Evolutionary Analysis and Comparative Genomics of Protein Superfamilies

National Library Of Medicine

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Abstract

The novel coronavirus (SARS-CoV-2) is the causative agent of an emergent severe respiratory disease (COVID-19) in humans that has resulted in a global health crisis. This pandemic, has emphasized the need for a better understanding of the evolution of virus-host interactions. ORF3a in both SARS-CoV-1 and SARS-CoV-2 are ion channels (viroporins) implicated in virion assembly and membrane budding. Using sensitive profile-based homology detection methods, Dr. Aravind working with Dr. Zhang's group at Saint Louis University unified the SARS-CoV ORF3a family with several families of viral proteins, including ORF5 from MERS-CoVs, proteins from beta-CoVs (ORF3c), alpha-CoVs (ORF3b), most importantly, the Matrix (M) proteins from CoVs, and more distant homologs from other nidoviruses. They presented computational evidence that these viral families might utilize specific conserved polar residues to constitute an aqueous pore within the membrane-spanning region. They reconstructed an evolutionary history of these families and objectively established the common origin of the M proteins of CoVs and Toroviruses. They also showed that the divergent ORF3 clade (ORF3a/ORF3b/ORF3c/ORF5 families) represents a duplication stemming from the M protein in alpha- and beta-CoVs. By phyletic profiling of major structural components of primary nidoviruses, they presented a hypothesis for their role in virion assembly of CoVs, ToroVs, and Arteriviruses. The unification of diverse M/ORF3 ion channel families in a wide range of nidoviruses, especially the typical M protein in CoVs, revealed a conserved, previously under-appreciated role of ion channels in virion assembly and membrane budding. They showed that M and ORF3 are under different evolutionary pressures; in contrast to the slow evolution of M as core structural component, the ORF3 clade is under selection for diversification, which suggested it might act at the interface with host molecules and/or immune attack. Jumbo phages have attracted much attention by virtue of their extraordinary genome size and unusual aspects of biology. By performing a comparative genomics analysis of 224 jumbo phages, Dr. Aravind and his team suggested an objective inclusion criterion based on genome size distributions and present a synthetic overview of their manifold adaptations across major biological systems. By means of clustering and principal component analysis of the phyletic patterns of conserved genes, all known jumbo phages can be classified into three higher-order groups, which include both myoviral and siphoviral morphologies indicating multiple independent origins from smaller predecessors. Their study uncovers several under-appreciated or unreported aspects of the DNA replication, recombination, transcription and virion maturation systems. Leveraging sensitive sequence analysis methods, they identified novel protein-modifying enzymes that might help hijack the host-machinery. Focusing on host-virus conflicts, they detected strategies used to counter different wings of the bacterial immune system, such as cyclic nucleotide- and NAD+-dependent effector-activation, and prevention of superinfection during pseudolysogeny. They reconstructed the RNA-repair systems of jumbo phages that counter the consequences of RNA-targeting host effectors. These findings also suggest that several jumbo phage proteins provide a snapshot of the systems found in ancient replicons preceding the last universal ancestor of cellular life. ABC ATPases form one of the largest clades of P-loop NTPase fold enzymes that catalyze ATP-hydrolysis and utilize its free energy for a staggering range of functions from transport to nucleoprotein dynamics. Using sensitive sequence and structure analysis with comparative genomics, for the first time Dr. Aravind and his team provided a comprehensive classification of the ABC ATPase superfamily. ABC ATPases developed structural hallmarks that unambiguously distinguish them from other P-loop NTPases such as an alternative to arginine-finger-based catalysis. At least five and up to eight distinct clades of ABC ATPases are reconstructed as being present in the last universal common ancestor. They underwent distinct phases of structural innovation with the emergence of inserts constituting conserved binding interfaces for proteins or nucleic acids and the adoption of a unique dimeric toroidal configuration for DNA-threading. Specifically, several clades have also extensively radiated in counter-invader conflict systems where they serve as nodal nucleotide-dependent sensory and energetic components regulating a diversity of effectors (including some previously unrecognized) acting independently or together with restriction-modification systems. They presented a unified mechanism for ABC ATPase function across disparate systems like RNA editing, translation, metabolism, DNA repair, and biological conflicts, and some unexpected recruitments, such as MutS ATPases in secondary metabolism. Nucleotide-activated effector deployment, prototyped by interferon-dependent immunity, is a common mechanistic theme shared by immune systems of several animals and prokaryotes. Prokaryotic versions include CRISPR-Cas with the CRISPR polymerase domain, their minimal variants, and systems with second messenger oligonucleotide or dinucleotide synthetase (SMODS). Cyclic or linear oligonucleotide signals in these systems help set a threshold for the activation of potentially deleterious downstream effectors in response to invader detection. Dr Aravind and his team established such a regulatory mechanism to be a more general principle of immune systems, which can also operate independently of such messengers. Using sensitive sequence analysis and comparative genomics, they identified 12 new prokaryotic immune systems, which we unify by this principle of threshold-dependent effector activation. These display regulatory mechanisms paralleling physiological signaling based on 3'-5' cyclic mononucleotides, NAD+-derived messengers, two- and one-component signaling that includes histidine kinase-based signaling, and proteolytic activation. Furthermore, these systems allowed the identification of multiple new sensory signal sensory components, such as a tetratricopeptide repeat (TPR) scaffold predicted to recognize NAD+-derived signals, unreported versions of the STING domain, prokaryotic YEATS domains, and a predicted nucleotide sensor related to receiver domains. We also identify previously unrecognized invader detection components and effector components, such as prokaryotic versions of the Wnt domain. Finally, they show that there have been multiple acquisitions of unidentified STING domains in eukaryotes, while the TPR scaffold was incorporated into the animal immunity/apoptosis signal-regulating kinase (ASK) signalosome. SpoIVA is a bacterial cytoskeletal protein that hydrolyzes ATP to polymerize irreversibly during Bacillus subtilis sporulation. SpoIVA evolved from a TRAFAC class of P-loop GTPases, but the evolutionary pressure that drove this change in nucleotide specificity is unclear. Dr Aravind and the Ramamurthi lab reengineered the nucleotide-binding pocket of SpoIVA to mimic its ancestral GTPase activity. SpoIVAGTPase functioned properly as a GTPase but failed to polymerize because it did not form an NDP-bound intermediate that they reported is required for polymerization. Further, incubation of SpoIVAGTPase with limiting ATP did not promote efficient polymerization. This approach revealed that the nucleotide base, in addition to the energy released from hydrolysis, can be critical in specific biological functions. They also presented data suggesting that increased levels of ATP relative to GTP at the end of sporulation was the evolutionary pressure that drove the change in nucleotide preference in SpoIVA.