A Study of the Evolution of Inverted-Topology Repeats from LeuT-Fold Transporters Using AlignMe

Abstract

X-ray crystal structures have revealed that numerous secondary transporter proteins originally categorized into different sequence families share similar structures, namely, the LeuT fold. The core of this fold consists of two units of five transmembrane helices, whose conformations have been proposed to exchange to form the two alternate states required for transport. That these two units are related implies that LeuT-like transporters evolved from gene-duplication and fusion events. Thus, the origins of this structural repeat may be relevant to the evolution of transport function. However, the lack of significant sequence similarity requires sensitive sequence search methods for analyzing their evolution. To this end, we developed a software application called AlignMe, which can use various types of input information, such as residue hydrophobicity, to perform pairwise alignments of sequences and/or of hydropathy profiles of (membrane) proteins. We used AlignMe to analyze the evolutionary relationships between repeats of the LeuT fold. In addition, we identified proteins from the so-called DedA family that potentially share a common ancestor with these repeats. DedA domains have been implicated in, e.g., selenite uptake; they are found widely distributed across all kingdoms of life; two or more DedA domains are typically found per genome, and some may adopt dual topologies. These results suggest that DedA proteins existed in ancient organisms and may function as dimers, as required for a would-be ancestor of the LeuT fold. In conclusion, we provide novel insights into the evolution of this important structural motif and thus potentially into the alternating-access mechanism of transport itself.

New Functions for the Ancient DedA Membrane Protein Family

“…It is important to note that the evolutionary relationship of the DedA and LeuT families is derived not from amino acid content but from hydrophobicity profiles and therefore would not turn up in a simple BLAST search. The data from the study by Khafizov et al also suggest that DedA family proteins may adopt dual topologies in the membrane (8).…”

“…An analysis of protein evolutionary relationships using a novel software program called AlignMe revealed that bacterial DedA family proteins may share structural motifs with the LeuT protein superfamily (8). LeuT is a bacterial homolog of the neurotransmitter sodium symporter (75) and vSGLT of the solute:sodium symporter family.…”

“…Despite these rather mundane origins, it is now appreciated that the DedA family is a highly conserved protein family represented in most sequenced genomes encoding membrane proteins of unknown function (5). There are virtually thousands of prokaryotic homologs of bacterial DedA proteins currently found in the NCBI protein database, and many sequenced bacterial genomes encode multiple family members (Table 1) (8). However, they remain difficult to classify, as the polypeptides do not resemble known enzymes, transporters, channels, or signaling proteins.…”

Converting the Yeast Arginine Can1 Permease to a Lysine Permease

“…This fold is shared by transporters of several different families (1). Structural studies of different "5 ϩ 5" transporters, including the bacterial Na ϩ /amino acid symporter LeuT, have provided major insights into the successive conformations adopted by this category of transporters during substrate transport via an alternative access mechanism (1,12,13).…”

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

“…The FIRL-fold is characterized by an internal twofold pseudosymmetry, with an axis running parallel to the membrane plane through the center of the transporter relating the first five transmembrane (TM) helices to the second set of five TM helices, so that their TM topologies are inverted with respect to one another (12,14). Because some of these transporters also contain additional TM helices either preceding or following the 5-TM repeat (N-or C-terminal helices, respectively), for simplicity of comparison we introduce a numbering scheme for the repeated helices, namely A1-5 for TM helices of the first repeat and B1-5 for helices belonging to the second repeat (Fig.…”