The evolutionary history of topological variations in the CPA/AT transporters.

CPA/AT transporters are made up of scaffold and a core domain. The core domain contains two non-canonical helices (broken or reentrant) that mediate the transport of ions, amino acids or other charged compounds. During evolution, these transporters have undergone substantial changes in structure, topology and function. To shed light on these structural transitions, we create models for all families using an integrated topology annotation method. We find that the CPA/AT transporters can be classified into four fold-types based on their structure; (1) the CPA-broken fold-type, (2) the CPA-reentrant fold-type, (3) the BART fold-type, and (4) a previously not described fold-type, the Reentrant-Helix-Reentrant fold-type. Several topological transitions are identified, including the transition between a broken and reentrant helix, one transition between a loop and a reentrant helix, complete changes of orientation, and changes in the number of scaffold helices. These transitions are mainly caused by gene duplication and shuffling events. Structural models, topology information and other details are presented in a searchable database, CPAfold (cpafold.bioinfo.se).

DisProt: intrinsic protein disorder annotation in 2020.

The Database of Protein Disorder (DisProt, URL: https://disprot.org) provides manually curated annotations of intrinsically disordered proteins from the literature. Here we report recent developments with DisProt (version 8), including the doubling of protein entries, a new disorder ontology, improvements of the annotation format and a completely new website. The website includes a redesigned graphical interface, a better search engine, a clearer API for programmatic access and a new annotation interface that integrates text mining technologies. The new entry format provides a greater flexibility, simplifies maintenance and allows the capture of more information from the literature. The new disorder ontology has been formalized and made interoperable by adopting the OWL format, as well as its structure and term definitions have been improved. The new annotation interface has made the curation process faster and more effective. We recently showed that new DisProt annotations can be effectively used to train and validate disorder predictors. We believe the growth of DisProt will accelerate, contributing to the improvement of function and disorder predictors and therefore to illuminate the 'dark' proteome.

Comparative analyses of quaternary arrangements in homo-oligomeric proteins in superfamilies: Functional implications.

A comprehensive analysis of the quaternary features of distantly related homo-oligomeric proteins is the focus of the current study. This study has been performed at the levels of quaternary state, symmetry, and quaternary structure. Quaternary state and quaternary structure refers to the number of subunits and spatial arrangements of subunits, respectively. Using a large dataset of available 3D structures of biologically relevant assemblies, we show that only 53% of the distantly related homo-oligomeric proteins have the same quaternary state. Considering these homologous homo-oligomers with the same quaternary state, conservation of quaternary structures is observed only in 38% of the pairs. In 36% of the pairs of distantly related homo-oligomers with different quaternary states the larger assembly in a pair shows high structural similarity with the entire quaternary structure of the related protein with lower quaternary state and it is referred as "Russian doll effect." The differences in quaternary state and structure have been suggested to contribute to the functional diversity. Detailed investigations show that even though the gross functions of many distantly related homo-oligomers are the same, finer level differences in molecular functions are manifested by differences in quaternary states and structures. Comparison of structures of biological assemblies in distantly and closely related homo-oligomeric proteins throughout the study differentiates the effects of sequence divergence on the quaternary structures and function. Knowledge inferred from this study can provide insights for improved protein structure classification and function prediction of homo-oligomers. Proteins 2016; 84:1190-1202. © 2016 Wiley Periodicals, Inc.

Evolutionary and structural analyses of heterodimeric proteins composed of subunits with same fold.

Heterodimeric proteins with homologous subunits of same fold are involved in various biological processes. The objective of this study is to understand the evolution of structural and functional features of such heterodimers. Using a non-redundant dataset of 70 such heterodimers of known 3D structure and an independent dataset of 173 heterodimers from yeast, we note that the mean sequence identity between interacting homologous subunits is only 23-24% suggesting that, generally, highly diverged paralogues assemble to form such a heterodimer. We also note that the functional roles of interacting subunits/domains are generally quite different. This suggests that, though the interacting subunits/domains are homologous, the high evolutionary divergence characterize their high functional divergence which contributes to a gross function for the heterodimer considered as a whole. The inverse relationship between sequence identity and RMSD of interacting homologues in heterodimers is not followed. We also addressed the question of formation of homodimers of the subunits of heterodimers by generating models of fictitious homodimers on the basis of the 3D structures of the heterodimers. Interaction energies associated with these homodimers suggests that, in overwhelming majority of the cases, such homodimers are unlikely to be stable. Majority of the homologues of heterodimers of known structures form heterodimers (51.8%) and a small proportion (14.6%) form homodimers. Comparison of 3D structures of heterodimers with homologous homodimers suggests that interfacial nature of residues is not well conserved. In over 90% of the cases we note that the interacting subunits of heterodimers are co-localized in the cell.

Structural and molecular basis of interaction of HCV non-structural protein 5A with human casein kinase 1α and PKR.

BACKGROUND: Interaction of non-structural protein 5A (NS5A) of Hepatitis C virus (HCV) with human kinases namely, casein kinase 1α (ck1α) and protein kinase R (PKR) have different functional implications such as regulation of viral replication and evasion of interferon induced immune response respectively. Understanding the structural and molecular basis of interactions of the viral protein with two different human kinases can be useful in developing strategies for treatment against HCV. RESULTS: Serine 232 of NS5A is known to be phosphorylated by human ck1α. A structural model of NS5A peptide containing phosphoacceptor residue Serine 232 bound to ck1α has been generated using the known 3-D structures of kinase-peptide complexes. The substrate interacting residues in ck1α has been identified from the model and these are found to be conserved well in the ck1 family. ck1α - substrate peptide complex has also been used to understand the structural basis of association between ck1α and its other viral stress induced substrate, tumour suppressor p53 transactivation domain which has a crystal structure available.Interaction of NS5A with another human kinase PKR is primarily genotype specific. NS5A from genotype 1b has been shown to interact and inhibit PKR whereas NS5A from genotype 2a/3a are unable to bind and inhibit PKR efficiently. This is one of the main reasons for the varied response to interferon therapy in HCV patients across different genotypes. Using PKR crystal structure, sequence alignment and evolutionary trace analysis some of the critical residues responsible for the interaction of NS5A 1b with PKR have been identified. CONCLUSIONS: The substrate interacting residues in ck1α have been identified using the structural model of kinase - substrate peptide. The PKR interacting NS5A 1b residues have also been predicted using PKR crystal structure, NS5A sequence analysis along with known experimental results. Functional significance and nature of interaction of interferon sensitivity determining region and variable region 3 of NS5A in different genotypes with PKR which was experimentally shown are also supported by the findings of evolutionary trace analysis. Designing inhibitors to prevent this interaction could enable the HCV genotype 1 infected patients respond well to interferon therapy.

A natural small molecule inhibitor corilagin blocks HCV replication and modulates oxidative stress to reduce liver damage.

Hepatitis C virus (HCV) infection causes chronic liver disease, which often leads to hepatocellular carcinoma. Earlier, we have demonstrated anti-HCV property of the methanolic extract of Phyllanthus amarus, an age-old folk-medicine against viral hepatitis. Here, we report identification of a principal bioactive component 'corilagin', which showed significant inhibition of the HCV key enzymes, NS3 protease and NS5B RNA-dependent-RNA-polymerase. This pure compound could effectively inhibit viral replication in the infectious cell culture system, displayed strong antioxidant activity by blocking HCV induced generation of reactive oxygen species and suppressed up-regulation of NOX4 and TGF-ß mRNA levels. Oral administration of corilagin in BALB/c mice demonstrated its better tolerability and systemic bioavailability. More importantly, corilagin could restrict serum HCV RNA levels, decrease collagen deposition and hepatic cell denaturation in HCV infected chimeric mice harbouring human hepatocytes. Taken together, results provide a basis towards developing a pure natural drug as an alternate therapeutic strategy for restricting viral replication and prevent liver damage towards better management of HCV induced pathogenesis.

Weak conservation of structural features in the interfaces of homologous transient protein-protein complexes.

Residue types at the interface of protein-protein complexes (PPCs) are known to be reasonably well conserved. However, we show, using a dataset of known 3-D structures of homologous transient PPCs, that the 3-D location of interfacial residues and their interaction patterns are only moderately and poorly conserved, respectively. Another surprising observation is that a residue at the interface that is conserved is not necessarily in the interface in the homolog. Such differences in homologous complexes are manifested by substitution of the residues that are spatially proximal to the conserved residue and structural differences at the interfaces as well as differences in spatial orientations of the interacting proteins. Conservation of interface location and the interaction pattern at the core of the interfaces is higher than at the periphery of the interface patch. Extents of variability of various structural features reported here for homologous transient PPCs are higher than the variation in homologous permanent homomers. Our findings suggest that straightforward extrapolation of interfacial nature and inter-residue interaction patterns from template to target could lead to serious errors in the modeled complex structure. Understanding the evolution of interfaces provides insights to improve comparative modeling of PPC structures.

An overview of recent advances in structural bioinformatics of protein-protein interactions and a guide to their principles.

Rich data bearing on the structural and evolutionary principles of protein-protein interactions are paving the way to a better understanding of the regulation of function in the cell. This is particularly the case when these interactions are considered in the framework of key pathways. Knowledge of the interactions may provide insights into the mechanisms of crucial 'driver' mutations in oncogenesis. They also provide the foundation toward the design of protein-protein interfaces and inhibitors that can abrogate their formation or enhance them. The main features to learn from known 3-D structures of protein-protein complexes and the extensive literature which analyzes them computationally and experimentally include the interaction details which permit undertaking structure-based drug discovery, the evolution of complexes and their interactions, the consequences of alterations such as post-translational modifications, ligand binding, disease causing mutations, host pathogen interactions, oligomerization, aggregation and the roles of disorder, dynamics, allostery and more to the protein and the cell. This review highlights some of the recent advances in these areas, including design, inhibition and prediction of protein-protein complexes. The field is broad, and much work has been carried out in these areas, making it challenging to cover it in its entirety. Much of this is due to the fast increase in the number of molecules whose structures have been determined experimentally and the vast increase in computational power. Here we provide a concise overview.

Small molecule inhibitors of HCV replication from pomegranate.

Hepatitis C virus (HCV) is the causative agent of end-stage liver disease. Recent advances in the last decade in anti HCV treatment strategies have dramatically increased the viral clearance rate. However, several limitations are still associated, which warrant a great need of novel, safe and selective drugs against HCV infection. Towards this objective, we explored highly potent and selective small molecule inhibitors, the ellagitannins, from the crude extract of Pomegranate (Punica granatum) fruit peel. The pure compounds, punicalagin, punicalin, and ellagic acid isolated from the extract specifically blocked the HCV NS3/4A protease activity in vitro. Structural analysis using computational approach also showed that ligand molecules interact with the catalytic and substrate binding residues of NS3/4A protease, leading to inhibition of the enzyme activity. Further, punicalagin and punicalin significantly reduced the HCV replication in cell culture system. More importantly, these compounds are well tolerated ex vivo and'no observed adverse effect level' (NOAEL) was established upto an acute dose of 5000 mg/kg in BALB/c mice. Additionally, pharmacokinetics study showed that the compounds are bioavailable. Taken together, our study provides a proof-of-concept approach for the potential use of antiviral and non-toxic principle ellagitannins from pomegranate in prevention and control of HCV induced complications.

Targeted modifications in adeno-associated virus serotype 8 capsid improves its hepatic gene transfer efficiency in vivo.

Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T→Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S→A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels (~9- to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector biodistribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8 mutant in therapeutic gene transfer, we delivered human coagulation factor IX (h.FIX) under the control of liver-specific promoters (LP1 or hAAT) into C57BL/6 mice. The circulating levels of h.FIX:Ag were higher in all the K137R-AAV8 treated groups up to 8 weeks post-hepatic gene transfer. These studies demonstrate the feasibility of the use of this novel AAV8 vectors for potential gene therapy of hemophilia B.

Bioengineering of AAV2 capsid at specific serine, threonine, or lysine residues improves its transduction efficiency in vitro and in vivo.

We hypothesized that the AAV2 vector is targeted for destruction in the cytoplasm by the host cellular kinase/ubiquitination/proteasomal machinery and that modification of their targets on AAV2 capsid may improve its transduction efficiency. In vitro analysis with pharmacological inhibitors of cellular serine/threonine kinases (protein kinase A, protein kinase C, casein kinase II) showed an increase (20-90%) on AAV2-mediated gene expression. The three-dimensional structure of AAV2 capsid was then analyzed to predict the sites of ubiquitination and phosphorylation. Three phosphodegrons, which are the phosphorylation sites recognized as degradation signals by ubiquitin ligases, were identified. Mutation targets comprising eight serine (S) or seven threonine (T) or nine lysine (K) residues were selected in and around phosphodegrons on the basis of their solvent accessibility, overlap with the receptor binding regions, overlap with interaction interfaces of capsid proteins, and their evolutionary conservation across AAV serotypes. AAV2-EGFP vectors with the wild-type (WT) capsid or mutant capsids (15 S/T→alanine [A] or 9 K→arginine [R] single mutant or 2 double K→R mutants) were then evaluated in vitro. The transduction efficiencies of 11 S/T→A and 7 K→R vectors were significantly higher (~63-90%) than the AAV2-WT vectors (~30-40%). Further, hepatic gene transfer of these mutant vectors in vivo resulted in higher vector copy numbers (up to 4.9-fold) and transgene expression (up to 14-fold) than observed from the AAV2-WT vector. One of the mutant vectors, S489A, generated ~8-fold fewer antibodies that could be cross-neutralized by AAV2-WT. This study thus demonstrates the feasibility of the use of these novel AAV2 capsid mutant vectors in hepatic gene therapy.