Assessing the ability of sequence-based methods to provide functional insight within membrane integral proteins: a case study analyzing the neurotransmitter/Na+ symporter family

Abstract

Background: Efforts to predict functional sites from globular proteins is increasingly common; however, the most successful of these methods generally require structural insight. Unfortunately, despite several recent technological advances, structural coverage of membrane integral proteins continues to be sparse. ConSequently, sequence-based methods represent an important alternative to illuminate functional roles. In this report, we critically examine the ability of several computational methods to provide functional insight within two specific areas. First, can phylogenomic methods accurately describe the functional diversity across a membrane integral protein family? And second, can sequence-based strategies accurately predict key functional sites? Due to the presence of a recently solved structure and a vast amount of experimental mutagenesis data, the neurotransmitter/Na + symporter (NSS) family is an ideal model system to assess the quality of our predictions.

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

“…Comparative modelling of LeuT with more than 300 prokaryotic and eukaryotic NSS sequences identified that the residues that interact with the substrates’ side-chains in the deeper regions of the binding pockets are not conserved [ 26 ]. Bioinformatics analyses, using functional site prediction strategies, anticipated key functional sites within the NSS family and correctly predicted 31/34 substrate-interacting residues in the LeuT structure [ 27 ]. Residues in the three “non-predicted” positions in LeuT all form van der Waals’ contacts with the substrate side-chain [ 3 ].…”

“…LeuT was subsequently crystallised bound to a series of amino acids with increasing side-chain size [ 28 ]. When LeuT is locked in the outward-open substrate-bound conformation, by interaction with the large indole ring of the non-transported inhibitor tryptophan [ 28 ], V104 in TM3, one of the non-conserved residues identified in the bioinformatics analyses [ 26 , 27 ], and the focus of the current investigation, occupies a deep position below the indole ring.…”

GATMD: -Aminobutyric Acid Transporter Mutagenesis Database

“…The GABA transporters belong to the large neurotransmitter/Na + symporter family (NSS; 2.A.22 according to the transporter classification system; SLC6 according to the Human Genome Organization classification) ( 2 , 9–12 ). Solute transport in these transporters is driven by the electrochemical potential gradient of Na + and Cl – and, in fact, 2 Na + ions and 1 Cl – ion are cotranslocated with GABA during each transport cycle ( 13–19 ).…”

Functional Consequences of Sulfhydryl Modification of the γ-Aminobutyric Acid Transporter 1 at a Single Solvent-Exposed Cysteine Residue

“…This observation is not consistent with the very high intrinsic reactivity of methanethiosulfonate reagents toward free thiol groups (Karlin and Akabas 1998 ) and suggests that C74 is only partially exposed to the extracellular fluid, such that accessibility of this site to MTSET is restricted (Yu et al 1998 ). Indeed, sequence alignments of GAT1 with the related bacterial leucine transporter (LeuT Aa ) and other members of the SLC6A family of transporters, as well as homology models of GAT1 built using the high-resolution structure of LeuT Aa as a template, strongly suggest that C74 is in the first transmembrane domain near the membrane–extracellular fluid interface and is partially buried in the membrane (Yamashita et al 2005 ; Beuming et al 2006 ; Livesay et al 2007 ). As a practical matter, the relatively short half-life of MTSET in physiological buffers (~10 min) also necessitates the use of experimental conditions that can achieve faster labeling of GAT1.…”