Lakshmipuram S. Swapna

Local admixture of amplified and diversified secreted pathogenesis determinants shapes mosaic Toxoplasma gondii genomes

Toxoplasma gondii is among the most prevalent parasites worldwide, infecting many wild and domestic animals and causing zoonotic infections in humans. T. gondii differs substantially in its broad distribution from closely related parasites that typically have narrow, specialized host ranges. To elucidate the genetic basis for these differences, we compared the genomes of 62 globally distributed T. gondii isolates to several closely related coccidian parasites. Our findings reveal that tandem amplification and diversification of secretory pathogenesis determinants is the primary feature that distinguishes the closely related genomes of these biologically diverse parasites. We further show that the unusual population structure of T. gondii is characterized by clade-specific inheritance of large conserved haploblocks that are significantly enriched in tandemly clustered secretory pathogenesis determinants. The shared inheritance of these conserved haploblocks, which show a different ancestry than the genome as a whole, may thus influence transmission, host range and pathogenicity.

Extent of Structural Asymmetry in Homodimeric Proteins: Prevalence and Relevance

Most homodimeric proteins have symmetric structure. Although symmetry is known to confer structural and functional advantage, asymmetric organization is also observed. Using a non-redundant dataset of 223 high-resolution crystal structures of biologically relevant homodimers, we address questions on the prevalence and significance of asymmetry. We used two measures to quantify global and interface asymmetry, and assess the correlation of several molecular and structural parameters with asymmetry. We have identified rare cases (11/223) of biologically relevant homodimers with pronounced global asymmetry. Asymmetry serves as a means to bring about 2∶1 binding between the homodimer and another molecule; it also enables cellular signalling arising from asymmetric macromolecular ligands such as DNA. Analysis of these cases reveals two possible mechanisms by which possible infinite array formation is prevented. In case of homodimers associating via non-topologically equivalent surfaces in their tertiary structures, ligand-dependent mechanisms are used. For stable dimers binding via large surfaces, ligand-dependent structural change regulates polymerisation/depolymerisation; for unstable dimers binding via smaller surfaces that are not evolutionarily well conserved, dimerisation occurs only in the presence of the ligand. In case of homodimers associating via interaction surfaces with parts of the surfaces topologically equivalent in the tertiary structures, steric hindrance serves as the preventive mechanism of infinite array. We also find that homodimers exhibiting grossly symmetric organization rarely exhibit either perfect local symmetry or high local asymmetry. Binding of small ligands at the interface does not cause any significant variation in interface asymmetry. However, identification of biologically relevant interface asymmetry in grossly symmetric homodimers is confounded by the presence of similar small magnitude changes caused due to artefacts of crystallisation. Our study provides new insights regarding accommodation of asymmetry in homodimers.

The role of palmitoylation for protein recruitment to the inner membrane complex of the malaria parasite

Background: Recruitment of peripheral proteins to the inner membrane complex (IMC) of the malaria parasite can be mediated by N-terminal acylation. Results: Characterization of substrate determinants and identification of an IMC-localized palmitoyl acyltransferase PfDHHC1. Conclusion: Residues close to palmitoylation sites interfere with specific IMC recruitment. PfDHHC1 represents an apicomplexan-specific PAT. Significance: Dissection of palmitoylation for protein recruitment to the inner membrane complex in P. falciparum. To survive and persist within its human host, the malaria parasite Plasmodium falciparum utilizes a battery of lineage-specific innovations to invade and multiply in human erythrocytes. With central roles in invasion and cytokinesis, the inner membrane complex, a Golgi-derived double membrane structure underlying the plasma membrane of the parasite, represents a unique and unifying structure characteristic to all organisms belonging to a large phylogenetic group called Alveolata. More than 30 structurally and phylogenetically distinct proteins are embedded in the IMC, where a portion of these proteins displays N-terminal acylation motifs. Although N-terminal myristoylation is catalyzed co-translationally within the cytoplasm of the parasite, palmitoylation takes place at membranes and is mediated by palmitoyl acyltransferases (PATs). Here, we identify a PAT (PfDHHC1) that is exclusively localized to the IMC. Systematic phylogenetic analysis of the alveolate PAT family reveals PfDHHC1 to be a member of a highly conserved, apicomplexan-specific clade of PATs. We show that during schizogony this enzyme has an identical distribution like two dual-acylated, IMC-localized proteins (PfISP1 and PfISP3). We used these proteins to probe into specific sequence requirements for IMC-specific membrane recruitment and their interaction with differentially localized PATs of the parasite.

Systems-Based Analysis of the Sarcocystis neurona Genome Identifies Pathways That Contribute to a Heteroxenous Life Cycle

ABSTRACT Sarcocystis neurona is a member of the coccidia, a clade of single-celled parasites of medical and veterinary importance including Eimeria, Sarcocystis, Neospora, and Toxoplasma. Unlike Eimeria, a single-host enteric pathogen, Sarcocystis, Neospora, and Toxoplasma are two-host parasites that infect and produce infectious tissue cysts in a wide range of intermediate hosts. As a genus, Sarcocystis is one of the most successful protozoan parasites; all vertebrates, including birds, reptiles, fish, and mammals are hosts to at least one Sarcocystis species. Here we sequenced Sarcocystis neurona, the causal agent of fatal equine protozoal myeloencephalitis. The S. neurona genome is 127 Mbp, more than twice the size of other sequenced coccidian genomes. Comparative analyses identified conservation of the invasion machinery among the coccidia. However, many dense-granule and rhoptry kinase genes, responsible for altering host effector pathways in Toxoplasma and Neospora, are absent from S. neurona. Further, S. neurona has a divergent repertoire of SRS proteins, previously implicated in tissue cyst formation in Toxoplasma. Systems-based analyses identified a series of metabolic innovations, including the ability to exploit alternative sources of energy. Finally, we present an S. neurona model detailing conserved molecular innovations that promote the transition from a purely enteric lifestyle (Eimeria) to a heteroxenous parasite capable of infecting a wide range of intermediate hosts. IMPORTANCE Sarcocystis neurona is a member of the coccidia, a clade of single-celled apicomplexan parasites responsible for major economic and health care burdens worldwide. A cousin of Plasmodium, Cryptosporidium, Theileria, and Eimeria, Sarcocystis is one of the most successful parasite genera; it is capable of infecting all vertebrates (fish, reptiles, birds, and mammals—including humans). The past decade has witnessed an increasing number of human outbreaks of clinical significance associated with acute sarcocystosis. Among Sarcocystis species, S. neurona has a wide host range and causes fatal encephalitis in horses, marine mammals, and several other mammals. To provide insights into the transition from a purely enteric parasite (e.g., Eimeria) to one that forms tissue cysts (Toxoplasma), we present the first genome sequence of S. neurona. Comparisons with other coccidian genomes highlight the molecular innovations that drive its distinct life cycle strategies. Sarcocystis neurona is a member of the coccidia, a clade of single-celled apicomplexan parasites responsible for major economic and health care burdens worldwide. A cousin of Plasmodium, Cryptosporidium, Theileria, and Eimeria, Sarcocystis is one of the most successful parasite genera; it is capable of infecting all vertebrates (fish, reptiles, birds, and mammals—including humans). The past decade has witnessed an increasing number of human outbreaks of clinical significance associated with acute sarcocystosis. Among Sarcocystis species, S. neurona has a wide host range and causes fatal encephalitis in horses, marine mammals, and several other mammals. To provide insights into the transition from a purely enteric parasite (e.g., Eimeria) to one that forms tissue cysts (Toxoplasma), we present the first genome sequence of S. neurona. Comparisons with other coccidian genomes highlight the molecular innovations that drive its distinct life cycle strategies.

Roles of residues in the interface of transient protein-protein complexes before complexation

Transient protein-protein interactions play crucial roles in all facets of cellular physiology. Here, using an analysis on known 3-D structures of transient protein-protein complexes, their corresponding uncomplexed forms and energy calculations we seek to understand the roles of protein-protein interfacial residues in the unbound forms. We show that there are conformationally near invariant and evolutionarily conserved interfacial residues which are rigid and they account for ∼65% of the core interface. Interestingly, some of these residues contribute significantly to the stabilization of the interface structure in the uncomplexed form. Such residues have strong energetic basis to perform dual roles of stabilizing the structure of the uncomplexed form as well as the complex once formed while they maintain their rigid nature throughout. This feature is evolutionarily well conserved at both the structural and sequence levels. We believe this analysis has general bearing in the prediction of interfaces and understanding molecular recognition.

The genome of Onchocerca volvulus, agent of river blindness.

Human onchocerciasis is a serious neglected tropical disease caused by the filarial nematode Onchocerca volvulus that can lead to blindness and chronic disability. Control of the disease relies largely on mass administration of a single drug, and the development of new drugs and vaccines depends on a better knowledge of parasite biology. Here, we describe the chromosomes of O. volvulus and its Wolbachia endosymbiont. We provide the highest-quality sequence assembly for any parasitic nematode to date, giving a glimpse into the evolution of filarial parasite chromosomes and proteomes. This resource was used to investigate gene families with key functions that could be potentially exploited as targets for future drugs. Using metabolic reconstruction of the nematode and its endosymbiont, we identified enzymes that are likely to be essential for O. volvulus viability. In addition, we have generated a list of proteins that could be targeted by Federal-Drug-Agency-approved but repurposed drugs, providing starting points for anti-onchocerciasis drug development.

Spastin oligomerizes into a hexamer and the mutant spastin (E442Q) redistribute the wild-type spastin into filamentous microtubule.

Spastin, a member of the ATPases associated with various cellular activities (AAA) family of proteins, is the most frequently mutated in hereditary spastic paraplegia. The defining feature of the AAA proteins is a structurally conserved AAA domain which assembles into an oligomer. By chemical cross‐linking and gel filtration chromatography, we show that spastin oligomerizes into a hexamer. Furthermore, to gain a comprehensive overview of the oligomeric structure of spastin, we generated a structural model of the AAA domain of spastin using template structure of VPS4B and p97/VCP. The generated model of spastin provided us with a framework to classify the identified missense mutations in the AAA domain from hereditary spastic paraplegia patients into different structural/functional groups. Finally, through co‐localization studies in mammalian cells, we show that E442Q mutant spastin acts in a dominant negative fashion and causes redistribution of both wild‐type spastin monomer and spastin interacting protein, RTN1 into filamentous microtubule bundles.

Protein flexibility in the light of structural alphabets

Protein structures are valuable tools to understand protein function. Nonetheless, proteins are often considered as rigid macromolecules while their structures exhibit specific flexibility, which is essential to complete their functions. Analyses of protein structures and dynamics are often performed with a simplified three-state description, i.e., the classical secondary structures. More precise and complete description of protein backbone conformation can be obtained using libraries of small protein fragments that are able to approximate every part of protein structures. These libraries, called structural alphabets (SAs), have been widely used in structure analysis field, from definition of ligand binding sites to superimposition of protein structures. SAs are also well suited to analyze the dynamics of protein structures. Here, we review innovative approaches that investigate protein flexibility based on SAs description. Coupled to various sources of experimental data (e.g., B-factor) and computational methodology (e.g., Molecular Dynamic simulation), SAs turn out to be powerful tools to analyze protein dynamics, e.g., to examine allosteric mechanisms in large set of structures in complexes, to identify order/disorder transition. SAs were also shown to be quite efficient to predict protein flexibility from amino-acid sequence. Finally, in this review, we exemplify the interest of SAs for studying flexibility with different cases of proteins implicated in pathologies and diseases.

Comparison of tertiary structures of proteins in protein-protein complexes with unbound forms suggests prevalence of allostery in signalling proteins

BackgroundMost signalling and regulatory proteins participate in transient protein-protein interactions during biological processes. They usually serve as key regulators of various cellular processes and are often stable in both protein-bound and unbound forms. Availability of high-resolution structures of their unbound and bound forms provides an opportunity to understand the molecular mechanisms involved. In this work, we have addressed the question “What is the nature, extent, location and functional significance of structural changes which are associated with formation of protein-protein complexes?”ResultsA database of 76 non-redundant sets of high resolution 3-D structures of protein-protein complexes, representing diverse functions, and corresponding unbound forms, has been used in this analysis. Structural changes associated with protein-protein complexation have been investigated using structural measures and Protein Blocks description. Our study highlights that significant structural rearrangement occurs on binding at the interface as well as at regions away from the interface to form a highly specific, stable and functional complex. Notably, predominantly unaltered interfaces interact mainly with interfaces undergoing substantial structural alterations, revealing the presence of at least one structural regulatory component in every complex.Interestingly, about one-half of the number of complexes, comprising largely of signalling proteins, show substantial localized structural change at surfaces away from the interface. Normal mode analysis and available information on functions on some of these complexes suggests that many of these changes are allosteric. This change is largely manifest in the proteins whose interfaces are altered upon binding, implicating structural change as the possible trigger of allosteric effect. Although large-scale studies of allostery induced by small-molecule effectors are available in literature, this is, to our knowledge, the first study indicating the prevalence of allostery induced by protein effectors.ConclusionsThe enrichment of allosteric sites in signalling proteins, whose mutations commonly lead to diseases such as cancer, provides support for the usage of allosteric modulators in combating these diseases.

PRODOC : a resource for the comparison of tethered protein domain architectures with in-built information on remotely related domain families

PROtein Domain Organization and Comparison (PRODOC) comprises several programs that enable convenient comparison of proteins as a sequence of domains. The in-built dataset currently consists of ∼698 000 proteins from 192 organisms with complete genomic data, and all the SWISSPROT proteins obtained from the Pfam database. All the entries in PRODOC are represented as a sequence of functional domains, assigned using hidden Markov models, instead of as a sequence of amino acids. On average 69% of the proteins in the proteomes and 49% of the residues are covered by functional domain assignments. Software tools allow the user to query the dataset with a sequence of domains and identify proteins with the same or a jumbled or circularly permuted arrangement of domains. As it is proposed that proteins with jumbled or the same domain sequences have similar functions, this search tool is useful in assigning the overall function of a multi-domain protein. Unique features of PRODOC include the generation of alignments between multi-domain proteins on the basis of the sequence of domains and in-built information on distantly related domain families forming superfamilies. It is also possible using PRODOC to identify domain sharing and gene fusion events across organisms. An exhaustive genome–genome comparison tool in PRODOC also enables the detection of successive domain sharing and domain fusion events across two organisms. The tool permits the identification of gene clusters involved in similar biological processes in two closely related organisms. The URL for PRODOC is .