Structural, functional and computational studies on the SLC7 family of amino acid transporters

Thesis

Amino acids are central components of the cell as protein building blocks, nitrogen sources and metabolic and cell signalling regulators. As such amino acid concentration in the cytosol and cellular compartments must be regulated. The SLC7 family of amino acid transporters provides a central gateway of amino acids to the cell. There is increasing understanding of mammalian SLC7 members, primarily, through structural and functional studies on prokaryotic and, recently, some human homologues. However, a member of the cationic amino acid transporter (CAT) subfamily remains of unknown function, prompting us to uncover its structure-function relationship. This thesis reports the first cryoEM structure of a eukaryotic SLC7A4 homologue, from Arabidopsis thaliana (AtCAT4), closely related to the mammalian SLC7A4 by conserving a key acidic residue, and other CATs responsible for the transport of L-arginine, L-lysine, and L-ornithine into the cell. AtCAT4 structural elucidation required the selection and generation of a nanomolar affinity sybody binder. The structure was solved in the apo outward-open state and showed AtCAT4 has an APC fold with two additional TM helices previously unobserved, likely involved in homodimerisation. Structural comparisons revealed an intricate electrostatic gating mechanism, which rearranges upon substrate binding, uncovering early steps of the transport cycle. AtCAT4 binding assays and MD simulation showed recognition of L-arginine, L-lysine, and L-ornithine with differential preferences for each. Human SLC7A4 however did not transport cationic amino acids, but preliminary data support a role for L-leucine transport. Structural analyses and mutagenesis suggested the root of the homologue discrepancy to be two bulky, hydrophobic residues absent from AtCAT4 and other CATs. The current structure serves as a template to further understand mammalian cationic amino acid transport via the SLC7 family. AtCAT4 was solved with a steroid lipid bound, similar to other SLC7s. With multiscale-MD and in vitro transport assays we suggest two cholesterol binding sites for HATs which stabilise transporter SLC7-SLC3 heterodimerisation and conformational state.

Authors: Kolokouris, D

• DOI: 10.5287/ora-5zbzzgm24
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: amino acid transporters; synthetic nanobodies; membrane proteins; protein-lipid interactions; CAT4; structural biology; SLC7 transporters; cholesterol interactions; SLC7A4; electron cryomicroscopy
• Subjects: SLC7A4; Membrane Proteins; SLC7 transporters; Electron cryomicroscopy; Structural Biology; Amino acid transporters; Cholesterol interactions; Synthetic Nanobodies; Protein-lipid interactions; CAT4