1) Natural history and classification of the beta-grasp fold domains.? The beta-grasp fold (beta-GF), prototyped by ubiquitin (UB), has been recruited for a strikingly diverse range of biochemical functions. These functions include providing a scaffold for different enzymatic active sites (e.g. NUDIX phosphohydrolases) and iron-sulfur clusters, RNA-soluble-ligand and co-factor-binding, sulfur transfer, adaptor functions in signaling, assembly of macromolecular complexes and post-translational protein modification. To understand the basis for the functional versatility of this small fold we undertook a comprehensive sequence-structure analysis of the fold and developed a natural classification for its members. As a result we were able to define the core distinguishing features of the fold and numerous elaborations, including several previously unrecognized variants. Systematic analysis of all known interactions of the fold showed that its manifold functional abilities arise primarily from the prominent beta-sheet, which provides an exposed surface for diverse interactions or additionally, by forming open barrel-like structures. We show that in the beta-GF both enzymatic activities and the binding of diverse co-factors (e.g. molybdopterin) have independently evolved on at least three occasions each, and iron-sulfur-cluster-binding on at least two independent occasions. Our analysis identified multiple previously unknown large monophyletic assemblages within the beta-GF, including one which unifies versions found in the fasciclin-1 superfamily, the ribosomal protein L25, the phosphoribosyl AMP cyclohydrolase (HisI) and glutamine synthetase. We also uncovered several new groups of beta-GF domains including a domain found in bacterial flagellar and fimbrial assembly components, and 5 new UB-like domains in the eukaryotes. Evolutionary reconstruction indicates that the beta-GF had differentiated into at least 7 distinct lineages by the time of the last universal common ancestor of all extant organisms, encompassing much of the structural diversity observed in extant versions of the fold. The earliest beta-GF members were probably involved in RNA metabolism and subsequently radiated into various functional niches. Most of the structural diversification occurred in the prokaryotes, whereas the eukaryotic phase was mainly marked by a specific expansion of the ubiquitin-like beta-GF members. The eukaryotic UB superfamily diversified into at least 67 distinct families, of which at least 19-20 families were already present in the eukaryotic common ancestor, including several protein and one lipid conjugated forms. Another key aspect of the eukaryotic phase of evolution of the beta-GF was the dramatic increase in domain architectural complexity of proteins related to the expansion of UB-like domains in numerous adaptor roles.? ? 2) The natural history of the WRKY-GCM1 zinc fingers and the relationship between transcription factors and transposons.? WRKY and GCM1 are metal chelating DNA-binding domains (DBD) which share a four stranded fold. Using sensitive sequence searches, we show that this WRKY-GCM1 fold is also shared by the FLYWCH Zn-finger domain and the DBDs of two classes of Mutator-like element (MULE) transposases. We present evidence that they share a stabilizing core, which suggests a possible origin from a BED finger-like intermediate that was in turn ultimately derived from a C2H2 Zn-finger domain. Through a systematic study of the phyletic pattern, we show that this WRKY-GCM1 superfamily is a widespread eukaryote-specific group of transcription factors (TFs). We identified several new members across diverse eukaryotic lineages, including potential TFs in animals, fungi and Entamoeba. By integrating sequence, structure, gene expression and transcriptional network data, we present evidence that at least two major global regulators belonging to this superfamily in Saccharomyces cerevisiae (Rcs1p and Aft2p) have evolved from transposons, and attained the status of transcription regulatory hubs in recent course of ascomycete yeast evolution. In plants, we show that the lineage-specific expansion of WRKY-GCM1 domain proteins acquired functional diversity mainly through expression divergence rather than by protein sequence divergence. We also use the WRKY-GCM1 superfamily as an example to illustrate the importance of transposons in the emergence of new TFs in different lineages.? ? 3) Apicomplexan transcription factors.? Apicomplexa have developed distinctive adaptations for invading and surviving within animal cells. Here a synthetic overview of the diversity and evolutionary history of cell membrane-associated, -secreted, and -exported proteins related to apicomplexan parasitism is presented. A notable feature in this regard was the early acquisition of adhesion protein domains and glycosylation systems through lateral transfer from animals. These were utilized in multiple contexts, including invasion of host cells and parasite-specific developmental processes. Apicomplexans possess a specialized version of the ancestral alveolate extrusion machinery, the rhoptries and micronemes, which are deployed in invasion and delivery of proteins into host cells. Each apicomplexan lineage has evolved a unique spectrum of extruded proteins that modify host molecules in diverse ways. Hematozoans, in particular, appear to have evolved novel systems for export of proteins into the host organelles and cell membrane during intracellular development. These exported proteins are an important aspect of the pathogenesis of Plasmodium and Theileria, being involved in response to fever and in leukocyte proliferation respectively. The complement of apicomplexan surface proteins has primarily diversified via massive lineage-specific expansions of certain protein families, which are often coded by subtelomeric gene arrays. Many of these families have been found to be central to immune evasion. Domain shuffling and accretion have resulted in adhesins with new domain architectures. In terms of individual genes, constant selective pressures from the host immune response has resulted in extensive protein polymorphisms and gene losses. Apicomplexans have also evolved complex regulatory mechanisms controlling expression and maturation of surface proteins at the chromatin, transcriptional, posttranscriptional, and posttranslational levels. Evolutionary reconstruction suggests that the ancestral apicomplexan had thrombospondin and EGF domain adhesins, which were linked to the parasite cytoskeleton, and played a central role in invasion through formation of the moving junction. It also suggests that the ancestral parasite had O-linked glycosylation of surface proteins which was partially or entirely lost in hematozoan lineages.
