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Structural characterization of a misfolded intermediate populated during the folding process of a PDZ domain

Nature Structural & Molecular Biology volume 17, pages 1431–1437 (2010)Cite this article

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Abstract

Incorrectly folded states transiently populated during the protein folding process are potentially prone to aggregation and have been implicated in a range of misfolding disorders that include Alzheimer's and Parkinson's diseases. Despite their importance, however, the structures of these states and the mechanism of their formation have largely escaped detailed characterization because of their short-lived nature. Here we present the structures of all the major states involved in the folding process of a PDZ domain, which include an off-pathway misfolded intermediate. By using a combination of kinetic, protein engineering, biophysical and computational techniques, we show that the misfolded intermediate is characterized by an alternative packing of the N-terminal β-hairpin onto an otherwise native-like scaffold. Our results suggest a mechanism of formation of incorrectly folded transient compact states by which misfolded structural elements are assembled together with more extended native-like regions.

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Figure 1: Folding kinetics of D1pPDZ.
Figure 2: Comparison between the chevron plots of D1pPDZ and of six representative mutants (Y169A, A183G, V198A, L210A, S211G and L245A).
Figure 3: Structural distribution of the ΦI values in the misfolding reaction of D1pPDZ.
Figure 4: Comparison of the structures of the intermediate and the native states.
Figure 5: Comparison of the spectroscopic and functional properties of D1pPDZ (gray) and three mutants that populate the intermediate state, V163A (blue), A193G (red) and L231A (black).
Figure 6: Comparison of the TSN and TSI structures of D1pPDZ.
Figure 7: Comparison of the solvent exposure of the aggregation-prone regions of D1pPDZ in the native and in the misfolded intermediate case.

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Acknowledgements

We acknowledge financial support from the Italian Ministero dell'Istruzione dell'Università e della Ricerca (2007B57EAB_004, 20074TJ3ZB_005, RBRN07BMCT_007) (S.G., Y.I., C.T.-A., M.B.), the Wenner Green Foundations (Y.I.), the Istituto Pasteur-Fondazione Cenci Bolognetti (Y.I.), European Molecular Biology Organization (Y.I.), Engineering and Physical Sciences Research Council (A.D.S.), Biotechnology and Biological Sciences Research Council (M.V.) and the Royal Society (M.V.).

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Author notes

  1. Ylva Ivarsson

    Present address: Present address: Department of Human Genetics, Katholieke Universiteit Leuven, Herestraat, Leuven, Belgium.,

  2. Stefano Gianni and Ylva Ivarsson: These authors contributed equally to this work.

Authors and Affiliations

  1. Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli', Istituto Pasteur–Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari del CNR, Università di Roma 'La Sapienza', Rome, Italy

    Stefano Gianni, Ylva Ivarsson, Carlo Travaglini-Allocatelli & Maurizio Brunori

  2. Department of Chemistry, University of Cambridge, Cambridge, UK

    Alfonso De Simone & Michele Vendruscolo

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Contributions

S.G., Y.I., C.T.-A. and M.B. conceived and designed the experimental work; A.D.S. and M.V. conceived and designed the computational work; Y.I. expressed and purified the protein samples; S.G. and Y.I. conducted the experiments; A.D.S. did the simulations; S.G., Y.I., A.D.S., C.T.-A., M.B. and M.V. analyzed the data and wrote the paper.

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Correspondence to Michele Vendruscolo.

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Gianni, S., Ivarsson, Y., De Simone, A. et al. Structural characterization of a misfolded intermediate populated during the folding process of a PDZ domain. Nat Struct Mol Biol 17, 1431–1437 (2010). https://doi.org/10.1038/nsmb.1956

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