Resculpting the binding pocket of APC superfamily LeuT-fold amino acid transporters

Abstract

Amino acid transporters are essential components of prokaryote and eukaryote cells, possess distinct physiological functions, and differ markedly in substrate specificity. Amino acid transporters can be both drug targets and drug transporters (bioavailability, targeting) with many monogenic disorders resulting from dysfunctional membrane transport. The largest collection of amino acid transporters (including the mammalian SLC6, SLC7, SLC32, SLC36, and SLC38 families), across all kingdoms of life, is within the Amino acid-Polyamine-organoCation (APC) superfamily. The LeuT-fold is a paradigm structure for APC superfamily amino acid transporters and carriers of sugars, neurotransmitters, electrolytes, osmolytes, vitamins, micronutrients, signalling molecules, and organic and fatty acids. Each transporter is specific for a unique sub-set of solutes, specificity being determined by how well a substrate fits into each binding pocket. However, the molecular basis of substrate selectivity remains, by and large, elusive. Using an integrated computational and experimental approach, we demonstrate that a single position within the LeuT-fold can play a crucial role in determining substrate specificity in mammalian and arthropod amino acid transporters within the APC superfamily. Systematic mutation of the amino acid residue occupying the equivalent position to LeuT V104 titrates binding pocket space resulting in dramatic changes in substrate selectivity in exemplar APC amino acid transporters including PAT2 (SLC36A2) and SNAT5 (SLC38A5). Our work demonstrates how a single residue/site within an archetypal structural motif can alter substrate affinity and selectivity within this important superfamily of diverse membrane transporters.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-017-2677-8) contains supplementary material, which is available to authorized users.

Trading amino acids at the aphid– Buchnera symbiotic interface

“…The Transporter Classification Database (36) groups the largest collection of amino acid transporters across all forms of life within the amino acid-polyamine-organocation (APC) superfamily (37). Within the APC superfamily, many eukaryotic amino acid transporters are grouped within the important amino acid/auxin permease (AAAP, TC# 2.A.18) family, expressed ubiquitously in animals, fungi, yeast, and plants (37–39). In mammals, 4 members of the AAAP family are found within the solute carrier (SLC) family SLC36 (38).…”

“…The SLC36 family is evolutionarily very old and likely had a single founding member conserved through evolution with duplications before the teleost lineage, before the separation of reptiles and birds, and a third, which is probably mammalian-specific (49). In invertebrates, this SLC36-related AAAP family has undergone extensive expansion with duplications in the common arthropod ancestor and more recent aphid-, psyllid-, whitefly-, and mealybug-specific expansions in Hemiptera (30, 31, 39, 50). In A. pisum , there are 14 putative, SLC36-related AAAP transporters (Fig.…”

“…Middle column: portion of a full sequence alignment (by PROMALS3D) showing the central section of TM3 with a representation of the variability at each residue position shown above as a Sequence Logo. The residues highlighted in bold are equivalent to both F159 in rat PAT2 (slc36a2) and V104 in LeuT (39, 51). ACYPI008971 has I161 (blue) at this residue position.…”

“…The substrate binding pocket of the carrier is formed by TM1, TM3, TM6, and TM8 (51, 52). Recently, we determined the importance of a single position in TM3 of the LeuT-fold in AAAP transporters, with the residue in that position shaping the bottom of the hydrophobic substrate binding pocket into which the substrate side-chain fits (39). The size of the residue occupying that single position in TM3, in exemplar mammalian and arthropod AAAP amino acid transporters, determines substrate selectivity by limiting the space available for the amino acid substrate side-chain (39).…”

Genome-Wide Association Study for Milk Protein Composition Traits in a Chinese Holstein Population Using a Single-Step Approach

“…Moreover, two common contiguous windows within SLC36A2 (64.56–64.57 Mbp) were associated with six milk protein composition traits (α s1 -CN, α s2 -CN, β-CN, κ-CN, protein yield, and protein percentage). SLC36A2 plays a key role in amino acid transport across the plasma membrane as well as the transport of glucose and other sugars, bile salts and organic acids, metal ions, and amine compounds (Edwards et al, 2018), and may therefore have pleiotropic effects for several milk protein composition traits.…”