Two aspartate residues at the putative p10 subunit of a type II metacaspase from Nicotiana tabacum L. may contribute to the substrate-binding pocket
| Autor: | Alexis Acosta-Maspons, Laura Sánchez-Baldoquín, Tirso Pons, Edgar Sepúlveda-García, Lien González, Junier Marrero-Gutiérrez, Mario Rocha-Sosa |
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| Rok vydání: | 2013 |
| Předmět: |
Models
Molecular Protein Folding DNA Complementary Saccharomyces cerevisiae Proteins Protein Conformation Protein subunit Nicotiana tabacum Molecular Sequence Data Trypanosoma brucei brucei Nicotiana benthamiana Sequence alignment Plant Science Protein structure Tobacco Genetics Escherichia coli Amino Acid Sequence Peptide sequence Plant Proteins Aspartic Acid Multiple sequence alignment Binding Sites biology fungi biology.organism_classification Metacaspase Protein Subunits Biochemistry Caspases Mutagenesis Site-Directed Sequence Alignment |
| Zdroj: | Planta. 239(1) |
| ISSN: | 1432-2048 |
| Popis: | Metacaspases are cysteine proteases present in plants, fungi, prokaryotes, and early branching eukaryotes, although a detailed description of their cellular function remains unclear. Currently, three-dimensional (3D) structures are only available for two metacaspases: Trypanosoma brucei (MCA2) and Saccharomyces cerevisiae (Yca1). Furthermore, metacaspases diverged from animal caspases of known structure, which limits straightforward homology-based interpretation of functional data. We report for the first time the identification and initial characterization of a metacaspase of Nicotiana tabacum L., NtMC1. By combining domain search, multiple sequence alignment (MSA), and protein fold-recognition studies, we provide compelling evidences that NtMC1 is a plant metacaspase type II, and predict its 3D structure using the crystal structure of two type I metacaspases (MCA2 and Yca1) and Gsu0716 protein from Geobacter sulfurreducens as template. Analysis of the predicted 3D structure allows us to propose Asp353, at the putative p10 subunit, as a new member of the aspartic acid triad that coordinates the P1 arginine/lysine residue of the substrate. Nevertheless, site-directed mutagenesis and expression analysis in bacteria and Nicotiana benthamiana indicate the functionality of both Asp348 and Asp353. Through the co-expression of mutant and wild-type proteins by transient expression in N. benthamiana leaves we found that polypeptide processing seems to be intramolecular. Our results provide the first evidence in plant metacaspases concerning the functionality of the putative p10 subunit. |
| Databáze: | OpenAIRE |
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