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Myotubularin or MTM1 is a lipid phosphatase that regulates vesicular trafficking in the cell. The MTM1 gene is mutated in a severe form of muscular disease, X-linked myotubular myopathy or XLMTM, affecting 1 in 50,000 newborn males worldwide. There have been several studies on the disease pathology of XLMTM, but the structural effects of missense mutations of MTM1 are underexplored due to the unavailability of a crystal structure. MTM1 consists of three domains- a lipid-binding N-terminal GRAM domain, the phosphatase domain and a coiled-coil domain which aids dimerization of Myotubularin homologs. While most mutations reported to date map to the phosphatase domain of MTM1, the other two domains on the sequence are also frequently mutated in XLMTM. To understand the overall structural and functional effects of missense mutations on MTM1, we curated several missense mutations and performed in silico and in vitro studies. Apart from significantly impaired binding to substrate, abrogation of phosphatase activity was observed for a few mutants. Possible long-range effects of mutations from non-catalytic domains on phosphatase activity were observed as well. Coiled-coil domain mutants have been characterised here for the first time in XLMTM literature.Author SummaryX-linked myotubular myopathy is a rare paediatric disorder and affected males suffer neonatal death or may live on only with ventilatory support. In this study, we employed a range of approaches to understand the molecular level effects of patient-derived mutations on an enzyme directly linked to the congenital muscular disorder. Using three-dimensional modelling and simulations, the effect of these mutations on the structure of the enzyme and its ability to bind its substrate was studied. To complement theoretical observations, experiments were performed with cells expressing this enzyme and its mutants. These studies reveal that each part of the protein may directly or indirectly affect its enzyme activity and most of the patient-derived mutations are expressed insufficiently in the cell. With the advent of genome sequencing technology, identification of congenital mutations is easier; computational studies of molecular consequences of mutations on a protein function such as this will prove immensely useful in understanding the disease and its prognosis.

Plants are essential facilitators of human life on planet earth. Plants play a critical functional role in mediating the quality of air, availability of food and the sustainability of agricultural resources. However, plants are in constant interaction with its environment and often hampered by various types of stresses like biotic and abiotic ones. Biotic stress is a significant reason for crop-loss and causes yield loss in the range of 31–42%, post-harvest loss due to biotic stress is in the range of 6–20%, and abiotic stress causes 6–20% of the crop damage. Recognizing the molecular factors driving plant stress-related events, and developing molecular strategies to aid plants to tolerate, resist or adapt to biotic and abiotic stress are critical for sustainable agriculture practice. In this review, we discuss how recent advances in bioinformatics, plant genomics, and data science could help to improve our understanding of plant stress biology and improve the scale of global food production. We present various areas of scientific and technological advances, such as increased availability of genomics data through whole genome sequencing that require attention. We also discuss emerging techniques including CRISPR-Cas9 based genome engineering systems to develop plant varieties that can handle combinatorial stress signals. Growing trend of converging multiple omics technologies and availability of accurate, multi-scale models of plant stress through the study of orthologs and synteny studies, would improve our knowledge of how plants perceive, respond, and manage stress to thrive as resilient crop species and thus help to reduce global food crisis.

A dominant rust resistance gene, VG 9514-Rgene was isolated through map-based cloning. Sequence analysis revealed non-synonymous mutations in the TIR, NBS and LRR region of the R-protein. Candidate gene-based markers from these SNPs revealed complete co-segregation of the isolated VG 9514-Rgene with rust resistance in a RIL population and confirmed their map position in between FRS 72 and SSR_GO340445 markers in arahy03 chromosome. Blastp search of VG 9514-Rprotein detected Arahy.T6DCA5 with80.0% identity that localized at 142,544,745.0.142,549,184 in arahy03 chromosome. K

Background Cardiomyopathies are genetic disorders resulting in an abnormal heart phenotype due to intrinsic cardiac muscle malfunction, which leads to heart failure. These are chronic disorders contributing more than 36% of heart failure in the patients. Cardiomyopathies also show co-morbidity with other diseases that frequently affect the elderly. Apart from this, many drugs used for treating other unrelated diseases cause cardio-toxicity and lead to cardiomyopathies. Unravelling the cross-talk between them is essential for useful characterization, subtyping, and better treatment of the cardiomyopathies. Results In this work, we applied a systems biology-based analysis to explore the associations between cardiomyopathies and other morbidities. For this, we built a cardiomyopathy disease-disease network based on the combine biological data relevant to cardiomyopathies. For this, we make use of publicly available genetic, non-coding, drug and PPI datasets related to cardiomyopathies and network science tools to identify new modifiers genes that may have a role in cardiomyopathies. These modifier genes are further enriched using mouse phenotype data. Lastly, we highlight key modifier genes that may be potentially associated with cardiomyopathies. Many predicted genes link cardiomyopathies to neoplasms, metabolic and neurological disorders; some of these have been explained as case studies in this analysis. Conclusions Traditional one drug–one target–one disease outlook alone in drug discovery is inefficient. We hereby develop an integrative approach based on primary cardiomyopathy target genes to discover new targets that provide clues in drug re-purposing, possible off-targets and hidden genetic overlap between cardiomyopathies and other morbidities. We report potential modifier genes like NOTCH4, SIRT1 that may be investigated for role in cardiomyopathy and other diseases.

Design of proteins has far-reaching potentials in diverse areas that span repurposing of the protein scaffold for reactions and substrates that they were not naturally meant for, to catching a glimpse of the ephemeral proteins that nature might have sampled during evolution. These non-natural proteins, either in synthesized or virtual form have opened the scope for the design of entities that not only rival their natural counterparts but also offer a chance to visualize the protein space continuum that might help to relate proteins and understand their associations. Here, we review the recent advances in protein engineering and design, in multiple areas, with a view to drawing attention to their future potential.

Protein functional annotation relies on the identification of accurate relationships, sequence divergence being a key factor. This is especially evident when distant protein relationships are demonstrated only with three-dimensional structures. To address this challenge, we describe a computational approach to purposefully bridge gaps between related protein families through directed design of protein-like ``linker'' sequences. For this, we represented SCOP domain families, integrated with sequence homologues, as multiple profiles and performed HMM-HMM alignments between related domain families. Where convincing alignments were achieved, we applied a roulette wheel-based method to design 3,611,010 protein-like sequences corresponding to 374 SCOP folds. To analyze their ability to link proteins in homology searches, we used 3024 queries to search two databases, one containing only natural sequences and another one additionally containing designed sequences. Our results showed that augmented database searches showed up to 30% improvement in fold coverage for over 74% of the folds, with 52 folds achieving all theoretically possible connections. Although sequences could not be designed between some families, the availability of designed sequences between other families within the fold established the sequence continuum to demonstrate 373 difficult relationships. Ultimately, as a practical and realistic extension, we demonstrate that such protein-like sequences can be ``plugged-into'' routine and generic sequence database searches to empower not only remote homology detection but also fold recognition. Our richly statistically supported findings show that complementary searches in both databases will increase the effectiveness of sequence-based searches in recognizing all homologues sharing a common fold. (C) 2013 Elsevier Ltd. All rights reserved.

Point mutations in genes encoding sarcomeric proteins often cause primary cardiomyopathies via changes in protein-protein interactions and/or alterations in calcium sensitivity. Cardiac troponin T (TnT) interacts with troponin I and troponin C (TnI, Tnc) and binds to tropomyosin (Tm) to provide control of contractility by calcium. A helical region of TnT (residues 80-150) is known to interact with Tm. There is a direct interaction of Tm residues at position d, g, a and e of the heptad repeat with residues e, a, d and g of TnT respectively. We hypothesize that the TnT mutations seen at the above mentioned positions will severe biochemical and biophysical changes than the residues at most other positions because of their direct interaction with Tm. To test the hypothesis, we have studied multiple biochemical parameters of 5 hypertrophic (HCM) and 2 dilated (DCM) cardiomyopathy-causing mutants at representative positions of the heptad repeat. Microscale thermophoresis revealed changes in binding patterns of mutants with Tm relative to wild-type. Alterations in calcium sensitivity were observed by ATPase assays. Significant changes were observed in velocities with in vitro motility analysis. We have also used the data from these mutants to better model the interaction between TnT and Tm. Our data illustrate multiple mechanistic routes by which single-residue changes in TnT alter contractility.

The common feature of receptors and antibodies against the ligand is that both display very specific, high affinity binding towards the ligand. Therefore, it can be hypothesized that the paratope of antibodies may exhibit homology with distinct domains of the receptor. By locating the hormone epitopes and determining the structure of the paratopes, it should be possible to identify the contact points between the ligand and the receptor. This hypothesis has been tested using hCG monoclonal antibodies (MAbs) recognizing different epitopes and having different effects on hormone binding and response. The beta subunit and heterodimer specific antibodies inhibited both hormone binding and response, while the alpha subunit specific antibodies inhibited response without affecting binding. The single chain fragment variables (ScFvs) produced from these antibodies also retained the properties of the parent antibodies. The amino acid sequences of the ScFvs exhibited homology to different regions of the receptor; the beta subunit specific antibody being homologous to the concave surface of the leucine rich repeats (LRR) of the receptor, particularly the concave surface of the LRRs, while the heterodimer specific antibody showed homology to the hinge region. The alpha subunit specific antibody showed homology to the transmembrane domain of the receptor. The exact locations of the epitopes of the monoclonal antibodies in the hormone molecule have also been identified. The data presented here also support the model of glycoprotein hormone-receptor interaction in which the hormone binds to the extracellular domain through the beta subunit and then the alpha subunit is brought in contact with the transmembrane domain leading to signal transduction.

The organophosphate degrading (opd) gene cluster of plasmid pPDL2 of Flavobacterium sp. ATCC27551 contains a novel open-reading frame, orf243. This was predicted to encode an alpha/beta hydrolase distantly related to the meta-fission product (MFP) hydrolases such as XylF, PhnD, and CumD. By homology modeling Orf243 has most of the structural features of MFP hydrolases including the characteristic active site catalytic triad. The purified protein (designated MfhA) is a homotetramer and shows similar affinity for 2-hydroxy-6-oxohepta-2,4-dienoate (HOHD), 2-hydroxymuconic semialdehyde (HMSA), and 2-hydroxy-5-methylmuconic semialdehyde (HMMSA), the meta-fission products of 3-methyl catechol, catechol, and 4-methyl catechol. The unique catalytic properties of MfhA and the presence near its structural gene of cis-elements required for transposition suggest that mfhA has evolved towards encoding a common hydrolase that can act on meta-fission products containing either aldehyde or ketone groups.

Intricate molecular signalling within cellular environment is manifested through phosphorylation of proteins. Regulation of the phosphorylation state is executed through complex networking among kinases and their biochemical antagonists, the protein phosphatases. Protein dephosphorylation in eukaryotic systems is largely performed through four structurally distinct Ser/Thr and Tyr O-protein phosphatase superfamilies. 555 O-protein phosphatases, belonging to the four distinct families, could be identified using sensitive sequence search techniques across five eukaryotic model organisms (yeast, fly, worm, mouse and humans). These phosphatases could be grouped into 49 subfamilies associated with distinct domain architecture and discrete biochemical function. Only five of the architectures are shared across the five eukaryotic genomes. Interestingly, the number of occurrence of tyrosine phosphatases is correlated to the complexity of the genome. Analysis of domain architectures suggests amenability of the tyrosine phosphatases to occur in complex architectures unlike Ser/Thr phosphatases. Domain duplication and shuffling is shown as the customary mechanism for the evolution of the phosphatases. Several architectures are common between humans and other genomes, which are probably non-linearly inherited in humans or specifically lost in several others.

Complex and diverse signal transduction circuits are responsible for the efficient functioning of cellular network. Protein kinases and O-protein phosphatases are primarily responsible for propagating such stimuli within a eukaryotic cell. However, there is limited understanding of O-protein phosphatases in the prokaryotic genomes. The availability of complete genome sequence information for several prokaryotes permits a genome-wide survey of O-protein phosphatases. The distribution of the various protein phosphatase families has been observed to be mosaic, with the exception of the members of the phospho protein family P (PPP), which is consistent with previous studies. The PPP family is ubiquitous in the prokaryotic world and undergoes the highest sequence divergence within a genome amongst phosphatases studied. The co-occurrence of low molecular mass tyrosine phosphatase (LMWPc) and PPP domain in a single polypeptide suggests that the protein present in Archaeoglobus fulgidus might represent the progenitor for all protein phosphatases. The curation of data on prokaryotic protein phosphatases provides a convenient framework for the analysis of domain architectures and for characterising structural and functional properties of this important family of signalling proteins.

A detailed knowledge of a protein's functional site is an absolute prerequisite for understanding its mode of action at the molecular level. However, the rapid pace at which sequence and structural information is being accumulated for proteins greatly exceeds our ability to determine their biochemical roles experimentally. As a result, computational methods are required which allow for the efficient processing of the evolutionary information contained in this wealth of data, in particular that related to the nature and location of functionally important sites and residues. The method presented here, referred to as conserved functional group (CFG) analysis, relies on a simplified representation of the chemical groups found in amino acid side-chains to identify functional sites from a single protein structure and a number of its sequence homologues. We show that CFG analysis can fully or partially predict the location of functional sites in approximately 96% of the 470 cases tested and that, unlike other methods available, it is able to tolerate wide variations in sequence identity. In addition, we discuss its potential in a structural genomics context, where automation, scalability and efficiency are critical, and an increasing number of protein structures are determined with no prior knowledge of function. This is exemplified by our analysis of the hypothetical protein Ydde_Ecoli, whose structure was recently solved by members of the North East Structural Genomics consortium. Although the proposed active site for this protein needs to be validated experimentally, this example illustrates the scope of CFG analysis as a general tool for the identification of residues likely to play an important role in a protein's biochemical function. Thus, our method offers a convenient solution to rapidly and automatically process the vast amounts of data that are beginning to emerge from structural genomics projects.

Primary cardiomyopathy is one of the most common cardiac disorders, affecting more than 1 in 500 individuals. Primary cardiomyopathies are most frequently caused by inherited single amino acid substitutions, in a single allele encoding one of the cardiac sarcomeric proteins.Alpha-tropomyosin is a key cardiac sarcomeric protein, which interacts structurally and functionally with all other components of the sarcomeric contractile apparatus and thereby regulates cardiac muscle contraction in response to Ca2+. There are more than fifteen substitutions identified throughout the length of alpha-tropomyosin which can result in cardiomyopathy. However, the fundamental biochemical and biophysical mechanism(s) by which these single amino acid substitutions affect sarcomeric function and cause cardiomyopathy is (are) unclear. Also, there is no clear relation between the location of these substitutions in alpha-tropomyosin and the nature of the resulting cardiomyopathy. Working with a collection of less-characterised, cardiomyopathy-associated mutations in human cardiac alpha-tropomyosin, we find that even if two mutations are associated with the same cardiomyopathy, the molecular dysfunction caused by the two mutations could be different. Previously it has been characterized that most HCM mutations in alpha-tropomyosin show weaker binding to actin. However, we find that the HCM-associated alpha-tropomyosin L185R mutant binds to F-actin with a greater affinity and co-operativity. In a co-sedimentation assay to measure the binding of alpha-tropomyosin and F-actin, the Kd decreases from 200 ± 20 nM (wild-type) to 100 ± 30 nM (L185R), and the Hill's co-efficient increases. This mutation, and some others, have been characterized in further detail, within the frame-work of the three-state model of the regulated thin filament, to provide novel insights into the mechanisms underlying cardiomyopathies.

Backgound: A database of globular domains, derived from a non-redundant set of proteins, is useful for the sequence analysis of aligned domains, for structural comparisons, for understanding domain stability and flexibility and for fold recognition procedures. Domains are defined by the program DIAL and classified structurally using the procedure SEA. Results: The DIAL-derived domain database (DDBASE) consists of 436 protein chains involving 695 protein domains. Of these, 206 are α -class, 191 are β -class and 294 α and β class. The domains, 63% from multidomain proteins and 73% less than 150 residues in length, were clustered automatically using both single-link cluster analysis and hierarchical clustering to give a quantitative estimate of similarity in the domain-fold space. Conclusion: Highly populated and well described folds (doubly wound α / β , singly wound α / β barrels, globins α , large Greek-key β and flavin-binding α / β ) are recognized at a SEA cut-off score of 0.55 in single-link clustering and at 0.65 in hierarchical clustering, although functionally related families are usually clearly distinguished at more stringent values.

One hundred years ago Emil Fischer proposed a descriptive but provocative analogy for molecular recognition: the lock and key hypothesis. At a time when little was known of the molecular structures of even the relatively simple substrates of enzymes, let alone the complex structures of proteins, this gave an extraordinarily useful visual image of enzyme action. Similar recognition processes, such as antigen-antibody, hormone or growth factor-receptor, lectin-sugar, repressor-DNA and so on, have since been identified in other classes of proteins. Can the Fischer hypothesis be applied to these systems? Has the hypothesis stood the test of time? In this paper, we examine the crystal structures of proteins complexed with their ligand molecules: the pentraxins bound to carbohydrate, several aspartic proteinases complexed with inhibitors, the SH3 domains bound to proline-rich peptide motifs, the periplasmic binding proteins and growth factor systems bound to cell surface receptors. We discuss the modes of binding in terms of surface rigidity, charge and shape complementarity. Such recognition processes are often accompanied by distinct conformational changes at the binding site. The ligand selectivity demonstrated in these systems supports a “soft” lock-and-key hypothesis.

A super-secondary structural motif comprising two orthogonally oriented β-strands connected by short linking segments of ≤5 residues has been identified from a data set of 65 independent protein crystal structures. Of the 42 examples from 14 proteins, a vast majority have only a single residue as the linking element. Analysis of the conformational angles at the junction reveals that the recently described type VIII β-turn occurs frequently at the connecting hinge, while the type II β-turn is also fairly common.

Resistin is a member of a secretory protein family, known as resistin-like molecules (RELMs) which were exclusively found in mammalian genomes. Though human resistin molecule has high sequence similarity with mouse, its structural and physiological roles differ considerably from mouse and the structural basis of this drastic functional diffrences are unknown The general objectives of our work are to address these issues and to progress towards a better understanding of the structural basis of resistin biology. Our primary focus is (i) to understand the similarities and differences between human and mouse resistin with respect to their structures and (ii) to infer, using computational approaches, putative functionally important residues. For that purpose we have applied known homology modelling approaches to build a comprehensive 3D model for human resistin using mouse crystallographic data as template. We further assessed the structural properties of this 3D model using molecular dynamics techniques. We importantly compared the properties of both mouse and human resistin structures. The structural status of conserved and non-conserved residues between mouse and human resistin were further investigated with particular emphasis on those residues involved in inter-chain contacts and those exposed on the surface. By identifying the few important residues from the above analysis, we further studied and compared the dynamic properties which provide important insights into structural and functional properties of resistin. Our work suggests that there are considerable differences in interchan interactions and contact surface area between human and mouse structures. Our work also suggests that considerable differences in N-terminal helical orientation in the human model.