EncoMPASS: An encyclopedia of membrane proteins analyzed by structure and symmetry.

Protein structure determination and prediction, active site detection, and protein sequence alignment techniques all exploit information about protein structure and structural relationships. For membrane proteins, however, there is limited agreement among available online tools for highlighting and mapping such structural similarities. Moreover, no available resource provides a systematic overview of quaternary and internal symmetries, and their orientation relative to the membrane, despite the fact that these properties can provide key insights into membrane protein function and evolution. Here, we describe the Encyclopedia of Membrane Proteins Analyzed by Structure and Symmetry (EncoMPASS), a database for relating integral membrane proteins of known structure from the points of view of sequence, structure, and symmetry. EncoMPASS is accessible through a web interface, and its contents can be easily downloaded. This allows the user not only to focus on specific proteins, but also to study general properties of the structure and evolution of membrane proteins.

Refining pairwise sequence alignments of membrane proteins by the incorporation of anchors.

The alignment of primary sequences is a fundamental step in the analysis of protein structure, function, and evolution, and in the generation of homology-based models. Integral membrane proteins pose a significant challenge for such sequence alignment approaches, because their evolutionary relationships can be very remote, and because a high content of hydrophobic amino acids reduces their complexity. Frequently, biochemical or biophysical data is available that informs the optimum alignment, for example, indicating specific positions that share common functional or structural roles. Currently, if those positions are not correctly matched by a standard pairwise sequence alignment procedure, the incorporation of such information into the alignment is typically addressed in an ad hoc manner, with manual adjustments. However, such modifications are problematic because they reduce the robustness and reproducibility of the aligned regions either side of the newly matched positions. Previous studies have introduced restraints as a means to impose the matching of positions during sequence alignments, originally in the context of genome assembly. Here we introduce position restraints, or "anchors" as a feature in our alignment tool AlignMe, providing an aid to pairwise global sequence alignment of alpha-helical membrane proteins. Applying this approach to realistic scenarios involving distantly-related and low complexity sequences, we illustrate how the addition of anchors can be used to modify alignments, while still maintaining the reproducibility and rigor of the rest of the alignment. Anchored alignments can be generated using the online version of AlignMe available at www.bioinfo.mpg.de/AlignMe/.

Global alignment and assessment of TRP channel transmembrane domain structures to explore functional mechanisms.

The recent proliferation of published TRP channel structures provides a foundation for understanding the diverse functional properties of this important family of ion channel proteins. To facilitate mechanistic investigations, we constructed a structure-based alignment of the transmembrane domains of 120 TRP channel structures. Comparison of structures determined in the absence or presence of activating stimuli reveals similar constrictions in the central ion permeation pathway near the intracellular end of the S6 helices, pointing to a conserved cytoplasmic gate and suggesting that most available structures represent non-conducting states. Comparison of the ion selectivity filters toward the extracellular end of the pore supports existing hypotheses for mechanisms of ion selectivity. Also conserved to varying extents are hot spots for interactions with hydrophobic ligands, lipids and ions, as well as discrete alterations in helix conformations. This analysis therefore provides a framework for investigating the structural basis of TRP channel gating mechanisms and pharmacology, and, despite the large number of structures included, reveals the need for additional structural data and for more functional studies to establish the mechanistic basis of TRP channel function.

MemSTATS: A Benchmark Set of Membrane Protein Symmetries and Pseudosymmetries.

In membrane proteins, symmetry and pseudosymmetry often have functional or evolutionary implications. However, available symmetry detection methods have not been tested systematically on this class of proteins because of the lack of an appropriate benchmark set. Here we present MemSTATS, a publicly available benchmark set of both quaternary- and internal-symmetries in membrane protein structures. The symmetries are described in terms of order, repeated elements, and orientation of the axis with respect to the membrane plane. Moreover, using MemSTATS, we compare the performance of four widely used symmetry detection algorithms and highlight specific challenges and areas for improvement in the future.

Intestinal serotonin and fluoxetine exposure modulate bacterial colonization in the gut.

The gut microbiota regulates levels of serotonin (5-hydroxytryptamine (5-HT)) in the intestinal epithelium and lumen1-5. However, whether 5-HT plays a functional role in bacteria from the gut microbiota remains unknown. We demonstrate that elevating levels of intestinal lumenal 5-HT by oral supplementation or genetic deficiency in the host 5-HT transporter (SERT) increases the relative abundance of spore-forming members of the gut microbiota, which were previously reported to promote host 5-HT biosynthesis. Within this microbial community, we identify Turicibacter sanguinis as a gut bacterium that expresses a neurotransmitter sodium symporter-related protein with sequence and structural homology to mammalian SERT. T. sanguinis imports 5-HT through a mechanism that is inhibited by the selective 5-HT reuptake inhibitor fluoxetine. 5-HT reduces the expression of sporulation factors and membrane transporters in T. sanguinis, which is reversed by fluoxetine exposure. Treating T. sanguinis with 5-HT or fluoxetine modulates its competitive colonization in the gastrointestinal tract of antibiotic-treated mice. In addition, fluoxetine reduces the membership of T. sanguinis in the gut microbiota of conventionally colonized mice. Host association with T. sanguinis alters intestinal expression of multiple gene pathways, including those important for lipid and steroid metabolism, with corresponding reductions in host systemic triglyceride levels and inguinal adipocyte size. Together, these findings support the notion that select bacteria indigenous to the gut microbiota signal bidirectionally with the host serotonergic system to promote their fitness in the intestine.

The Molecular Mechanism of Transport by the Mitochondrial ADP/ATP Carrier.

Mitochondrial ADP/ATP carriers transport ADP into the mitochondrial matrix for ATP synthesis, and ATP out to fuel the cell, by cycling between cytoplasmic-open and matrix-open states. The structure of the cytoplasmic-open state is known, but it has proved difficult to understand the transport mechanism in the absence of a structure in the matrix-open state. Here, we describe the structure of the matrix-open state locked by bongkrekic acid bound in the ADP/ATP-binding site at the bottom of the central cavity. The cytoplasmic side of the carrier is closed by conserved hydrophobic residues, and a salt bridge network, braced by tyrosines. Glycine and small amino acid residues allow close-packing of helices on the matrix side. Uniquely, the carrier switches between states by rotation of its three domains about a fulcrum provided by the substrate-binding site. Because these features are highly conserved, this mechanism is likely to apply to the whole mitochondrial carrier family. VIDEO ABSTRACT.

EncoMPASS: an online database for analyzing structure and symmetry in membrane proteins.

The EncoMPASS online database (http://encompass.ninds.nih.gov) collects, organizes, and presents information about membrane proteins of known structure, emphasizing their structural similarities as well as their quaternary and internal symmetries. Unlike, e.g. SCOP, the EncoMPASS database does not aim for a strict classification of membrane proteins, but instead is organized as a protein chain-centric network of sequence and structural homologues. The online server for the EncoMPASS database provides tools for comparing the structural features of its entries, making it a useful resource for homology modeling and active site identification studies. The database can also be used for inferring functionality, which for membrane proteins often involves symmetry-related mechanisms. To this end, the online database also provides a comprehensive description of both the quaternary and internal symmetries in known membrane protein structures, with a particular focus on their orientation relative to the membrane.

Structural elements required for coupling ion and substrate transport in the neurotransmitter transporter homolog LeuT.

The coupled transport of ions and substrates allows transporters to accumulate substrates using the energy of transmembrane ion gradients and electrical potentials. During transport, conformational changes that switch accessibility of substrate and ion binding sites from one side of the membrane to the other must be controlled so as to prevent uncoupled movement of ions or substrates. In the neurotransmitter:sodium symporter (NSS) family, Na+ stabilizes the transporter in an outward-open state, thus decreasing the likelihood of uncoupled Na+ transport. Substrate binding, in a step essential for coupled transport, must overcome the effect of Na+, allowing intracellular substrate and Na+ release from an inward-open state. However, the specific elements of the protein that mediate this conformational response to substrate binding are unknown. Previously, we showed that in the prokaryotic NSS transporter LeuT, the effect of Na+ on conformation requires the Na2 site, where it influences conformation by fostering interaction between two domains of the protein. Here, we used cysteine accessibility to measure conformational changes of LeuT in Escherichia coli membranes. We identified a conserved tyrosine residue in the substrate binding site required for substrate to convert LeuT to inward-open states by establishing an interaction between the two transporter domains. We further identify additional required interactions between the two transporter domains in the extracellular pathway. Together with our previous work on the conformational effect of Na+, these results identify mechanistic components underlying ion-substrate coupling in NSS transporters.

The transport mechanism of the mitochondrial ADP/ATP carrier.

The mitochondrial ADP/ATP carrier imports ADP from the cytosol and exports ATP from the mitochondrial matrix, which are key transport steps for oxidative phosphorylation in eukaryotic organisms. The transport protein belongs to the mitochondrial carrier family, a large transporter family in the inner membrane of mitochondria. It is one of the best studied members of the family and serves as a paradigm for the molecular mechanism of mitochondrial carriers. Structurally, the carrier consists of three homologous domains, each composed of two transmembrane α-helices linked with a loop and short α-helix on the matrix side. The transporter cycles between a cytoplasmic and matrix state in which a central substrate binding site is alternately accessible to these compartments for binding of ADP or ATP. On both the cytoplasmic and matrix side of the carrier are networks consisting of three salt bridges each. In the cytoplasmic state, the matrix salt bridge network is formed and the cytoplasmic network is disrupted, opening the central substrate binding site to the intermembrane space and cytosol, whereas the converse occurs in the matrix state. In the transport cycle, tighter substrate binding in the intermediate states allows the interconversion of conformations by lowering the energy barrier for disruption and formation of these networks, opening and closing the carrier to either side of the membrane in an alternating way. Conversion between cytoplasmic and matrix states might require the simultaneous rotation of three domains around a central translocation pathway, constituting a unique mechanism among transport proteins. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.

Machine-learning scoring functions to improve structure-based binding affinity prediction and virtual screening.

Docking tools to predict whether and how a small molecule binds to a target can be applied if a structural model of such target is available. The reliability of docking depends, however, on the accuracy of the adopted scoring function (SF). Despite intense research over the years, improving the accuracy of SFs for structure-based binding affinity prediction or virtual screening has proven to be a challenging task for any class of method. New SFs based on modern machine-learning regression models, which do not impose a predetermined functional form and thus are able to exploit effectively much larger amounts of experimental data, have recently been introduced. These machine-learning SFs have been shown to outperform a wide range of classical SFs at both binding affinity prediction and virtual screening. The emerging picture from these studies is that the classical approach of using linear regression with a small number of expert-selected structural features can be strongly improved by a machine-learning approach based on nonlinear regression allied with comprehensive data-driven feature selection. Furthermore, the performance of classical SFs does not grow with larger training datasets and hence this performance gap is expected to widen as more training data becomes available in the future. Other topics covered in this review include predicting the reliability of a SF on a particular target class, generating synthetic data to improve predictive performance and modeling guidelines for SF development. WIREs Comput Mol Sci 2015, 5:405-424. doi: 10.1002/wcms.1225 For further resources related to this article, please visit the WIREs website.