Alternative splicing and protein structure evolution

Alternative splicing is thought to be one of the major sources for functional diversity in higher eukaryotes. Interestingly, when mapping splicing events onto protein structures, about half of the events affect structured and even highly conserved regions i.e. are non-trivial on the structure level. This has led to the controversial hypothesis that such splice variants result in nonsense-mediated mRNA decay or non-functional, unstructured proteins, which do not contribute to the functional diversity of an organism. Here we show in a comprehensive study on alternative splicing that proteins appear to be much more tolerant to structural deletions, insertions and replacements than previously thought. We find literature evidence that such non-trivial splicing isoforms exhibit different functional properties compared to their native counterparts and allow for interesting regulatory patterns on the protein network level. We provide examples that splicing events may represent transitions between different folds in the protein sequence–structure space and explain these links by a common genetic mechanism. Taken together, those findings hint to a more prominent role of splicing in protein structure evolution and to a different view of phenotypic plasticity of protein structures.     

Using de novo assembly to identify structural variation of eight complex immune system gene regions

Driven by the necessity to survive environmental pathogens, the human immune system has evolved exceptional diversity and plasticity, to which several factors contribute including inheritable structural polymorphism of the underlying genes. Characterizing this variation is challenging due to the complexity of these loci, which contain extensive regions of paralogy, segmental duplication and high copy-number repeats, but recent progress in long-read sequencing and optical mapping techniques suggests this problem may now be tractable. Here we assess this by using long-read sequencing platforms from PacBio and Oxford Nanopore, supplemented with short-read sequencing and Bionano optical mapping, to sequence DNA extracted from CD14⁺ monocytes and peripheral blood mononuclear cells from a single European individual identified as HV31. We use this data to build a de novo assembly of eight genomic regions encoding four key components of the immune system, namely the human leukocyte antigen, immunoglobulins, T cell receptors, and killer-cell immunoglobulin-like receptors. Validation of our assembly using k-mer based and alignment approaches suggests that it has high accuracy, with estimated base-level error rates below 1 in 10 kb, although we identify a small number of remaining structural errors. We use the assembly to identify heterozygous and homozygous structural variation in comparison to GRCh38. Despite analyzing only a single individual, we find multiple large structural variants affecting core genes at all three immunoglobulin regions and at two of the three T cell receptor regions. Several of these variants are not accurately callable using current algorithms, implying that further methodological improvements are needed. Our results demonstrate that assessing haplotype variation in these regions is possible given sufficiently accurate long-read and associated data. Continued reductions in the cost of these technologies will enable application of these methods to larger samples and provide a broader catalogue of germline structural variation at these loci, an important step toward making these regions accessible to large-scale genetic association studies.     

Verification of alternative splicing variants based on domain integrity, truncation length and intrinsic protein disorder

According to current estimations ～95% of multi-exonic human protein-coding genes undergo alternative splicing (AS). However, for 4000 human proteins in PDB, only 14 human proteins have structures of at least two alternative isoforms. Surveying these structural isoforms revealed that the maximum insertion accommodated by an isoform of a fully ordered protein domain was 5 amino acids, other instances of domain changes involved intrinsic structural disorder. After collecting 505 minor isoforms of human proteins with evidence for their existence we analyzed their length, protein disorder and exposed hydrophobic surface. We found that strict rules govern the selection of alternative splice variants aimed to preserve the integrity of globular domains: alternative splice sites (i) tend to avoid globular domains or (ii) affect them only marginally or (iii) tend to coincide with a location where the exposed hydrophobic surface is minimal or (iv) the protein is disordered. We also observed an inverse correlation between the domain fraction lost and the full length of the minor isoform containing the domain, possibly indicating a buffering effect for the isoform protein counteracting the domain truncation effect. These observations provide the basis for a prediction method (currently under development) to predict the viability of splice variants.     

Genome-wide analyses of human noroviruses provide insights on evolutionary dynamics and evidence of coexisting viral populations evolving under recombination constraints

Norovirus is a major cause of acute gastroenteritis worldwide. Over 30 different genotypes, mostly from genogroup I (GI) and II (GII), have been shown to infect humans. Despite three decades of genome sequencing, our understanding of the role of genomic diversification across continents and time is incomplete. To close the spatiotemporal gap of genomic information of human noroviruses, we conducted a large-scale genome-wide analyses that included the nearly full-length sequencing of 281 archival viruses circulating since the 1970s in over 10 countries from four continents, with a major emphasis on norovirus genotypes that are currently underrepresented in public genome databases. We provided new genome information for 24 distinct genotypes, including the oldest genome information from 12 norovirus genotypes. Analyses of this new genomic information, together with those publicly available, showed that (i) noroviruses evolve at similar rates across genomic regions and genotypes; (ii) emerging viruses evolved from transiently-circulating intermediate viruses; (iii) diversifying selection on the VP1 protein was recorded in genotypes with multiple variants; (iv) non-structural proteins showed a similar branching on their phylogenetic trees; and (v) contrary to the current understanding, there are restrictions on the ability to recombine different genomic regions, which results in co-circulating populations of viruses evolving independently in human communities. This study provides a comprehensive genetic analysis of diverse norovirus genotypes and the role of non-structural proteins on viral diversification, shedding new light on the mechanisms of norovirus evolution and transmission.     

VarSite: Disease variants and protein structure

VarSite is a web server mapping known disease‐associated variants from UniProt and ClinVar, together with natural variants from gnomAD, onto protein 3D structures in the Protein Data Bank. The analyses are primarily image‐based and provide both an overview for each human protein, as well as a report for any specific variant of interest. The information can be useful in assessing whether a given variant might be pathogenic or benign. The structural annotations for each position in the protein include protein secondary structure, interactions with ligand, metal, DNA/RNA, or other protein, and various measures of a given variant's possible impact on the protein's function. The 3D locations of the disease‐associated variants can be viewed interactively via the 3dmol.js JavaScript viewer, as well as in RasMol and PyMOL. Users can search for specific variants, or sets of variants, by providing the DNA coordinates of the base change(s) of interest. Additionally, various agglomerative analyses are given, such as the mapping of disease and natural variants onto specific Pfam or CATH domains. The server is freely accessible to all at: https://www.ebi.ac.uk/thornton-srv/databases/VarSite .     

Characterizing complex structural variation in germline and somatic genomes

Genome structural variation (SV) is a major source of genetic diversity in mammals and a hallmark of cancer. Although SV is typically defined by its canonical forms (duplication, deletion, insertion, inversion and translocation), recent breakpoint mapping studies have revealed a surprising number of 'complex' variants that evade simple classification. Complex SVs are defined by clustered breakpoints that arose through a single mutation but cannot be explained by one simple end-joining or recombination event. Some complex variants exhibit profoundly complicated rearrangements between distinct loci from multiple chromosomes, whereas others involve more subtle alterations at a single locus. These diverse and unpredictable features present a challenge for SV mapping experiments. Here, we review current knowledge of complex SV in mammals, and outline techniques for identifying and characterizing complex variants using next-generation DNA sequencing.     

Prediction of the structure of proteins using related structures,energy minimization and computer graphics

Insight into the functions and interactions of proteins may be gained by correlating a variety of types of experimental data (including kinetics, spectroscopy, biophysical measurements, among others) with three-dimensional structural models displayed and manipulated using interactive computer graphics. Although tertiary structures have been determined for a large number of proteins, one limiting factor in structure-function studies is the lack of availability of the structural coordinates of specific proteins for which other types of detailed experimental data are known. However, as the data base of known structures grows, it becomes more and more likely that the structure of a closely related protein will be available. Here we present a method for predicting structures by ( 1 ) careful alteration of a known structure of a homologous, functionally analogous protein followed by (2) energy minimization to optimize the predicted structure. This method provides a rapid and effective solution to the initial problem of obtaining a working structure for modeling studies.     

Structure-based functional inference in structural genomics

The dramatically increasing number of new protein sequences arising from genomics 4 proteomics requires the need for methods to rapidly and reliably infer the molecular and cellular functions of these proteins. One such approach, structural genomics, aims to delineate the total repertoire of protein folds in nature, thereby providing three-dimensional folding patterns for all proteins and to infer molecular functions of the proteins based on the combined information of structures and sequences. The goal of obtaining protein structures on a genomic scale has motivated the development of high throughput technologies and protocols for macromolecular structure determination that have begun to produce structures at a greater rate than previously possible. These new structures have revealed many unexpected functional inferences and evolutionary relationships that were hidden at the sequence level. Here, we present samples of structures determined at Berkeley Structural Genomics Center and collaborators laboratories to illustrate how structural information provides and complements sequence information to deduce the functional inferences of proteins with unknown molecular functions.Two of the major premises of structural genomics are to discover a complete repertoire of protein folds in nature and to find molecular functions of the proteins whose functions are not predicted from sequence comparison alone. To achieve these objectives on a genomic scale, new methods, protocols, and technologies need to be developed by multi-institutional collaborations worldwide. As part of this effort, the Protein Structure Initiative has been launched in the United States (PSI; www.nigms.nih.gov/funding/psi.html). Although infrastructure building and technology development are still the main focus of structural genomics programs [1–6], a considerable number of protein structures have already been produced, some of them coming directly out of semi-automated structure determination pipelines [6–10]. The Berkeley Structural Genomics Center (BSGC) has focused on the proteins of Mycoplasma or their homologues from other organisms as its structural genomics targets because of the minimal genome size of the Mycoplasmas as well as their relevance to human and animal pathogenicity (http://www.strgen.org). Here we present several protein examples encompassing a spectrum of functional inferences obtainable from their three-dimensional structures in five situations, where the inferences are new and testable, and are not predictable from protein sequence information alone.     

Can Predicted Protein 3D Structures Provide Reliable Insights into whether Missense Variants Are Disease Associated?

Knowledge of protein structure can be used to predict the phenotypic consequence of a missense variant. Since structural coverage of the human proteome can be roughly tripled to over 50% of the residues if homology-predicted structures are included in addition to experimentally determined coordinates, it is important to assess the reliability of using predicted models when analyzing missense variants. Accordingly, we assess whether a missense variant is structurally damaging by using experimental and predicted structures. We considered 606 experimental structures and show that 40% of the 1965 disease-associated missense variants analyzed have a structurally damaging change in the mutant structure. Only 11% of the 2134 neutral variants are structurally damaging. Importantly, similar results are obtained when 1052 structures predicted using Phyre2 algorithm were used, even when the model shares low (< 40%) sequence identity to the template. Thus, structure-based analysis of the effects of missense variants can be effectively applied to homology models. Our in-house pipeline, Missense3D, for structurally assessing missense variants was made available at http://www.sbg.bio.ic.ac.uk/~missense3d     

Next Generation Protein Structure Predictions and Genetic Variant Interpretation

The need to make sense of the thousands of genetic variants uncovered every day in terms of pathology or biological mechanism is acute. Many insights into how genetic changes impact protein function can be gleaned if three-dimensional structures of the associated proteins are available. The availability of a highly accurate method of predicting structures from amino acid sequences (e.g. Alphafold2) is thus potentially a great boost to those wanting to understand genetic changes. In this paper we discuss the current state of protein structures known for the human and other proteomes and how Alphafold2 might impact on variant interpretation efforts. For the human proteome in particular, the state of the available structural data suggests that the impact on variant interpretation might be less than anticipated. We also discuss additional efforts in structure prediction that could further aid the understanding of genetic variants.     

Mapping of antigenic epitopes on the alpha 1 subunit of the inhibitory glycine receptor.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel protein that occurs in developmentally regulated isoforms in the vertebrate central nervous system. Monoclonal antibodies (mAbs) against the GlyR distinguish neonatal and adult GlyR proteins by identifying distinct alpha subunit variants within these receptor isoforms. Here, bacterially expressed fusion proteins of the rat GlyR alpha 1 subunit were used to localize the major antigenic epitopes of this protein within its N-terminal 105 amino acids. Synthetic peptides allowed further fine mapping of two mAb binding domains. MAb 2b, specific for the adult alpha 1 subunit, bound to a peptide corresponding to amino acids 1-10, whereas mAb 4a, which recognizes both neonatal and adult GlyR isoforms, reacted with a peptide representing residues 96-105 of the alpha 1 polypeptide. These data define unique and common antigenic epitopes on GlyR alpha subunit variants.     

Proteomic tools for the investigation of human hair structural proteins and evidence of weakness sites on hair keratin coil segments

Human hair is principally composed of hair keratins and keratin-associated proteins (KAPs) that form a complex network giving the hair its rigidity and mechanical properties. However, during their growth, hairs are subject to various treatments that can induce irreversible damage. For a better understanding of the human hair protein structures, proteomic mass spectrometry (MS)-based strategies could assist in characterizing numerous isoforms and posttranslational modifications of human hair fiber proteins. However, due to their physicochemical properties, characterization of human hair proteins using classical proteomic approaches is still a challenge. To address this issue, we have used two complementary approaches to analyze proteins from the human hair cortex. The multidimensional protein identification technology (MudPit) approach allowed identifying all keratins and the major KAPs present in the hair as well as posttranslational modifications in keratins such as cysteine trioxidation, lysine, and histidine methylation. Then two-dimensional gel electrophoresis coupled with MS (2-DE gel MS) allowed us to obtain the most complete 2-DE gel pattern of human hair proteins, revealing an unexpected heterogeneity of keratin structures. Analyses of these structures by differential peptide mapping have brought evidence of cleaved species in hair keratins and suggest a preferential breaking zone in α-helical segments.     

The actinome of Dictyostelium discoideum in comparison to actins and actin-related proteins from other organisms

Actin belongs to the most abundant proteins in eukaryotic cells which harbor usually many conventional actin isoforms as well as actin-related proteins (Arps). To get an overview over the sometimes confusing multitude of actins and Arps, we analyzed the Dictyostelium discoideum actinome in detail and compared it with the genomes from other model organisms. The D. discoideum actinome comprises 41 actins and actin-related proteins. The genome contains 17 actin genes which most likely arose from consecutive gene duplications, are all active, in some cases developmentally regulated and coding for identical proteins (Act8-group). According to published data, the actin fraction in a D. discoideum cell consists of more than 95% of these Act8-type proteins. The other 16 actin isoforms contain a conventional actin motif profile as well but differ in their protein sequences. Seven actin genes are potential pseudogenes. A homology search of the human genome using the most typical D. discoideum actin (Act8) as query sequence finds the major actin isoforms such as cytoplasmic beta-actin as best hit. This suggests that the Act8-group represents a nearly perfect actin throughout evolution. Interestingly, limited data from D. fasciculatum, a more ancient member among the social amoebae, show different relationships between conventional actins. The Act8-type isoform is most conserved throughout evolution. Modeling of the putative structures suggests that the majority of the actin-related proteins is functionally unrelated to canonical actin. The data suggest that the other actin variants are not necessary for the cytoskeleton itself but rather regulators of its dynamical features or subunits in larger protein complexes.     

The murine GABA(B) receptor 1: cDNA cloning, tissue distribution, structure of the Gabbr1 gene, and mapping to chromosome 17

GABA (gamma-aminobutyric acid) is a major inhibitory neurotransmitter in the central nervous system (CNS) which activates both ionotropic (GABA(A)/GABA(C)) and metabotropic (GABA(B)) receptor systems. We identified two alternatively spliced cDNA variants of the murine GABA(B) receptor 1 that are predominantly expressed in the CNS. Deduced protein structures are highly homologous to the previously characterized rat and human receptors. Comparison of the genomic structures of mouse and human revealed that alternative splicing occurred at the same position, whereas the mouse gene has an additional 5' exon. Radiation hybrid mapping, combined with database searches, indicated that the GABA(B) receptor gene (Gabbr1) is located on mouse chromosome 17, adjacent to the marker D17Mit24 in a region homologous to human chromosome 6p21.3.     

Beyond history and “on a roll”: The list of the most well‐studied human protein structures and overall trends in the protein data bank

Of the roughly 20,000 canonical human protein sequences, as of January 20, 2021, 7,077 proteins have had their full or partial, medium‐ to high‐resolution structures determined by x‐ray crystallography or other methods. Which of these proteins dominate the protein data bank (the PDB) and why? In this paper, we list the 273 top human protein structures based on the number of their PDB entries. This set of proteins accounts for more than 40% of all available human PDB entries and represent past trends as well as current status for protein structural biology. We briefly discuss the relationship which some of the prominent protein structures have with protein research as a whole and mention their relevance to human diseases. The top‐10 soluble and membrane proteins are all well‐known (most of their first structures being deposited more than 30 years ago). Overall, there is no dramatic change in recent trends in the PDB. Remarkably, the number of structure depositions has grown nearly exponentially over the last 10 or more years (with a doubling time of 7 years for proteins, obtained from any organism). Growth in human protein structures is slightly faster (at 5.9 years). The information in this paper may be informative to senior scientists but also inspire researchers who are new to protein science, providing the year 2021 snap‐shot for the state of protein structural biology.     

GWYRE: A Resource for Mapping Variants onto Experimental and Modeled Structures of Human Protein Complexes

Rapid progress in structural modeling of proteins and their interactions is powered by advances in knowledge-based methodologies along with better understanding of physical principles of protein structure and function. The pool of structural data for modeling of proteins and protein–protein complexes is constantly increasing due to the rapid growth of protein interaction databases and Protein Data Bank. The GWYRE (Genome Wide PhYRE) project capitalizes on these developments by advancing and applying new powerful modeling methodologies to structural modeling of protein–protein interactions and genetic variation. The methods integrate knowledge-based tertiary structure prediction using Phyre2 and quaternary structure prediction using template-based docking by a full-structure alignment protocol to generate models for binary complexes. The predictions are incorporated in a comprehensive public resource for structural characterization of the human interactome and the location of human genetic variants. The GWYRE resource facilitates better understanding of principles of protein interaction and structure/function relationships. The resource is available at http://www.gwyre.org.     

Recent developments in computational proteomics.

The mapping of the human genome was completed earlier this year and efforts are underway to understand the role of gene products (i.e. proteins) in biological pathways and human disease and to exploit their functional roles to derive protein therapeutics and protein-based drugs. A key component to the next revolution in the 'post-genomic' era will be the increasingly widespread use of protein structure in rational experimental design. Improvements in quality, availability and utility of large-scale 3D and 4D protein structural information are enabling a revolution in rational design, having particular impact on drug discovery and optimization. New computational methodologies now yield modeled structures that are, in many cases, quantitatively comparable with crystal structures, at a fraction of the cost.