Evolutionary Analysis and Comparative Genomics of Protein Superfamilies

National Library Of Medicine

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Abstract

The systematic analysis and discovery of biological conflict systems have been a major focus of research in the Aravind lab. Biological replicators, from genes within a genome to whole organisms, are locked in conflicts. Comparative genomics has revealed a staggering diversity of molecular armaments and mechanisms regulating their deployment, collectively termed biological conflict systems. These encompass toxins used in inter- and intraspecific interactions, self/nonself discrimination, antiviral immune mechanisms, and counter-host effectors deployed by viruses and intragenomic selfish elements. These systems possess shared syntactical features in their organizational logic and a set of effectors targeting genetic information flow through the Central Dogma, certain membranes, and key molecules like NAD+. These principles can be exploited to discover new conflict systems through sensitive computational analyses. This has led to significant advances in our understanding of the biology of these systems and furnished new biotechnological reagents for genome editing, sequencing, and beyond. Dr. Aravind and his team applied this to discover novel toxin-, restriction-modification, apoptosis, CRISPR/second messenger-regulated systems, and other enigmatic nucleic acid-targeting systems. Over the past two decades, studies have revealed profound evolutionary connections between prokaryotic and eukaryotic immune systems, challenging the notion of their unrelatedness. Dr. Aravind's work showed that immune systems across the tree of life share an operational framework, shaping their biochemical logic and evolutionary trajectories. The diversification of immune genes in the prokaryotic superkingdoms, followed by lateral transfer to eukaryotes, was central to the emergence of innate immunity in the latter. These include protein domains related to nucleotide second messenger-dependent systems, NAD+/nucleotide degradation, and P-loop NTPase domains of the STAND and GTPase clades playing pivotal roles in eukaryotic immunity and inflammation. Moreover, Dr. Aravind's work showed that several domains orchestrating programmed cell death, ultimately of prokaryotic provenance, suggest an intimate link between immunity and the emergence of multicellularity in eukaryotes such as animals. While eukaryotes directly adopted some proteins from bacterial immune systems, they repurposed others for new immune functions from bacterial interorganismal conflict systems. These emerging immune components hold substantial biotechnological potential. In collaborative work with the Kumaran Ramamurthi lab at NCI, the Aravind lab showed how an enzyme originally involved in anti-viral activity transmogrified into a cell division regulator. The spherical bacterium Staphylococcus aureus, a leading cause of nosocomial infections, undergoes binary fission by dividing in two alternating orthogonal planes, but the mechanism by which S. aureus correctly selects the next cell division plane is not known. To identify cell division placement factors, the Ramamurthi lab performed a chemical genetic screen that revealed a gene termed pcdA. The Aravind lab showed that PcdA is a member of the McrB family of AAA+ NTPases that has undergone structural changes and a concomitant functional shift from a restriction enzyme subunit to an early cell division protein. PcdA directly interacts with the tubulin-like central divisome component FtsZ and localizes to future cell division sites before membrane invagination initiates. This parallels the action of another McrB family protein, CTTNBP2, which stabilizes microtubules in animals. Their work showed that PcdA also interacts with the structural protein DivIVA and proposed that the DivIVA/PcdA complex recruits unpolymerized FtsZ to assemble along the proper cell division plane. Deletion of pcdA conferred abnormal, non-orthogonal division plane selection, increased sensitivity to cell wall-targeting antibiotics, and reduced virulence in a murine infection model. Targeting PcdA could therefore highlight a treatment strategy for combating antibiotic-resistant strains of S. aureus. In a collaborative work with Dr. Gisela Storz and Doreen Matthies at NICHD/NIH, Dr. Aravind characterized the P-type ATPase superfamily and its role in Mg2+ transport. Magnesium (Mg2+) uptake systems are present in all domains of life, consistent with the vital role of this ion. P-type ATPase Mg2+ importers are required for bacterial growth when Mg2+ is limiting or during pathogenesis. However, insights into their mechanisms of action are missing. Dr. Matthies' lab solved the cryo-EM structure of the Mg2+ transporter MgtA from Escherichia coli and obtained high-resolution structures of both homodimeric (2.9 Å) and monomeric (3.6 Å) forms. Dr. Aravind's work on them helped identify mechanistic details relating to catalytic activity, domain movement and nucleotide binding. The dimer structure was shown to be formed by multiple contacts between residues in adjacent soluble N and P subdomains. The structures revealed an ion, assigned as Mg2+, in the transmembrane segment. Moreover, this work detected two cytoplasmic ion-binding sites with a potential allosteric role. Interestingly, it was established that an N-terminal tail identified by Dr. Aravind played a key regulatory role in the ATPase action. Sequence conservation analysis, mutagenesis, and ATPase assays based on his predictions indicated dimerization, the ion-binding sites and the N-terminal tail facilitate cation transport or serve regulatory roles. In a collaborative work with Dr. Philip Adams' lab, Dr. Aravind identified a key protein in flagellar biogenesis. Flagella propel pathogens through their environments, yet are expensive to synthesize and are immunogenic. Thus, complex hierarchical regulatory networks control flagellar gene expression. Spirochetes are highly motile bacteria, but peculiarly, the archetypal flagellar regulator σ28 is absent in the Lyme spirochete Borrelia burgdorferi. Dr. Aravind's work showed that the gene bb0268 (flgV) in B. burgdorferi, previously and incorrectly annotated to encode the RNA-binding protein Hfq, is instead a structural flagellar component that modulates flagellar assembly. Working with the Adams labs, Dr. Aravind and colleagues established that the flgV gene is broadly conserved in the flagellar superoperon alongside σ28 in many Spirochaetae, Firmicutes and other phyla, with distant homologs in Epsilonproteobacteria. They were able to show that B. burgdorferi FlgV is localized within flagellar basal bodies, and strains lacking flgV produce fewer and shorter flagellar filaments and are defective in cell division and motility. During the enzootic cycle, flgV-deficient B. burgdorferi survive and replicate in Ixodes ticks but are attenuated for infection and dissemination in mice. This work defined infection timepoints when spirochete motility is most crucial and implicated FlgV as a broadly distributed structural flagellar component that modulates flagellar assembly. Dr. Aravind's lab worked in conjunction with Dr. Allen Buskirk's lab at Johns Hopkins University to identify the mechanism by which collided ribosomes are cleared by cells. Although many antibiotics inhibit bacterial ribosomes, loss of known factors that rescue stalled ribosomes does not lead to robust antibiotic sensitivity in E. coli. This suggested the existence of additional unknown mechanisms. A screen by Dr. Buskirk's lab showed that the protein HrpA rescues stalled ribosomes in E. coli. Dr. Aravind's lab characterized its catalytic RNA helicase and additionally, accessory domains fused to it. Acting selectively on ribosomes that have collided, HrpA uses ATP hydrolysis by the helicase domain to split stalled ribosomes into subunits. Cryo-EM structures collaboratively done by the Beckmann lab reveal how HrpA simultaneously binds to two collided ribosomes, explaining its selectivity, and how its helicase module engages downstream mRNA, such that by exerting a pulling force on the mRNA, it would destabilize the stalled ribosome. These studies show that ribosome splitting is a conserved mechanism that allows proteobacteria to tolerate ribosome-targeting antibiotics. Dr. Aravind is working on a collaborative project with the lab of Dr. Matthias Machner at NICHD/NIH on a new enzyme that removes lipid conjugates in the form of palmitoylate from cysteine side chains in proteins. Dr. Aravind's work helped identify the active site of this enzyme and its mode of action. The hypotheses generated from his analysis are now being tested in the Machner lab. Dr. Aravind is also working in collaboration with Philip Adams' lab in characterizing a novel flagellar and cell-division proteins he identified in the Lyme disease pathogen, Borrelia burgdorferi.