Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis

ABSTRACT Identification 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...

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Autores principales:	Yushen Du, Nicholas C. Wu, Lin Jiang, Tianhao Zhang, Danyang Gong, Sara Shu, Ting-Ting Wu, Ren Sun
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2016
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/8867eb0a00a8416e92cd0993397b84bb
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spelling	oai:doaj.org-article:8867eb0a00a8416e92cd0993397b84bb2021-11-15T15:50:16ZAnnotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis10.1128/mBio.01801-162150-7511https://doaj.org/article/8867eb0a00a8416e92cd0993397b84bb2016-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01801-16https://doaj.org/toc/2150-7511ABSTRACT Identification 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. IMPORTANCE To fully comprehend the diverse functions of a protein, it is essential to understand the functionality of individual residues. Current methods are highly dependent on evolutionary sequence conservation, which is usually limited by sampling size. Sequence conservation-based methods are further confounded by structural constraints and multifunctionality of proteins. Here we present a method that can systematically identify and annotate functional residues of a given protein. We used a high-throughput functional profiling platform to identify essential residues. Coupling it with homologous-structure comparison, we were able to annotate multiple functions of proteins. We demonstrated the method with the PB1 protein of influenza A virus and identified novel functional residues in addition to its canonical function as an RNA-dependent RNA polymerase. Not limited to virology, this method is generally applicable to other proteins that can be functionally selected and about which homologous-structure information is available.Yushen DuNicholas C. WuLin JiangTianhao ZhangDanyang GongSara ShuTing-Ting WuRen SunAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 7, Iss 6 (2016)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Yushen Du Nicholas C. Wu Lin Jiang Tianhao Zhang Danyang Gong Sara Shu Ting-Ting Wu Ren Sun Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis
description	ABSTRACT Identification 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. IMPORTANCE To fully comprehend the diverse functions of a protein, it is essential to understand the functionality of individual residues. Current methods are highly dependent on evolutionary sequence conservation, which is usually limited by sampling size. Sequence conservation-based methods are further confounded by structural constraints and multifunctionality of proteins. Here we present a method that can systematically identify and annotate functional residues of a given protein. We used a high-throughput functional profiling platform to identify essential residues. Coupling it with homologous-structure comparison, we were able to annotate multiple functions of proteins. We demonstrated the method with the PB1 protein of influenza A virus and identified novel functional residues in addition to its canonical function as an RNA-dependent RNA polymerase. Not limited to virology, this method is generally applicable to other proteins that can be functionally selected and about which homologous-structure information is available.
format	article
author	Yushen Du Nicholas C. Wu Lin Jiang Tianhao Zhang Danyang Gong Sara Shu Ting-Ting Wu Ren Sun
author_facet	Yushen Du Nicholas C. Wu Lin Jiang Tianhao Zhang Danyang Gong Sara Shu Ting-Ting Wu Ren Sun
author_sort	Yushen Du
title	Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis
title_short	Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis
title_full	Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis
title_fullStr	Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis
title_full_unstemmed	Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis
title_sort	annotating protein functional residues by coupling high-throughput fitness profile and homologous-structure analysis
publisher	American Society for Microbiology
publishDate	2016
url	https://doaj.org/article/8867eb0a00a8416e92cd0993397b84bb
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Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis

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