Structure-function study of heteromeric amino acid transporter, LAT1-CD98hc

The L-type neutral amino acid transporter 1 (LAT1/SLC7A5) is one of 7 light chains that can form a heteromeric amino acid transporter (HAT) with the type II single pass glycoprotein CD98hc (SLC3A2). LAT1-CD98hc transports essential amino acids and some of their catabolites, such as tryptophan, methionine and kynurenine, across the plasma membranes of normal and cancer cells. It is also a drug transporter, carrying drugs such as gabapentin and L-DOPA across the blood brain barrier. The atypical heterodimeric nature of LAT1-CD98hc and its role in disease and drug delivery, motivate the structural characterisation of the HAT. Sequence analysis revealed two putative cholesterol binding motifs conserved between dDAT and LAT1 as well as 32 putative CRAC/CARC motifs. The crystal structures of various bacterial homologues of LAT1 were used for structure prediction, in order to visualise these putative cholesterol binding motifs and assess their plausibility. Here is presented the first binding mode analysis of ligands to the inward facing occluded conformation of LAT1. Substrates had lower predicted free energies of binding to the inward facing conformation compared to the outward open. The putative gating residue F252 may play a role in binding to aromatic substrates via p-p stacking in the outward open conformation and with all substrates via p-cation bonding with their amino termini in the inward facing occluded conformation. Based on the docking analysis, inhibitors of LAT1, JPH203 and SKN203 are predicted to transportable substrates of the transporter and KMH233 a non-transportable competitive inhibitor with a unique binding mode. LAT1 was overexpressed in HEK293 cells and co-purified with CD98hc to a sufficient biochemical homogeneity for structural characterisation. The role of cholesterol hemisuccinate in stabilizing detergent solubilized LAT1-CD98hc was established. Detergent solubilized and purified LAT1-CD98hc was subject to structural analysis by single particle electron cryo-microscopy to a resolution of 12 Å. Multibody 3D auto-refinement and principal component analysis revealed flexibility and limited interaction between CD98hc ectodomain and LAT1, contrary to predictions based on homology to LAT2-CD98hc. Docking of CD98hc allowed for visualisation and generation of molecular movies of the structural dynamics of LAT1- CD98hc ectodomain, based on these the ectodomain of CD98hc seems tethered to LAT1 via the inter-subunit disulphide bond and interaction between their transmembrane domains

Modulation of LAT1 (SLC7A5) transporter activity and stability by membrane cholesterol.

LAT1 (SLC7A5) is a transporter for both the uptake of large neutral amino acids and a number of pharmaceutical drugs. It is expressed in numerous cell types including T-cells, cancer cells and brain endothelial cells. However, mechanistic knowledge of how it functions and its interactions with lipids are unknown or limited due to inability of obtaining stable purified protein in sufficient quantities. Our data show that depleting cellular cholesterol reduced the Vmax but not the Km of the LAT1 mediated uptake of a model substrate into cells (L-DOPA). A soluble cholesterol analogue was required for the stable purification of the LAT1 with its chaperon CD98 (4F2hc,SLC3A2) and that this stabilised complex retained the ability to interact with a substrate. We propose cholesterol interacts with the conserved regions in the LAT1 transporter that have been shown to bind to cholesterol/CHS in Drosophila melanogaster dopamine transporter. In conclusion, LAT1 is modulated by cholesterol impacting on its stability and transporter activity. This novel finding has implications for other SLC7 family members and additional eukaryotic transporters that contain the LeuT fold

LAT1 (SLC7A5) and CD98hc (SLC3A2) complex dynamics revealed by single-particle cryo-EM

Solute carriers are a large class of transporters that play key roles in normal and disease physiology. Among the solute carriers, heteromeric amino-acid transporters (HATs) are unique in their quaternary structure. LAT1–CD98hc, a HAT, transports essential amino acids and drugs across the blood–brain barrier and into cancer cells. It is therefore an important target both biologically and therapeutically. During the course of this work, cryo-EM structures of LAT1–CD98hc in the inward-facing conformation and in either the substrate-bound or apo states were reported to 3.3–3.5 Å resolution [Yan et al. (2019), Nature (London), 568, 127–130]. Here, these structures are analyzed together with our lower resolution cryo-EM structure, and multibody 3D auto-refinement against single-particle cryo-EM data was used to characterize the dynamics of the interaction of CD98hc and LAT1. It is shown that the CD98hc ectodomain and the LAT1 extracellular surface share no substantial interface. This allows the CD98hc ectodomain to have a high degree of movement within the extracellular space. The functional implications of these aspects are discussed together with the structure determination

Dimeric structures of quinol-dependent nitric oxide reductases (qNORs) revealed by cryo–electron microscopy

Quinol-dependent nitric oxide reductases (qNORs) are membrane-integrated, iron-containing enzymes of the denitrification pathway, which catalyze the reduction of nitric oxide (NO) to the major ozone destroying gas nitrous oxide (N2O). Cryo–electron microscopy structures of active qNOR from Alcaligenes xylosoxidans and an activity-enhancing mutant have been determined to be at local resolutions of 3.7 and 3.2 Å, respectively. They unexpectedly reveal a dimeric conformation (also confirmed for qNOR from Neisseria meningitidis) and define the active-site configuration, with a clear water channel from the cytoplasm. Structure-based mutagenesis has identified key residues involved in proton transport and substrate delivery to the active site of qNORs. The proton supply direction differs from cytochrome c–dependent NOR (cNOR), where water molecules from the cytoplasm serve as a proton source similar to those from cytochrome c oxidase