins with long disordered regions tended to be more similar between organisms of similar habitats than between organisms of similar taxonomy. For example, predictions tended to detect substantially more proteins with long disordered regions in prokaryotic halophiles (survive high salt) than in their taxonomic neighbors. Another peculiar environment is that of high radiation survived, e.g. by <i>Deinococcus radiodurans</i>. The relatively high fraction of disorder predicted in this extremophile might provide a shield against mutations. Although our analysis fails to establish causation, the observed correlation between such a simplistic, coarse-grained, microscopic molecular feature (disorder content) and a macroscopic variable (habitat) remains stunning.</p></div

Distribution of disorder content in different organisms.

<p>Fractions of proteins with long regions of disorder (here ≥30 consecutive residues) were predicted by three prediction methods (MD, NORSnet and IUPred). <b>(A)</b> The raw values are standardized using the Z-scores (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133990#pone.0133990.e001" target="_blank">Eq 1</a>; mean and standard deviation σ from a 1613 prokaryotes calculated for each method; positive: higher than the mean; negative: below the mean; integers +/- N imply N*σ above/below the mean). The top panel shows the extremophiles; the lower panel shows the closest phylogenetic relative for each extremophile in the top panel (for relatives discussed in the text and left out for clarity from the figure, for all studied organisms <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133990#pone.0133990.s003" target="_blank">S3 Fig</a>). The archaeal halophiles <i>Haloarcula marismortui ATCC 43049</i> and <i>Halobacterium sp</i>. <i>NRC-1</i> were predicted with the highest content of proteins with long disorder. Conversely, the archaeal thermophile <i>Aeropyrum pernix K1</i> was one of the organisms predicted with the lowest disorder. The taxonomic neighbors section compares the disorder predicted for the closest relatives of the extremophiles. <b>(B-D)</b> Mapping of disorder protein content predictions for all organisms for each prediction method (B: MD [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133990#pone.0133990.ref042" target="_blank">42</a>], C: NORSnet [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133990#pone.0133990.ref006" target="_blank">6</a>], and D: IUPred Clearly, all three methods put the thermophiles on the left (less disorder), while the halophiles appear on the right (high disorder). The blue curves are Gaussian fits based on the mean and σ of our data.</p

Protein disorder content differs for habitat, not for phyla.

<p>We represent the protein disorder content for the organisms in similar habitats (left panel) and those in the same phyla (right panel). The y-axes give the percentage of proteins with at least one region of ≥30 consecutive residues predicted as disordered by MD (A), NORSnet (B) and IUPred (C). The x-axis on the left side marks the different environmental groups (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133990#pone.0133990.s010" target="_blank">S2 Table</a>); on the right side marks the studied phylogenetic groups (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133990#pone.0133990.s022" target="_blank">S14 Table</a>). The groups which are significant for a paired Wilcoxon Test are marked with * (P<0.05) or ** (P<0.005).</p

Structure-Based Identification of Inhibitors for the SLC13 Family of Na⁺/Dicarboxylate Cotransporters

In mammals, citric acid cycle intermediates play a key role in regulating various metabolic processes, such as fatty acid synthesis and glycolysis. Members of the sodium-dependent SLC13 transporter family mediate the transport of di- and tricarboxylates into cells. SLC13 family members have been implicated in lifespan extension and resistance to high-fat diets; thus, they are emerging drug targets for aging and metabolic disorders. We previously characterized key structural determinants of substrate and cation binding for the human NaDC3/SLC13A3 transporter using a homology model. Here, we combine computational modeling and virtual screening with functional and biochemical testing, to identify nine previously unknown inhibitors for multiple members of the SLC13 family from human and mouse. Our results reveal previously unknown substrate selectivity determinants for the SLC13 family, including key residues that mediate ligand binding and transport, as well as promiscuous and specific SLC13 small molecule ligands. The newly discovered ligands can serve as chemical tools for further characterization of the SLC13 family or as lead molecules for the future development of potent inhibitors for the treatment of metabolic diseases and aging. Our results improve our understanding of the structural components that are important for substrate specificity in this physiologically important family as well as in other structurally related transport systems

Mapping Functionally Important Residues in the Na⁺/Dicarboxylate Cotransporter, NaDC1

Transporters from the SLC13 family couple the transport of two to four Na⁺ ions with a di- or tricarboxylate, such as succinate or citrate. We have previously modeled mammalian members of the SLC13 family, including the Na⁺/dicarboxylate cotransporter NaDC1 (SLC13A2), based on a structure of the bacterial homologue VcINDY in an inward-facing conformation with one sodium ion bound at the Na1 site. In the study presented here, we modeled the outward-facing conformation of rabbit and human NaDC1 (rbNaDC1 and hNaDC1, respectively) using an outward-facing model of VcINDY as a template and identified residues in or near the putative Na2 and Na3 cation binding sites. Guided by the structural models in both conformations, we performed site-directed mutagenesis in rbNaDC1 for residues proposed to be in the Na⁺ or substrate binding sites. Cysteine substitution of T474 in the predicted Na2 binding site results in an inactive protein. The M539C mutant has a low apparent affinity for both sodium and lithium cations, suggesting that M539 may form part of the putative Na3 binding site. The Y432C and T86C mutants have increased <i>K</i>_m values for succinate, supporting their proposed location in the outward-facing substrate binding site. In addition, cysteine labeling by MTSEA-biotin shows that Y432C is accessible from the outside of the cell, and the accessibility changes in the presence or absence of Na⁺. The results of this study improve our understanding of substrate and ion recognition in the mammalian members of the SLC13 family and provide a framework for developing conformationally specific inhibitors against these transporters

Human Concentrative Nucleoside Transporter 3 (hCNT3, SLC28A3) Forms a Cyclic Homotrimer

Many anticancer and antiviral drugs are purine or pyrimidine analogues, which use membrane transporters to cross cellular membranes. Concentrative nucleoside transporters (CNTs) mediate the salvage of nucleosides and the transport of therapeutic nucleoside analogues across plasma membranes by coupling the transport of ligands to the sodium gradient. Of the three members of the human CNT family, CNT3 has the broadest selectivity and the widest expression profile. However, the molecular mechanisms of the transporter, including how it interacts with and translocates structurally diverse nucleosides and nucleoside analogues, are unclear. Recently, the crystal structure of vcCNT showed that the prokaryotic homologue of CNT3 forms a homotrimer. In this study, we successfully expressed and purified the wild type human homologue, hCNT3, demonstrating the homotrimer by size exclusion profiles and glutaraldehyde cross-linking. Further, by creating a series of cysteine mutants at highly conserved positions guided by comparative structure models, we cross-linked hCNT3 protomers in a cell-based assay, thus showing the existence of hCNT3 homotrimers in human cells. The presence and absence of cross-links at specific locations along TM9 informs us of important structural differences between vcCNT and hCNT3. Comparative modeling of the trimerization domain and sequence coevolution analysis both indicate that oligomerization is critical to the stability and function of hCNT3. In particular, trimerization appears to shorten the translocation path for nucleosides across the plasma membrane and may allow modulation of the transport function via allostery

Human Concentrative Nucleoside Transporter 3 (hCNT3, SLC28A3) Forms a Cyclic Homotrimer

Many anticancer and antiviral drugs are purine or pyrimidine analogues, which use membrane transporters to cross cellular membranes. Concentrative nucleoside transporters (CNTs) mediate the salvage of nucleosides and the transport of therapeutic nucleoside analogues across plasma membranes by coupling the transport of ligands to the sodium gradient. Of the three members of the human CNT family, CNT3 has the broadest selectivity and the widest expression profile. However, the molecular mechanisms of the transporter, including how it interacts with and translocates structurally diverse nucleosides and nucleoside analogues, are unclear. Recently, the crystal structure of vcCNT showed that the prokaryotic homologue of CNT3 forms a homotrimer. In this study, we successfully expressed and purified the wild type human homologue, hCNT3, demonstrating the homotrimer by size exclusion profiles and glutaraldehyde cross-linking. Further, by creating a series of cysteine mutants at highly conserved positions guided by comparative structure models, we cross-linked hCNT3 protomers in a cell-based assay, thus showing the existence of hCNT3 homotrimers in human cells. The presence and absence of cross-links at specific locations along TM9 informs us of important structural differences between vcCNT and hCNT3. Comparative modeling of the trimerization domain and sequence coevolution analysis both indicate that oligomerization is critical to the stability and function of hCNT3. In particular, trimerization appears to shorten the translocation path for nucleosides across the plasma membrane and may allow modulation of the transport function via allostery