DynBench3D,a Web-Resource to Dynamically Generate Benchmark Sets of Large Heteromeric Protein Complexes

Multi-protein machines are responsible for most cellular tasks, and many efforts have been invested in the systematic identification and characterization of thousands of these macromolecular assemblies. However, unfortunately, the (quasi) atomic details necessary to understand their function are available only for a tiny fraction of the known complexes. The computational biology community is developing strategies to integrate structural data of different nature, from electron microscopy to X-ray crystallography, to model large molecular machines, as it has been done for individual proteins and interactions with remarkable success. However, unlike for binary interactions, there is no reliable gold-standard set of three-dimensional (3D) complexes to benchmark the performance of these methodologies and detect their limitations. Here, we present a strategy to dynamically generate non-redundant sets of 3D heteromeric complexes with three or more components. By changing the values of sequence identity and component overlap between assemblies required to define complex redundancy, we can create sets of representative complexes with known 3D structure (i.e., target complexes). Using an identity threshold of 20% and imposing a fraction of component overlap of < 0.5, we identify 495 unique target complexes, which represent a real non-redundant set of heteromeric assemblies with known 3D structure. Moreover, for each target complex, we also identify a set of assemblies, of varying degrees of identity and component overlap, that can be readily used as input in a complex modeling exercise (i.e., template subcomplexes). We hope that resources like this will significantly help the development and progress assessment of novel methodologies, as docking benchmarks and blind prediction contests did. The interactive resource is accessible at https://DynBench3D.irbbarcelona.org.     

Domain-Based Protein Docking with Extremely Large Conformational Changes

Proteins are key components in many processes in living cells, and physical interactions with other proteins and nucleic acids often form key parts of their functions. In many cases, large flexibility of proteins as they interact is key to their function. To understand the mechanisms of these processes, it is necessary to consider the 3D structures of such protein complexes. When such structures are not yet experimentally determined, protein docking has long been present to computationally generate useful structure models. However, protein docking has long had the limitation that the consideration of flexibility is usually limited to very small movements or very small structures. Methods have been developed which handle minor flexibility via normal mode or other structure sampling, but new methods are required to model ordered proteins which undergo large-scale conformational changes to elucidate their function at the molecular level. Here, we present Flex-LZerD, a framework for docking such complexes. Via partial assembly multidomain docking and an iterative normal mode analysis admitting curvilinear motions, we demonstrate the ability to model the assembly of a variety of protein–protein and protein-nucleic acid complexes.     

Asymmetry in membrane protein sequence and structure: glycine outside rule

The growth in the number of known membrane protein structures enables us to conduct new statistical analyses that were not possible before. Using the recently developed Orientation of Protein in Membrane database that has explicit orientation information, here we address the asymmetry in sequence and structure of transmembrane helical proteins. We found that the Gly is markedly more populated at the outer side, the noncytoplasmic side, rather than the inner side, and that Lys, Arg, Leu, and Ile are biased toward the inner side. The asymmetry in Gly distribution is partly attributed to the fact that the coil is more populated in the outer side. Moreover, the frequency of Gly in the coil, but not in the helix, is weakly biased toward the outer side. The asymmetry in Gly distribution is confirmed by a genome-wide sequence-based analysis of membrane proteins accompanied by transmembrane helix prediction.     

Making the Most of Fusion Tags Technology in Structural Characterization of Membrane Proteins

Membrane proteins can be investigated at various structural levels, including the topological structure, the high-resolution three-dimensional structure, and the organization and assembly of membrane protein complexes. Gene fusion technology makes it possible to insert a polynucleotide encoding a protein or polypeptide tag into the gene encoding a membrane protein of interest. Resultant recombinant proteins may possess the functions of the original membrane proteins, together with the biochemical properties of the imported fusion tag, greatly enhancing functional and structural studies of membrane proteins. In this article, the latest literature is reviewed in relation to types, applications, strategies, and approaches to fusion tag technology for structural investigations of membrane proteins.     

The evolution of structural databases

Starting with the Protein Data Bank (PDB) as a common ancestor, the evolution of structural databases has been driven by the rapprochement of the structural world and the practical applications. The result is an impressive number of secondary structural databases that is welcomed by structural biologists and bioinformaticians but runs the risk of producing an embarrassment of riches among non-specialist users. Given that any profit depends on the number of customers, efficient interfaces between many structural data banks must be available to make their contents easily accessible. Increasing the information content of central structural repositories might be the best way to guide users through the many, sometimes overlapping databases.     

Enhanced validation of small-molecule ligands and carbohydrates in the Protein Data Bank

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双向BN/SDS-PAGE分离鉴定痢疾杆菌福氏5型M90T株毒力相关膜蛋白复合物

通过蓝色非变性凝胶电泳(BN-PAGE)比较痢疾杆菌福氏5型野生株M90T和大质粒缺失株M90T△T的膜蛋白复合物,发现一个野生株特有的复合物,其分子量的为290 kD,命名为M90T-290.通过第二向SDS-PAGE分离M90T-290得到6个蛋白亚基,质谱鉴定为:一个由大质粒编码的毒力蛋白Apyrase(ATP-二磷酸水解酶)和5个染色体编码的蛋白,这些蛋白可能以膜复合物的形式影响毒力蛋白IcsA的单极性分布和痢疾杆菌在细胞间的扩散.这个新发现的毒力相关膜复合物在痢疾杆菌的致病过程中可能发挥重要作用.     

Lipid-dependent Generation of Dual Topology for a Membrane Protein

The mechanism by which membrane proteins exhibit structural and functional duality in the same membrane or different membranes is unknown. We posit that such duality is determined by both the protein sequence and the membrane lipid composition wherein a spatial or temporal change in the latter can result in a post-assembly change in protein structure and function. To investigate whether co-existence of multiple topological conformers is dependent on the membrane lipid composition, we determined the topological organization of lactose permease in an Escherichia coli model cell system in which phosphatidylethanolamine membrane content can be systematically varied. At intermediate levels of phosphatidylethanolamine a mixture of native and topologically mis-oriented conformers co-existed. There was no threshold level of phosphatidylethanolamine determining a sharp transition from one conformer to the other. Co-existing conformers were not in rapid equilibrium at a static lipid composition indicating that duality of topology is established during an early folding step. Depletion of intermediate levels of phosphatidylethanolamine after final protein assembly resulted in complete mis-orientation of the native conformer. Combined with previous results, such topological dynamics are reversible in both directions. We propose a thermodynamically based model for how lipid-protein interactions can result in a mixed topological organization and how changes in lipid composition can result in changes in the ratio of topologically distinct conformers of proteins. These observations demonstrate a potential lipid-dependent biological switch for generating dynamic structural and functional heterogeneity for a protein within the same membrane or between different membranes in more complex eukaryotic cells.     

Monitoring Shifts in the Conformation Equilibrium of the Membrane Protein Cytochrome P450 Reductase (POR) in Nanodiscs

Nanodiscs are self-assembled ∼50-nm² patches of lipid bilayers stabilized by amphipathic belt proteins. We demonstrate that a well ordered dense film of nanodiscs serves for non-destructive, label-free studies of isolated membrane proteins in a native like environment using neutron reflectometry (NR). This method exceeds studies of membrane proteins in vesicle or supported lipid bilayer because membrane proteins can be selectively adsorbed with controlled orientation. As a proof of concept, the mechanism of action of the membrane-anchored cytochrome P450 reductase (POR) is studied here. This enzyme is responsible for catalyzing the transfer of electrons from NADPH to cytochrome P450s and thus is a key enzyme in the biosynthesis of numerous primary and secondary metabolites in plants. Neutron reflectometry shows a coexistence of two different POR conformations, a compact and an extended form with a thickness of 44 and 79 Å, respectively. Upon complete reduction by NADPH, the conformational equilibrium shifts toward the compact form protecting the reduced FMN cofactor from engaging in unspecific electron transfer reaction.     

Subunit Organization in the TatA Complex of the Twin Arginine Protein Translocase: A SITE-DIRECTED EPR SPIN LABELING STUDY*

The Tat system is used to transport folded proteins across the cytoplasmic membrane in bacteria and archaea and across the thylakoid membrane of plant chloroplasts. Multimers of the integral membrane TatA protein are thought to form the protein-conducting element of the Tat pathway. Nitroxide radicals were introduced at selected positions within the transmembrane helix of Escherichia coli TatA and used to probe the structure of detergent-solubilized TatA complexes by EPR spectroscopy. A comparison of spin label mobilities allowed classification of individual residues as buried within the TatA complex or exposed at the surface and suggested that residues Ile¹² and Val¹⁴ are involved in interactions between helices. Analysis of inter-spin distances suggested that the transmembrane helices of TatA subunits are arranged as a single-walled ring containing a contact interface between Ile¹² on one subunit and Val¹⁴ on an adjacent subunit. Experiments in which labeled and unlabeled TatA samples were mixed demonstrate that TatA subunits are exchanged between TatA complexes. This observation is consistent with the TatA dynamic polymerization model for the mechanism of Tat transport.     

Continuous Evaluation of Ligand Protein Predictions: A Weekly Community Challenge for Drug Docking

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Genome-wide Membrane Protein Structure Prediction

Transmembrane proteins allow cells to extensively communicate with the external world in a very accurate and specific way. They form principal nodes in several signaling pathways and attract large interest in therapeutic intervention, as the majority pharmaceutical compounds target membrane proteins. Thus, according to the current genome annotation methods, a detailed structural/functional characterization at the protein level of each of the elements codified in the genome is also required. The extreme difficulty in obtaining high-resolution three-dimensional structures, calls for computational approaches. Here we review to which extent the efforts made in the last few years, combining the structural characterization of membrane proteins with protein bioinformatics techniques, could help describing membrane proteins at a genome-wide scale. In particular we analyze the use of comparative modeling techniques as a way of overcoming the lack of high-resolution three-dimensional structures in the human membrane proteome.     

融合标签技术在膜蛋白结构研究中的应用

膜蛋白高级结构的研究包括不同的层次,即膜蛋白拓扑学结构的研究、利用核磁共振技术和蛋白质晶体衍射技术对三维结构的研究,以及膜蛋白复合体的研究。在研究过程中,如果能够基于膜蛋白的拓扑学结构预测,选择合适的蛋白质或多肽融合标签,利用基因融合技术在基因水平上对膜蛋白进行改造,可以产生含有融合标签的重组膜蛋自,不仅具有原有膜蛋白的功能活性,还具有融合标签所特有的生理生化特性,将会极大地促进膜蛋白结构和功能的研究。我们就目前膜蛋白结构研究中所涉及的融合标签技术及其应用策略和所取得的进展做一简述。     

Solution Structure,Membrane Interactions,and Protein Binding Partners of the Tetraspanin Sm-TSP-2, a Vaccine Antigen from the Human Blood Fluke Schistosoma mansoni

The tetraspanins (TSPs) are a family of integral membrane proteins that are ubiquitously expressed at the surface of eukaryotic cells. TSPs mediate a range of processes at the surface of the plasma membrane by providing a scaffold for the assembly of protein complexes known as tetraspanin-enriched microdomains (TEMs). We report here the structure of the surface-exposed EC2 domain from Sm-TSP-2, a TSP from Schistosoma mansoni and one of the better prospects for the development of a vaccine against schistosomiasis. This is the first solution structure of this domain, and our investigations of its interactions with lipid micelles provide a general model for interactions between TSPs, membranes, and other proteins. Using chemical cross-linking, eight potential protein constituents of Sm-TSP-2-mediated TEMs were also identified. These include proteins important for membrane maintenance and repair, providing further evidence for the functional role of Sm-TSP-2- and Sm-TSP-2-mediated TEMs. The identification of calpain, Sm29, and fructose-bisphosphate aldolase, themselves potential vaccine antigens, suggests that the Sm-TSP-2-mediated TEMs could be disrupted via multiple targets. The identification of further Sm-TSP-2-mediated TEM proteins increases the available candidates for multiplex vaccines and/or novel drugs targeting TEMs in the schistosome tegument.     

Hierarchical Description and Extensive Classification of Protein Structural Changes by Motion Tree

The structures of the same protein, determined under different conditions, provide clues toward understanding the role of structural changes in the protein's function. Structural changes are usually identified as rigid-body motions, which are defined using a particular threshold of rigidity, such as domain motions. However, each protein actually undergoes motions with various size and magnitude ranges. In this study, to describe protein structural changes more comprehensively, we propose a method based on hierarchical clustering. This method enables the illustration of a wide range of protein motions in a single tree diagram, named the “Motion Tree”. We applied the method to 432 proteins exhibiting large structural changes and classified their Motion Trees in terms of the characteristic indices of the trees. This classification of the Motion Trees revealed clear relationships to their protein functions. Especially, complex structural changes are significantly correlated with multi-step protein functions.     

Saposin Lipid Nanoparticles: A Highly Versatile and Modular Tool for Membrane Protein Research

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A Conformational Switch in the Scaffolding Protein NHERF1 Controls Autoinhibition and Complex Formation

The mammalian Na⁺/H⁺ exchange regulatory factor 1 (NHERF1) is a multidomain scaffolding protein essential for regulating the intracellular trafficking and macromolecular assembly of transmembrane ion channels and receptors. NHERF1 consists of tandem PDZ-1, PDZ-2 domains that interact with the cytoplasmic domains of membrane proteins and a C-terminal (CT) domain that binds the membrane-cytoskeleton linker protein ezrin. NHERF1 is held in an autoinhibited state through intramolecular interactions between PDZ2 and the CT domain that also includes a C-terminal PDZ-binding motif (-SNL). We have determined the structures of the isolated and tandem PDZ2CT domains by high resolution NMR using small angle x-ray scattering as constraints. The PDZ2CT structure shows weak intramolecular interactions between the largely disordered CT domain and the PDZ ligand binding site. The structure reveals a novel helix-turn-helix subdomain that is allosterically coupled to the putative PDZ2 domain by a network of hydrophobic interactions. This helical subdomain increases both the stability and the binding affinity of the extended PDZ structure. Using NMR and small angle neutron scattering for joint structure refinement, we demonstrate the release of intramolecular domain-domain interactions in PDZ2CT upon binding to ezrin. Based on the structural information, we show that human disease-causing mutations in PDZ2, R153Q and E225K, have significantly reduced protein stability. Loss of NHERF1 expressed in cells could result in failure to assemble membrane complexes that are important for normal physiological functions.