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



Chiduza, GN
(2019) Structure-function study of heteromeric amino acid transporter, LAT1-CD98hc. PhD thesis, University of Liverpool.

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Abstract

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.

Item Type: Thesis (PhD)
Divisions: Fac of Health & Life Sciences > Institute of Integrative Biology
Depositing User: Symplectic Admin
Date Deposited: 23 Jul 2019 08:18
Last Modified: 08 May 2020 23:48
DOI: 10.17638/03037423
Supervisors:
URI: http://livrepository.liverpool.ac.uk/id/eprint/3037423