Abstract
ABSTRACTIdentification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available.
Highlights
Amino acid residues in a protein have two roles: providing a structural framework and mediating interactions with other biomolecules
Multimerization of hepatitis C virus (HCV) Viral RNA-dependent RNA polymerase (vRdRp) is essential for viral replication
Noncanonical functional residues in vRdRp are difficult to determine by commonly used methods and are not as well studied as the key residues for polymerase catalytic functions
Summary
Amino acid residues in a protein have two roles: providing a structural framework (structural residues) and mediating interactions with other biomolecules (functional residues). A more direct and systematic method needs to be used for the accurate identification and annotation of functional residues Because of their compact genome, viruses usually encode multifunctional proteins, including viral polymerase proteins. We previously developed a method to systematically identify functional residues by coupling experimental fitness measurement with protein stability prediction [16]. We extended this method to annotate functional residues in combination with structural comparison of homologous proteins. We identified noncanonical functional residues, which are exemplified by a cluster of residues located in the loop region of the PB1 -ribbon These previously uncharacterized residues were shown to be important for PB1 protein nuclear import by interacting with Ranbinding protein 5 (RanBP5) [32]
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