L amino acid transporter structure and molecular bases for the asymmetry of substrate interaction

Abstract

L-amino acid transporters (LATs) play key roles in human physiology and are implicated in several human pathologies. LATs are asymmetric amino acid exchangers where the low apparent affinity cytoplasmic side controls the exchange of substrates with high apparent affinity on the extracellular side. Here, we report the crystal structures of an LAT, the bacterial alanine-serine-cysteine exchanger (BasC), in a non-occluded inward-facing conformation in both apo and substrate-bound states. We crystallized BasC in complex with a nanobody, which blocks the transporter from the intracellular side, thus unveiling the sidedness of the substrate interaction of BasC. Two conserved residues in human LATs, Tyr 236 and Lys 154, are located in equivalent positions to the Na1 and Na2 sites of sodium-dependent APC superfamily transporters. Functional studies and molecular dynamics (MD) calculations reveal that these residues are key for the asymmetric substrate interaction of BasC and in the homologous human transporter Asc-1.

Asc-1 Transporter (SLC7A10): Homology Models And Molecular Dynamics Insights Into The First Steps Of The Transport Mechanism

“…Among these new templates, two are bacterial including apo BasC (PDB ID: 6F2G) and BasC bound to α-aminoisobutyric acid (AIB) substrate (PDB ID: 6F2W). The others are human LAT1 linked to the heavy-chain including LAT1-CD98hc (PDB ID: 6JMQ) and LAT1-4F2hc (PDB IDs: 6IRS, 6IRT) and were solved by Cryo-EM 20,30,31 .…”

“…2) [22][23][24][25] . Five recent structures (see details in the Methods section) with better sequence identity scores were published but these potential templates were solved in the inward-open state 20,30,31 . Sequence comparison of the human Asc-1 versus bacterial BasC bound to AIB substrate (PDB ID: 6F2W) and versus the human apo LAT1-4F2hc (PDB ID: 6IRS) show 26% and 47% of sequence identity, respectively.…”

“…Unfortunately, atomic-level intricacies of human Asc-1 regulation and shuttle properties remain elusive as no X-ray crystallographic structure has been published to date. Although, the bacterial alanine-serine-cysteine exchanger (BasC) has been solved very recently, this template doesn't fit our target homology models (see below) 20 . This lack of success in crystallizing human Asc-1 is of little surprise given the historic difficulties in solving the structures of membrane-bound proteins in general.…”

Signaling Role of Glutamate in Plants

“…In general, the redistribution of Glu can be achieved by transporters locating in the plasma membrane. In Arabidopsis, the Glu transporters include cationic amino acid transporters (CAT) family (amino acid-polyamine-choline facilitator, APC superfamily), g-aminobutyric acid (GABA) permease-related (APC superfamily), and amino acid transporter family (ATF superfamily) (Fischer et al, 1998;Yang et al, 2014;Errasti-Murugarren et al, 2019). Glu redistribution can alter plant growth and development, which in turn improve the resistance of plants to adverse environments (Yang et al, 2014).…”

Cryo-EM structure of the human heteromeric amino acid transporter b 0,+ AT-rBAT

“…5B), via a traditional mechanism, which is similar to the LAT1-4F2hc complex and also the prokaryotic homologs BasC and GkApcT, with a little difference that the side chain of Arg substrate mainly interacted with residues in TM6b ( fig. S9) (30,31). A water molecule is found near pocket 1, which hydrogen bonds with Ser 46 , Ser 131 , and Trp 230 , thus connecting TM1, TM3, and TM6 (fig.…”