Finding Protein Sequence Motifs--Methods And Applications

National Library Of Medicine

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Abstract

The rapid accumulation of genome sequences and protein structures during the last decade has been paralleled by major advances in sequence database search methods as well as protein structure prediction. The powerful Position-Specific Iterating BLAST (PSI-BLAST) method developed at the NCBI forms the basis of our work on protein motif analysis. In addition, Hidden Markov Models (HMM), protein profile-against-profile comparison implemented in the HHSearch method, protein structure comparison methods, homology modeling of protein structure and genome context analysis were extensively and increasingly applied. Furthermore, custom libraries of protein domain profiles as well as computational pipelines for novel domain identification have been developed and applied. Lately, these methods for protein motif search are being complemented by deep learning computational methods, in particular, AlphaFold2, a powerful method for protein structure modeling. During the year under review, we have continued our investigation of the proteins domains, particularly, those that are encoded in the genomes of viruses of prokaryotes and eukaryotes as well as domains involved in the defense of bacteria against viruses. The scope of these studies was substantially expanded through extensive use of AlphaFold2. As a case study for the application of AlphaFold2 to predict structures and functions of fast-evolving virus proteins, we modeled the proteome of orthopoxviruses, an important group of viruses that includes major human pathogens. Viruses with large, double-stranded DNA genomes captured the majority of their genes from their hosts at different stages of evolution. The origins of many virus genes are readily detected through significant sequence similarity with cellular homologs. In particular, this is the case for virus enzymes, such as DNA and RNA polymerases or nucleotide kinases, that retain their catalytic activity after capture by an ancestral virus. However, a large fraction of virus genes have no readily detectable cellular homologs, meaning that their origins remain enigmatic. We explored the potential origins of such proteins that are encoded in the genomes of orthopoxviruses, a thoroughly studied virus genus that includes major human pathogens. To this end, we used AlphaFold2 to predict the structures of all 214 proteins that are encoded by orthopoxviruses. Among the proteins of unknown provenance, structure prediction yielded clear indications of origin for 14 of them and validated several inferences that were previously made via sequence analysis. A notable emerging trend is the exaptation of enzymes from cellular organisms for nonenzymatic, structural roles in virus reproduction that is accompanied by the disruption of catalytic sites and by an overall drastic divergence that precludes homology detection at the sequence level. Among the 16 orthopoxvirus proteins that were found to be inactivated enzyme derivatives are the poxvirus replication processivity factor A20, which is an inactivated NAD-dependent DNA ligase; the major core protein A3, which is an inactivated deubiquitinase; F11, which is an inactivated prolyl hydroxylase; and more similar cases. For nearly one-third of the orthopoxvirus virion proteins, no significantly similar structures were identified, suggesting exaptation with subsequent major structural rearrangement that yielded unique protein folds. In another study, we set out to identify the origins of anti-CRISPR proteins (ACRs) encoded by bacterial and archaeal viruses. The majority of the Acrs are small, non-enzymatic proteins that abrogate CRISPR activity by binding to Cas effector proteins. The Acrs evolve fast, due to the arms race with the respective CRISPR-Cas systems, which hampers the elucidation of their evolutionary origins by sequence comparison. We performed comprehensive structural modeling using AlphaFold2 for 3693 experimentally characterized and predicted Acrs, followed by a comparison to the protein structures in the Protein Data Bank database. After clustering the Acrs by sequence similarity, 363 high-quality structural models were obtained that accounted for 102 Acr families. Structure comparisons allowed the identification of homologs for 13 of these families that could be ancestors of the Acrs. Despite the limited extent of structural conservation, the inferred origins of Acrs show distinct trends, in particular, recruitment of toxins and antitoxins and SOS repair system components for the Acr function. In collaboration with the laboratory of Professor Feng Zhang at the Broad Insitute of MIT and Harvard, we investigated the structures and functional of target selector proteins of Tn7-like transposons. To spread, transposons must integrate into target sites without disruption of essential genes while avoiding host defense systems. Tn7-like transposons employ multiple mechanisms for target-site selection, including protein-guided targeting and, in CRISPR-associated transposons (CASTs), RNA-guided targeting. Combining phylogenomic and structural analyses, we conducted a broad survey of target selectors, revealing diverse mechanisms used by Tn7 to recognize target sites, including previously uncharacterized target-selector proteins found in newly discovered transposable elements (TEs). We experimentally characterized a CAST I-D system and a Tn6022-like transposon that uses TnsF, which contains an inactivated tyrosine recombinase domain, to target the comM gene. Additionally, we identified a non-Tn7 transposon, Tsy, encoding a homolog of TnsF with an active tyrosine recombinase domain, which we show also inserts into comM. Our findings show that Tn7 transposons employ modular architecture and co-opt target selectors from various sources to optimize target selection and drive transposon spread.