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.
Highlights
Non-globular proteins, which contain repeated structural motifs arranged in tandem, are ubiquitous in nature
Analytical ultracentrifugation experiments estimate that the Tetratricopeptide Repeat (TPR) domain exists predominantly as a monomer
Isothermal calorimetry studies indicate that the domain interacts with the C-terminal regions of Hsp70 and Hsp90 with similar micromolar affinity and 1:1 stoichiometry
Summary
Non-globular proteins, which contain repeated structural motifs arranged in tandem, are ubiquitous in nature. We focus on Tetratricopeptide Repeat (TPR) repeats and their versatility with respect to ligand recognition. These structural domains act as interaction scaffolds and mediators of multi-protein complexes and are found in all kingdoms of life [7]. TPR repeats form the 20 most common folds in the Pfam database [8] They consist of multiple repeats of degenerate 34 amino acids, forming the canonical helix-turn-helix fold. Proteins with this motif contain 3–16 sequential TPR motifs, arranged in a tandem array. Proteins with TPR domains play an essential role in the import of proteins into mitochondria [17], chloroplast [18] and peroxisomes [19]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
