Kamil Khafizov

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

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.

Trends in structural coverage of the protein universe and the impact of the Protein Structure Initiative

Significance The Protein Structure Initiative and related worldwide efforts are engaged in the large-scale structural annotation of proteins. In this work, we investigated the dynamic changes that have occurred in this complex race, where sequence databases double every 1.5 y but are becoming increasingly redundant and have exhibited profound changes in taxonomic composition over the last 5 y. Meanwhile, the number of known protein structures is approximately 200 times smaller, and the pace of discovery of new folds is slowing. Nevertheless, the overall structural coverage of proteins has increased from 30% to 40% over the last 10 y. Assuming current trends, ∼55% coverage will be achieved within 15 y, a level considered sufficient to fully characterize the metabolic network of an organism. The exponential growth of protein sequence data provides an ever-expanding body of unannotated and misannotated proteins. The National Institutes of Health-supported Protein Structure Initiative and related worldwide structural genomics efforts facilitate functional annotation of proteins through structural characterization. Recently there have been profound changes in the taxonomic composition of sequence databases, which are effectively redefining the scope and contribution of these large-scale structure-based efforts. The faster-growing bacterial genomic entries have overtaken the eukaryotic entries over the last 5 y, but also have become more redundant. Despite the enormous increase in the number of sequences, the overall structural coverage of proteins—including proteins for which reliable homology models can be generated—on the residue level has increased from 30% to 40% over the last 10 y. Structural genomics efforts contributed ∼50% of this new structural coverage, despite determining only ∼10% of all new structures. Based on current trends, it is expected that ∼55% structural coverage (the level required for significant functional insight) will be achieved within 15 y, whereas without structural genomics efforts, realizing this goal will take approximately twice as long.

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

Sodium-coupled substrate transport plays a central role in many biological processes. However, despite knowledge of the structures of several sodium-coupled transporters, the location of the sodium-binding site(s) often remains unclear. Several of these structures have the five transmembrane-helix inverted-topology repeat, LeuT-like (FIRL) fold, whose pseudosymmetry has been proposed to facilitate the alternating-access mechanism required for transport. Here, we provide biophysical, biochemical, and computational evidence for the location of the two cation-binding sites in the sodium-coupled betaine symporter BetP. A recent X-ray structure of BetP in a sodium-bound closed state revealed that one of these sites, equivalent to the Na2 site in related transporters, is located between transmembrane helices 1 and 8 of the FIRL-fold; here, we confirm the location of this site by other means. Based on the pseudosymmetry of this fold, we hypothesized that the second site is located between the equivalent helices 6 and 3. Molecular dynamics simulations of the closed-state structure suggest this second sodium site involves two threonine sidechains and a backbone carbonyl from helix 3, a phenylalanine from helix 6, and a water molecule. Mutating the residues proposed to form the two binding sites increased the apparent Km and Kd for sodium, as measured by betaine uptake, tryptophan fluorescence, and 22Na+ binding, and also diminished the transient currents measured in proteoliposomes using solid supported membrane-based electrophysiology. Taken together, these results provide strong evidence for the identity of the residues forming the sodium-binding sites in BetP.

Alignment of helical membrane protein sequences using AlignMe.

Few sequence alignment methods have been designed specifically for integral membrane proteins, even though these important proteins have distinct evolutionary and structural properties that might affect their alignments. Existing approaches typically consider membrane-related information either by using membrane-specific substitution matrices or by assigning distinct penalties for gap creation in transmembrane and non-transmembrane regions. Here, we ask whether favoring matching of predicted transmembrane segments within a standard dynamic programming algorithm can improve the accuracy of pairwise membrane protein sequence alignments. We tested various strategies using a specifically designed program called AlignMe. An updated set of homologous membrane protein structures, called HOMEP2, was used as a reference for optimizing the gap penalties. The best of the membrane-protein optimized approaches were then tested on an independent reference set of membrane protein sequence alignments from the BAliBASE collection. When secondary structure (S) matching was combined with evolutionary information (using a position-specific substitution matrix (P)), in an approach we called AlignMePS, the resultant pairwise alignments were typically among the most accurate over a broad range of sequence similarities when compared to available methods. Matching transmembrane predictions (T), in addition to evolutionary information, and secondary-structure predictions, in an approach called AlignMePST, generally reduces the accuracy of the alignments of closely-related proteins in the BAliBASE set relative to AlignMePS, but may be useful in cases of extremely distantly related proteins for which sequence information is less informative. The open source AlignMe code is available at https://sourceforge.net/projects/alignme/, and at http://www.forrestlab.org, along with an online server and the HOMEP2 data set.

AlignMe—a membrane protein sequence alignment web server

We present a web server for pair-wise alignment of membrane protein sequences, using the program AlignMe. The server makes available two operational modes of AlignMe: (i) sequence to sequence alignment, taking two sequences in fasta format as input, combining information about each sequence from multiple sources and producing a pair-wise alignment (PW mode); and (ii) alignment of two multiple sequence alignments to create family-averaged hydropathy profile alignments (HP mode). For the PW sequence alignment mode, four different optimized parameter sets are provided, each suited to pairs of sequences with a specific similarity level. These settings utilize different types of inputs: (position-specific) substitution matrices, secondary structure predictions and transmembrane propensities from transmembrane predictions or hydrophobicity scales. In the second (HP) mode, each input multiple sequence alignment is converted into a hydrophobicity profile averaged over the provided set of sequence homologs; the two profiles are then aligned. The HP mode enables qualitative comparison of transmembrane topologies (and therefore potentially of 3D folds) of two membrane proteins, which can be useful if the proteins have low sequence similarity. In summary, the AlignMe web server provides user-friendly access to a set of tools for analysis and comparison of membrane protein sequences. Access is available at http://www.bioinfo.mpg.de/AlignMe

The role of trimerization in the osmoregulated betaine transporter BetP

The osmoregulated betaine transporter BetP is a stable trimer. Structural studies have shown that individual protomers can adopt distinct transport conformations, implying a functional role for the trimeric state in transport, although the role of trimerization in regulation is not yet understood. We designed putative monomeric mutants by molecular‐dynamics simulations and in silico alanine‐scanning mutagenesis. Several mutants including BetP‐W101A/T351A were monomeric in detergent as well as in the membrane, as shown by blue native gel electrophoresis, crosslinking and electron microscopy. This monomeric form retains the ability to accumulate betaine, but is no longer regulated by hyperosmotic shock.

G protein inactive and active forms investigated by simulation methods

Molecular dynamics and computational alanine scanning techniques have been used to investigate G proteins in their inactive state (the Gαi1β1γ2 heterotrimer) as well as in their empty and monomeric active states (Gαi1 subunit). We find that: (i) the residue Q204 of Gαi1 plays a key role for binding Gβ1γ2 and is classified among the most relevant in the interaction with a key cellular partner, the so‐called regulator of G protein signaling protein. The mutation of this residue to L, which is observed in a variety of diseases, provides still fair stability to the inactive state because of the formation of van der Waals interactions. (ii) The empty state turns out to adopt some structural features of the active one, including a previously unrecognized rearrangement of a key residue (K46). (iii) The so‐called Switch IV region increases its mobility on passing from the empty to the active state, and, even more, to the inactive state. Such change in mobility could be important for its several structural and functional roles. (iv) A large scale motion of the helical domain in the inactive state might be important for GDP release upon activation by GPCR, consistently with experimental data. Proteins 2009.

Highly conserved tyrosine 37 stabilizes desensitized states and restricts calcium permeability of ATP-gated P2X3 receptor.

J. Neurochem. (2011) 119, 676–685.

Role of the Ectodomain Serine 275 in Shaping the Binding Pocket of the ATP-Gated P2X3 Receptor

ATP-activated P2X3 receptors expressed in nociceptive sensory neurons play an important role in pain signaling. Basic properties of this receptor subtype, including very strong desensitization, depend on the rate of dissociation of the agonist from the binding site. Even though the rough structure of the ATP binding site has been proposed on the basis of the X-ray structure of the zebrafish P2X4 receptor and mutagenesis studies, the fine subunit-specific structural properties predisposing the receptor to tight capture of the agonist inside the binding pocket have not been elucidated. In this work, by exploring in silico the functional role for the left flipper located in the ectodomain region, we identified within this loop a candidate residue S275, which could contribute to the closure of the agonist-binding pocket. Testing of the S275 mutants using the patch-clamp technique revealed a crucial role for S275 in agonist binding and receptor desensitization. The S275A mutant showed a reduced rate of onset of desensitization and accelerated resensitization and was weakly inhibited by nanomolar agonist. Extracellular calcium application produced inhibition instead of facilitation of membrane currents. Moreover, some full agonists became only partial agonists when applied to the S275A receptor. These effects were stronger with the more hydrophobic mutants S275C and S275V. Taken together, our data suggest that S275 contributes to the closure of the agonist-binding pocket and that effective capture of the agonist provided by the left flipper in calcium-dependent manner determines the high rate of desensitization, slow recovery, and sensitivity to nanomolar agonist of the P2X3 receptor.

GoLoco motif proteins binding to Gαi1: insights from molecular simulations

Molecular dynamics simulations, computational alanine scanning and sequence analysis were used to investigate the structural properties of the Gαi1/GoLoco peptide complex. Using these methodologies, binding of the GoLoco motif peptide to the Gαi1 subunit was found to restrict the relative movement of the helical and catalytic domains in the Gαi1 subunit, which is in agreement with a proposed mechanism of GDP dissociation inhibition by GoLoco motif proteins. In addition, the results provide further insights into the role of the “Switch IV” region located within the helical domain of Gα, the conformation of which might be important for interactions with various Gα partners.