title: Ligand recognition by the TPR domain of the import factor Toc64 from Arabidopsis thaliana.
abstract: The specific targeting of protein to organelles is achieved by targeting signals being recognised by their cognate receptors. Cytosolic chaperones, bound to precursor proteins, are recognized by specific receptors of the import machinery enabling transport into the specific organelle. The aim of this study was to gain greater insight into the mode of recognition of the C-termini of Hsp70 and Hsp90 chaperones by the Tetratricopeptide Repeat (TPR) domain of the chloroplast import receptor Toc64 from Arabidopsis thaliana (At). The monomeric TPR domain binds with 1∶1 stoichiometry in similar micromolar affinity to both Hsp70 and Hsp90 as determined by isothermal titration calorimetry (ITC). Mutations of the terminal EEVD motif caused a profound decrease in affinity. Additionally, this study considered the contributions of residues upstream as alanine scanning experiments of these residues showed reduced binding affinity. Molecular dynamics simulations of the TPR domain helices upon peptide binding predicted that two helices within the TPR domain move backwards, exposing the cradle surface for interaction with the peptide. Our findings from ITC and molecular dynamics studies suggest that AtToc64_TPR does not discriminate between C-termini peptides of Hsp70 and Hsp90.
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia, Australia.
authors.forename: Rashmi
authors.initials: R
authors.lastname: Panigrahi
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia, Australia.
authors.forename: Abdussalam
authors.initials: A
authors.lastname: Adina-Zada
authors.affiliation: ARC Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley, Western Australia, Australia ; Department of Botany, School of Life Science, La Trobe University, Bundoora, Victoria, Australia.
authors.forename: James
authors.initials: J
authors.lastname: Whelan
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia, Australia.
authors.forename: Alice
authors.initials: A
authors.lastname: Vrielink
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid
chemical_list: D029681:Arabidopsis Proteins; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D008024:Ligands; D008565:Membrane Proteins; D011994:Recombinant Proteins; C488374:Toc64 protein, Arabidopsis