BTB domain mutations perturbing KCTD15 oligomerisation cause a distinctive frontonasal dysplasia syndrome.

INTRODUCTION: KCTD15 encodes an oligomeric BTB domain protein reported to inhibit neural crest formation through repression of Wnt/beta-catenin signalling, as well as transactivation by TFAP2. Heterozygous missense variants in the closely related paralogue KCTD1 cause scalp-ear-nipple syndrome. METHODS: Exome sequencing was performed on a two-generation family affected by a distinctive phenotype comprising a lipomatous frontonasal malformation, anosmia, cutis aplasia of the scalp and/or sparse hair, and congenital heart disease. Identification of a de novo missense substitution within KCTD15 led to targeted sequencing of DNA from a similarly affected sporadic patient, revealing a different missense mutation. Structural and biophysical analyses were performed to assess the effects of both amino acid substitutions on the KCTD15 protein. RESULTS: A heterozygous c.310G>C variant encoding p.(Asp104His) within the BTB domain of KCTD15 was identified in an affected father and daughter and segregated with the phenotype. In the sporadically affected patient, a de novo heterozygous c.263G>A variant encoding p.(Gly88Asp) was present in KCTD15. Both substitutions were found to perturb the pentameric assembly of the BTB domain. A crystal structure of the BTB domain variant p.(Gly88Asp) revealed a closed hexameric assembly, whereas biophysical analyses showed that the p.(Asp104His) substitution resulted in a monomeric BTB domain likely to be partially unfolded at physiological temperatures. CONCLUSION: BTB domain substitutions in KCTD1 and KCTD15 cause clinically overlapping phenotypes involving craniofacial abnormalities and cutis aplasia. The structural analyses demonstrate that missense substitutions act through a dominant negative mechanism by disrupting the higher order structure of the KCTD15 protein complex.

Dissecting and predicting different types of binding sites in nucleic acids based on structural information.

The biological functions of DNA and RNA generally depend on their interactions with other molecules, such as small ligands, proteins and nucleic acids. However, our knowledge of the nucleic acid binding sites for different interaction partners is very limited, and identification of these critical binding regions is not a trivial work. Herein, we performed a comprehensive comparison between binding and nonbinding sites and among different categories of binding sites in these two nucleic acid classes. From the structural perspective, RNA may interact with ligands through forming binding pockets and contact proteins and nucleic acids using protruding surfaces, while DNA may adopt regions closer to the middle of the chain to make contacts with other molecules. Based on structural information, we established a feature-based ensemble learning classifier to identify the binding sites by fully using the interplay among different machine learning algorithms, feature spaces and sample spaces. Meanwhile, we designed a template-based classifier by exploiting structural conservation. The complementarity between the two classifiers motivated us to build an integrative framework for improving prediction performance. Moreover, we utilized a post-processing procedure based on the random walk algorithm to further correct the integrative predictions. Our unified prediction framework yielded promising results for different binding sites and outperformed existing methods.

Improving the clinical interpretation of missense variants in X linked genes using structural analysis.

BACKGROUND: Improving the clinical interpretation of missense variants can increase the diagnostic yield of genomic testing and lead to personalised management strategies. Currently, due to the imprecision of bioinformatic tools that aim to predict variant pathogenicity, their role in clinical guidelines remains limited. There is a clear need for more accurate prediction algorithms and this study aims to improve performance by harnessing structural biology insights. The focus of this work is missense variants in a subset of genes associated with X linked disorders. METHODS: We have developed a protein-specific variant interpreter (ProSper) that combines genetic and protein structural data. This algorithm predicts missense variant pathogenicity by applying machine learning approaches to the sequence and structural characteristics of variants. RESULTS: ProSper outperformed seven previously described tools, including meta-predictors, in correctly evaluating whether or not variants are pathogenic; this was the case for 11 of the 21 genes associated with X linked disorders that met the inclusion criteria for this study. We also determined gene-specific pathogenicity thresholds that improved the performance of VEST4, REVEL and ClinPred, the three best-performing tools out of the seven that were evaluated; this was the case in 11, 11 and 12 different genes, respectively. CONCLUSION: ProSper can form the basis of a molecule-specific prediction tool that can be implemented into diagnostic strategies. It can allow the accurate prioritisation of missense variants associated with X linked disorders, aiding precise and timely diagnosis. In addition, we demonstrate that gene-specific pathogenicity thresholds for a range of missense prioritisation tools can lead to an increase in prediction accuracy.

cAMP is an allosteric modulator of DNA-binding specificity in the cAMP receptor protein from Mycobacterium tuberculosis.

Allosteric proteins with multiple subunits and ligand-binding sites are central in regulating biological signals. The cAMP receptor protein from Mycobacterium tuberculosis (CRPMTB) is a global regulator of transcription composed of two identical subunits, each one harboring structurally conserved cAMP- and DNA-binding sites. The mechanisms by which these four binding sites are allosterically coupled in CRPMTB remain unclear. Here, we investigate the binding mechanism between CRPMTB and cAMP, and the linkage between cAMP and DNA interactions. Using calorimetric and fluorescence-based assays, we find that cAMP binding is entropically driven and displays negative cooperativity. Fluorescence anisotropy experiments show that apo-CRPMTB forms high-order CRPMTB-DNA oligomers through interactions with nonspecific DNA sequences or preformed CRPMTB-DNA complexes. Moreover, we find that cAMP prevents and reverses the formation of CRPMTB-DNA oligomers, reduces the affinity of CRPMTB for nonspecific DNA sequences, and stabilizes a 1-to-1 CRPMTB-DNA complex, but does not increase the affinity for DNA like in the canonical CRP from Escherichia coli (CRPEcoli). DNA-binding assays as a function of cAMP concentration indicate that one cAMP molecule per homodimer dissociates high-order CRPMTB-DNA oligomers into 1-to-1 complexes. These cAMP-mediated allosteric effects are lost in the double-mutant L47P/E178K found in CRP from Mycobacterium bovis Bacille Calmette-Guérin (CRPBCG). The functional behavior, thermodynamic stability, and dimerization constant of CRPBCG are not due to additive effects of L47P and E178K, indicating long-range interactions between these two sites. Altogether, we provide a previously undescribed archetype of cAMP-mediated allosteric regulation that differs from CRPEcoli, illustrating that structural homology does not imply allosteric homology.

Functional and structural analysis of rare SLC2A2 variants associated with Fanconi-Bickel syndrome and metabolic traits.

Deleterious variants in SLC2A2 cause Fanconi-Bickel Syndrome (FBS), a glycogen storage disorder, whereas less common variants in SLC2A2 associate with numerous metabolic diseases. Phenotypic heterogeneity in FBS has been observed, but its causes remain unknown. Our goal was to functionally characterize rare SLC2A2 variants found in FBS and metabolic disease-associated variants to understand the impact of these variants on GLUT2 activity and expression and establish genotype-phenotype correlations. Complementary RNA-injected Xenopus laevis oocytes were used to study mutant transporter activity and membrane expression. GLUT2 homology models were constructed for mutation analysis using GLUT1, GLUT3, and XylE as templates. Seventeen FBS variants were characterized. Only c.457_462delCTTATA (p.Leu153_Ile154del) exhibited residual glucose uptake. Functional characterization revealed that only half of the variants were expressed on the plasma membrane. Most less common variants (except c.593 C>A (p.Thr198Lys) and c.1087 G>T (p.Ala363Ser)) exhibited similar GLUT2 transport activity as the wild type. Structural analysis of GLUT2 revealed that variants affect substrate-binding, steric hindrance, or overall transporter structure. The mutant transporter that is associated with a milder FBS phenotype, p.Leu153_Ile154del, retained transport activity. These results improve our overall understanding of the underlying causes of FBS and impact of GLUT2 function on various clinical phenotypes ranging from rare to common disease.

Structural Homology Between Nucleoproteins of ssRNA Viruses.

Our understanding of the viral world changed just after the first structures of icosahedral viral particles were unveiled. The structural similarities between capsid proteins of distant viral groups were not anticipated, and the findings suggested the existence of common ancestors for viruses with different host range, genomic structure and multiplication strategies. This way, diverse viruses with icosahedral particles can now be grouped based on the structural homology between their capsid proteins. In the last years, the presence of conserved folds between viral proteins in non-icosahedral viruses has also emerged. Viral particles with radically different morphologies, ranging from naked and filamentous to enveloped and pleomorphic, have shown structural homology between the nucleoproteins that bind directly to their genomes. This chapter overviews recent findings regarding the similar structure found between nucleoproteins of eukaryotic ssRNA viruses. The structural homology includes the coat proteins from all known families of flexible filamentous plant viruses, a group with monopartite (+)ssRNA genomes. Their coat proteins share a core domain with nucleoproteins of previously unrelated families of enveloped viruses that have segmented (-)ssRNA genomes. This last group consists of mostly animals viruses, including influenza virus.

Identification of a new nucleotide binding site by structural alignment and site directed mutagenesis.

Nucleotide binding proteins are involved in many important cellular processes and form one of the largest protein families. Traditionally, the identification of nucleotide binding motif, such as the ATP binding P-loop, has relied on the comparison of protein sequences, consideration of the function of each of the proteins and the identification of signature motifs within the sequence. Sometimes, it is difficult to identify nucleotide binding proteins based on sequence alignment because of increased evolutionary distances. In such cases, structural alignments can provide a better guide for comparing specific features of sequences because the overall structures of these motifs are conserved despite low sequence identity. In the present study, on the basis of bioinformatics and structural comparison of three representative protein structures of Ham1 superfamily, YjjX, YggV, and YhdE, previously identified as nucleotide binding proteins, we have identified a novel nucleotide binding motif (T/SXXXXK/R). The importance of this signature motif in binding of nucleotides was validated using site directed mutagenesis. Mutations of conserved residues of the loop either decreased or completely abolished the nucleotide binding activity of the protein. We used the conserved motif identified in the study to search for other proteins having a similar motif. Two proteins, GTP cyclohydrolase II and dephospho-CoA pyrophosphorylase showed presence of the loop, suggesting that this nucleotide binding motif is not unique in the Ham1 superfamily, but represents a novel NTP recognition motif.

An overview of combined D-2- and L-2-hydroxyglutaric aciduria: functional analysis of CIC variants.

Combined D-2- and L-2-hydroxyglutaric aciduria (D/L-2-HGA) is a devastating neurometabolic disorder, usually lethal in the first years of life. Autosomal recessive mutations in the SLC25A1 gene, which encodes the mitochondrial citrate carrier (CIC), were previously detected in patients affected with combined D/L-2-HGA. We showed that transfection of deficient fibroblasts with wild-type SLC25A1 restored citrate efflux and decreased intracellular 2-hydroxyglutarate levels, confirming that deficient CIC is the cause of D/L-2-HGA. We developed and implemented a functional assay and applied it to all 17 missense variants detected in a total of 26 CIC-deficient patients, including eight novel cases, showing reduced activities of varying degrees. In addition, we analyzed the importance of residues affected by these missense variants using our existing scoring system. This allowed not only a clinical and biochemical overview of the D/L-2-HGA patients but also phenotype-genotype correlation studies.

The Defensins Consist of Two Independent, Convergent Protein Superfamilies.

The defensin and defensin-like proteins are an extensive group of small, cationic, disulfide-rich proteins found in animals, plants, and fungi and mostly perform roles in host defense. The term defensin was originally used for small mammalian proteins found in neutrophils and was subsequently applied to insect proteins and plant γ-thionins based on their perceived sequence and structural similarity. Defensins are often described as ancient innate immunity molecules and classified as a single superfamily and both sequence alignments and phylogenies have been constructed. Here, we present evidence that the defensins have not all evolved from a single ancestor. Instead, they consist of two analogous superfamilies, and extensive convergent evolution is the source of their similarities. Evidence of common origin necessarily gets weaker for distantly related genes, as is the case for defensins, which are both divergent and small. We show that similarities that have been used as evidence for common origin are all expected by chance in short, constrained, disulfide-rich proteins. Differences in tertiary structure, secondary structure order, and disulfide bond connectivity indicate convergence as the likely source of the similarity. We refer to the two evolutionarily independent groups as the cis-defensins and trans-defensins based on the orientation of the most conserved pair of disulfides.

Cryo-EM near-atomic structure of a dsRNA fungal virus shows ancient structural motifs preserved in the dsRNA viral lineage.

Viruses evolve so rapidly that sequence-based comparison is not suitable for detecting relatedness among distant viruses. Structure-based comparisons suggest that evolution led to a small number of viral classes or lineages that can be grouped by capsid protein (CP) folds. Here, we report that the CP structure of the fungal dsRNA Penicillium chrysogenum virus (PcV) shows the progenitor fold of the dsRNA virus lineage and suggests a relationship between lineages. Cryo-EM structure at near-atomic resolution showed that the 982-aa PcV CP is formed by a repeated α-helical core, indicative of gene duplication despite lack of sequence similarity between the two halves. Superimposition of secondary structure elements identified a single "hotspot" at which variation is introduced by insertion of peptide segments. Structural comparison of PcV and other distantly related dsRNA viruses detected preferential insertion sites at which the complexity of the conserved α-helical core, made up of ancestral structural motifs that have acted as a skeleton, might have increased, leading to evolution of the highly varied current structures. Analyses of structural motifs only apparent after systematic structural comparisons indicated that the hallmark fold preserved in the dsRNA virus lineage shares a long (spinal) α-helix tangential to the capsid surface with the head-tailed phage and herpesvirus viral lineage.

RNA3DB: A structurally-dissimilar dataset split for training and benchmarking deep learning models for RNA structure prediction.

With advances in protein structure prediction thanks to deep learning models like AlphaFold, RNA structure prediction has recently received increased attention from deep learning researchers. RNAs introduce substantial challenges due to the sparser availability and lower structural diversity of the experimentally resolved RNA structures in comparison to protein structures. These challenges are often poorly addressed by the existing literature, many of which report inflated performance due to using training and testing sets with significant structural overlap. Further, the most recent Critical Assessment of Structure Prediction (CASP15) has shown that deep learning models for RNA structure are currently outperformed by traditional methods. In this paper we present RNA3DB, a dataset of structured RNAs, derived from the Protein Data Bank (PDB), that is designed for training and benchmarking deep learning models. The RNA3DB method arranges the RNA 3D chains into distinct groups (Components) that are non-redundant both with regard to sequence as well as structure, providing a robust way of dividing training, validation, and testing sets. Any split of these structurally-dissimilar Components are guaranteed to produce test and validations sets that are distinct by sequence and structure from those in the training set. We provide the RNA3DB dataset, a particular train/test split of the RNA3DB Components (in an approximate 70/30 ratio) that will be updated periodically. We also provide the RNA3DB methodology along with the source-code, with the goal of creating a reproducible and customizable tool for producing structurally-dissimilar dataset splits for structural RNAs.

Rhizobium etli has two L-asparaginases with low sequence identity but similar structure and catalytic center.

The genome of Rhizobium etli, a nitrogen-fixing bacterial symbiont of legume plants, encodes two L-asparaginases, ReAIV and ReAV, that have no similarity to the well characterized enzymes of class 1 (bacterial type) and class 2 (plant type). It has been hypothesized that ReAIV and ReAV might belong to the same structural class 3 despite their low level of sequence identity. When the crystal structure of the inducible and thermolabile protein ReAV was solved, this hypothesis gained a stronger footing because the key residues of ReAV are also present in the sequence of the constitutive and thermostable ReAIV protein. High-resolution crystal structures of ReAIV now confirm that it is a class 3 L-asparaginase that is structurally similar to ReAV but with important differences. The most striking differences concern the peculiar hydration patterns of the two proteins, the presence of three internal cavities in ReAIV and the behavior of the zinc-binding site. ReAIV has a high pH optimum (9-11) and a substrate affinity of ∼1.3 mM at pH 9.0. These parameters are not suitable for the direct application of ReAIV as an antileukemic drug, although its thermal stability and lack of glutaminase activity would be of considerable advantage. The five crystal structures of ReAIV presented in this work allow a possible enzymatic scenario to be postulated in which the zinc ion coordinated in the active site is a dispensable element. The catalytic nucleophile seems to be Ser47, which is part of two Ser-Lys tandems in the active site. The structures of ReAIV presented here may provide a basis for future enzyme-engineering experiments to improve the kinetic parameters for medicinal applications.

Structural analysis of hypothetical proteins from Helicobacter pylori: an approach to estimate functions of unknown or hypothetical proteins.

Helicobacter pylori (H. pylori) have a unique ability to survive in extreme acidic environments and to colonize the gastric mucosa. It can cause diverse gastric diseases such as peptic ulcers, chronic gastritis, mucosa-associated lymphoid tissue (MALT) lymphoma, gastric cancer, etc. Based on genomic research of H. pylori, over 1600 genes have been functionally identified so far. However, H. pylori possess some genes that are uncharacterized since: (i) the gene sequences are quite new; (ii) the function of genes have not been characterized in any other bacterial systems; and (iii) sometimes, the protein that is classified into a known protein based on the sequence homology shows some functional ambiguity, which raises questions about the function of the protein produced in H. pylori. Thus, there are still a lot of genes to be biologically or biochemically characterized to understand the whole picture of gene functions in the bacteria. In this regard, knowledge on the 3D structure of a protein, especially unknown or hypothetical protein, is frequently useful to elucidate the structure-function relationship of the uncharacterized gene product. That is, a structural comparison with known proteins provides valuable information to help predict the cellular functions of hypothetical proteins. Here, we show the 3D structures of some hypothetical proteins determined by NMR spectroscopy and X-ray crystallography as a part of the structural genomics of H. pylori. In addition, we show some successful approaches of elucidating the function of unknown proteins based on their structural information.

Deltamethrin-Induced Respiratory and Behavioral Effects and Adverse Outcome Pathways (AOP) in Short-Term Exposed Mozambique Tilapia, Oreochromis mossambicus.

Disrupted behavior and respiratory distress effects of 96-h acute deltamethrin exposures in adult Mozambique tilapia, Oreochromis mossambicus, were investigated using behavioral indices and opercular movement, respectively. Deltamethrin concentrations were found to be associated with toxicological (lethal and sublethal) responses. At 24, 48, 72, and 96 h, the LC50 values and 95% confidence limits were 12.290 (11.174-14.411 µg/L), 12.671 (11.334-15.649 µg/L), 10.172 (9.310-11.193 µg/L), and 8.639 (7.860-9.417 µg/L), respectively. The GUTS-model analysis showed that GUTS-SD (stochastic death) with a narrow tolerance distribution in deltamethrin exposed O. mossambicus populations was more sensitive than the GUTS-IT (individual tolerance) model. Prior to death, exposed fish demonstrated concentration-dependent mortality and disturbed behavioral responses, including uncoordinated swim motions, increased mucus secretion, unbalanced and unpredictable swimming patterns, and inactivity. The altered behavioral patterns and increased opercular movement with increased deltamethrin levels and exposure time are strongly suggestive of neurotoxicity and respiratory distress, respectively. Adverse Outcome Pathways (AOPs), describing biological mechanisms and plausible pathways, highlighted oxidative stress and cholinergic effects as intermediate steps linked to respiratory distress and behavioral toxicity.

Structural analysis of PpSP15 and PsSP9 sand fly salivary proteins designed with a self-cleavable linker as a live vaccine candidate against cutaneous leishmaniasis.

BACKGROUND: Leishmania parasites are deposited in the host through sand fly bites along with sand fly saliva. Therefore, salivary proteins are promising vaccine candidates for controlling leishmaniasis. Herein, two immunogenic salivary proteins, PpSP15 from Phlebotomus papatasi and PsSP9 from Phlebotomus sergenti, were selected as vaccine candidates to be delivered by live Leishmania tarentolae as vector. The stepwise in silico protocol advantaged in this study for multi-protein design in L. tarentolae is then described in detail. METHODS: All possible combinations of two salivary proteins, PpSP15 and PsSP9, with or without T2A peptide were designed at the mRNA and protein levels. Then, the best combination for the vaccine candidate was selected based on mRNA and protein stability along with peptide analysis. RESULTS: At the mRNA level, the most favored secondary structure was PpSP15-T2A-PsSP9. At the protein level, the refined three-dimensional models of all combinations were structurally valid; however, local quality estimation showed that the PpSp15-T2A-PsSP9 fusion had higher stability for each amino acid position, with low root-mean-square deviation (RMSD), compared with the original proteins. In silico evaluation confirmed the PpSP15-T2A-PsSP9 combination as a good Th1-polarizing candidate in terms of high IFN-γ production and low IL-10/TGF-ß ratio in response to three consecutive immunizations. Potential protein expression was then confirmed by Western blotting. CONCLUSIONS: The approach presented herein is among the first studies to have privileged protein homology modeling along with mRNA analysis for logical live vaccine design-coding multi-proteins.

Comparative Reverse Vaccinology of Piscirickettsia salmonis, Aeromonas salmonicida, Yersinia ruckeri, Vibrio anguillarum and Moritella viscosa, Frequent Pathogens of Atlantic Salmon and Lumpfish Aquaculture.

Marine finfish aquaculture is affected by diverse infectious diseases, and they commonly occur as co-infection. Some of the most frequent and prevalent Gram-negative bacterial pathogens of the finfish aquaculture include Piscirickettsia salmonis, Aeromonas salmonicida, Yersinia ruckeri, Vibrio anguillarum and Moritella viscosa. To prevent co-infections in aquaculture, polyvalent or universal vaccines would be ideal. Commercial polyvalent vaccines against some of these pathogens are based on whole inactivated microbes and their efficacy is controversial. Identification of common antigens can contribute to the development of effective universal or polyvalent vaccines. In this study, we identified common and unique antigens of P. salmonis, A. salmonicida, Y. ruckeri, V. anguillarum and M. viscosa based on a reverse vaccinology pipeline. We screened the proteome of several strains using complete available genomes and identified a total of 154 potential antigens, 74 of these identified antigens corresponded to secreted proteins, and 80 corresponded to exposed outer membrane proteins (OMPs). Further analysis revealed the outer membrane antigens TonB-dependent siderophore receptor, OMP assembly factor BamA, the LPS assembly protein LptD and secreted antigens flagellar hook assembly protein FlgD and flagellar basal body rod protein FlgG are present in all pathogens used in this study. Sequence and structural alignment of these antigens showed relatively low percentage sequence identity but good structural homology. Common domains harboring several B-cells and T-cell epitopes binding to major histocompatibility (MHC) class I and II were identified. Selected peptides were evaluated for docking with Atlantic salmon (Salmo salar) and Lumpfish MHC class II. Interaction of common peptide-MHC class II showed good in-silico binding affinities and dissociation constants between -10.3 to -6.5 kcal mol-1 and 5.10 × 10-9 to 9.4 × 10-6 M. This study provided the first list of antigens that can be used for the development of polyvalent or universal vaccines against these Gram-negative bacterial pathogens affecting finfish aquaculture.

The Rad9-Rad1-Hus1 DNA Repair Clamp is Found in Microsporidia.

DNA repair is an important component of genome integrity and organisms with reduced repair capabilities tend to accumulate mutations at elevated rates. Microsporidia are intracellular parasites exhibiting high levels of genetic divergence postulated to originate from the lack of several proteins, including the heterotrimeric Rad9-Rad1-Hus1 DNA repair clamp. Microsporidian species from the Encephalitozoonidae have undergone severe streamlining with small genomes coding for about 2,000 proteins. The highly divergent sequences found in Microsporidia render functional inferences difficult such that roughly half of these 2,000 proteins have no known function. Using a structural homology-based annotation approach combining protein structure prediction and tridimensional similarity searches, we found that the Rad9-Rad1-Hus1 DNA clamp is present in Microsporidia, together with many other components of the DNA repair machinery previously thought to be missing from these organisms. Altogether, our results indicate that the DNA repair machinery is present and likely functional in Microsporidia.

Molecular Mimicry Analyses Unveiled the Human Herpes Simplex and Poxvirus Epitopes as Possible Candidates to Incite Autoimmunity.

Clinical epidemiological studies have reported that viral infections cause autoimmune pathology in humans. Host-pathogen protein sequences and structure-based molecular mimicry cause autoreactive T cells to cross-activate. The aim of the current study was to implement immunoinformatics approaches to infer sequence- and structure-based molecular mimicry between viral and human proteomic datasets. The protein sequences of all the so far known human-infecting viruses were obtained from the VIPR database, and complete human proteome data were retrieved from the NCBI repository. Based on a predefined, stringent threshold of comparative sequence analyses, 24 viral proteins were identified with significant sequence similarity to human proteins. PathDIP identified the enrichment of these homologous proteins in nine metabolic pathways with a p-value < 0.0001. Several viral and human mimic epitopes from these homologous proteins were predicted as strong binders of human HLA alleles, with IC50 < 50 nM. Downstream molecular docking analyses identified that lead virus-human homologous epitopes feasibly interact with HLA and TLR4 types of immune receptors. The vast majority of these top-hit homolog epitopic peptides belong to the herpes simplex and poxvirus families. These lead epitope biological sequences and 3D structural-based molecular mimicry may be promising for interpreting herpes simplex virus and poxvirus infection-mediated autoimmune disorders in humans.

Functional Annotation from Structural Homology.

With the nexus of super computing and the biotech revolution, it seems an era of predictive biology through systems biology may be at hand. Modern omics capabilities enable examination of the state of biological system in exquisite detail. The genome, transcriptome, proteome, and metabolome may all be largely knowable, at least for some model systems, providing a basis for modeling and simulation of molecular mechanisms, or pathways, that could capture a biological system's emergent properties. However, there are significant challenges remaining that impede the realization of this vision, perhaps the most significant being the missing functional annotation of genes and gene products. For even the most well-studied organisms as much as a third of called genes for a given genome are not annotated and more than half may be tenuous. Homology inferred from sequence similarity is the basis for much of genome annotation. Homology inferred from structural similarity could be a powerful complement to sequence-based annotation methods. Structural biology or structural informatics can be used to assign molecular function and may have increasing utility with the rapid growth of gene sequence databases and emerging methods for structure determination, like structure prediction based on coevolution. Here we describe tools and provide example cases using structural similarity at the level of quaternary structure, domain content, domain topology, and small 3D motifs to infer homology and posit function. Ultimately annotation by similarity, be it 3D structure homology or more classically primary sequence homology, must be founded by accurate annotation of one ortholog in the group-understanding every function encoded by a genome remains a major challenge to life science.

The Viral Class II Membrane Fusion Machinery: Divergent Evolution from an Ancestral Heterodimer.

A key step during the entry of enveloped viruses into cells is the merger of viral and cell lipid bilayers. This process is driven by a dedicated membrane fusion protein (MFP) present at the virion surface, which undergoes a membrane-fusogenic conformational change triggered by interactions with the target cell. Viral MFPs have been extensively studied structurally, and are divided into three classes depending on their three-dimensional fold. Because MFPs of the same class are found in otherwise unrelated viruses, their intra-class structural homology indicates horizontal gene exchange. We focus this review on the class II fusion machinery, which is composed of two glycoproteins that associate as heterodimers. They fold together in the ER of infected cells such that the MFP adopts a conformation primed to react to specific clues only upon contact with a target cell, avoiding premature fusion in the producer cell. We show that, despite having diverged in their 3D fold during evolution much more than the actual MFP, the class II accompanying proteins (AP) also derive from a distant common ancestor, displaying an invariant core formed by a ß-ribbon and a C-terminal immunoglobulin-like domain playing different functional roles-heterotypic interactions with the MFP, and homotypic AP/AP contacts to form spikes, respectively. Our analysis shows that class II APs are easily identifiable with modern structural prediction algorithms, providing useful information in devising immunogens for vaccine design.