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Structural determinants of chaperone function

Research Project
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01.08.2016
 - 31.07.2019

A majority of proteins require folding into a three-dimensional structure in order to obtain their proper functionality. At the same time, protein misfolding and aggregation pose severe threats to the health of cells and organisms. Protein folding in vivo is therefore closely guided by molecular chaperones, which interact with substrate polypeptides to increase folding yields, resolve misfolding, or transport them to a target location. Despite the availability of many high-resolution crystal structures of chaperones in their substrate-free apo forms, structural information about how substrates are bound by chaperones and thus protected from misfolding and aggregation is sparse. Consequently, the structural determinants for chaperone functions are still very poorly understood. This lack of information is particularly remarkable, because chaperone functions are essential in all kingdoms of life. Due to the disordered nature of unfolded and partially folded substrates and the highly dynamic ways of interaction between chaperones and substrates, solution NMR spectroscopy is ideally suited to characterize these interactions. Here, we propose to characterize conformation and dynamics of selected chaperone-substrate systems at the atomic level. These studies will contribute to recognizing fundamental principles of chaperone function. We will then apply our experience to characterize the functionally highly related, eukaryotic unfolded protein response (UPR) at the atomic level. In particular, we will pursue three projects A-C:Project A: Molecular determinants of Skp chaperone function. We have previously obtained a complete description of the chaperone Skp in complex with its aggregation-prone substrate OmpX. The dynamic conformational ensemble of the substrate randomly populates a spherical volume in first order approximation. However, additional data show that this ensemble contains non-random substructures. We will now characterize these substructures at the atomic level and compare them with preparations of the Omp in aqueous solution. We will thus be able to directly observe the effect that Skp-binding has on an aggregation-prone substrate. Furthermore, we will determine the contributions of two structural features of Skp on its function: Its symmetry and its cavity, resulting from homooligomerization. Project B: Molecular determinants of Spy chaperone function. The recently discovered periplasmic chaperone Spy presents an ideal model system to study the interaction of a holdase chaperone with both soluble and non-soluble substrates. An atomic resolution description of Spy function is lacking. We will characterize structure and dynamics of the chaperone Spy in its complexes with the soluble Im7 and the insoluble OmpX at the atomic level. We will also investigate how the Spy chaperone distinguishes between folded and unfolded segments of its substrates. Furthermore, we will search for a structural explanation for the Superspy variants. These are single point-mutants of Spy that have been created by directed evolution, leading to increased chaperone activity. Defining the mechanism for a generally enhanced chaperone activity would undoubtedly be very valuable in understanding chaperone action.Project C: Mechanisms for substrate recognition in the eukaryotic UPR system. The endoplasmatic reticulum (ER) contains a signaling system that recognizes unfolded protein in the ER lumen (unfolded protein response; UPR). The UPR is of high relevance for eukaryotic life and its malfunction or overload is involved in metabolic diseases including diabetes and cancer. Its functional mechanism remains however poorly understood. We hypothesize that the basic biophysical principles underlying holdase chaperone function are related to mechanisms of UPR function. We want to reconstitute relevant parts of the UPR system in vitro and reveal the mechanism of unfolded protein by the UPR structurally and functionally.

Collaborations & Cooperations

2099 - Participation or Organization of Collaborations on a national level
Müller, Daniel J., Prof., ETH D-BSSE, Research cooperation

Funding

Structural determinants of chaperone function

SNF Projekt (GrantsTool), 08.2016-07.2019 (36)
PI : Hiller, Sebastian.

Publications

Burmann, Björn M. et al. (2020) ‘Regulation of α-synuclein by chaperones in mammalian cells’, Nature, 577(7788), pp. 127–132. Available at: https://doi.org/10.1038/s41586-019-1808-9.

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Kohl, Bastian et al. (2020) ‘Protocol for High-Yield Production of Photo-Leucine-Labeled Proteins in Escherichia coli’, Journal of Proteome Research, 19(8), pp. 3100–3108. Available at: https://doi.org/10.1021/acs.jproteome.0c00105.

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Mas, Guillaume et al. (2020) ‘Regulation of chaperone function by coupled folding and oligomerization’, Science advances, 6(43), p. eabc5822. Available at: https://doi.org/10.1126/sciadv.abc5822.

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He, Lichun and Hiller, Sebastian (2019) ‘Frustrated Interfaces Facilitate Dynamic Interactions between Native Client Proteins and Holdase Chaperones’, Chembiochem : a European journal of chemical biology, 20(22), pp. 2803–2806. Available at: https://doi.org/10.1002/cbic.201900215.

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Hiller, Sebastian (2019) ‘Chaperone-Bound Clients: The Importance of Being Dynamic’, Trends in Biochemical Sciences, 44(6), pp. 517–527. Available at: https://doi.org/10.1016/j.tibs.2018.12.005.

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Mas, Guillaume, Thoma, Johannes and Hiller, Sebastian (2019) ‘The Periplasmic Chaperones Skp and SurA’, in Kuhn, A. (ed.) Bacterial Cell Walls and Membranes. Cham: Springer Nature (Subcellular Biochemistry), pp. 169–186. Available at: https://doi.org/10.1007/978-3-030-18768-2_6.

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He, Lichun and Hiller, Sebastian (2018) ‘Common Patterns in Chaperone Interactions with a Native Client Protein’, Angewandte Chemie International Edition, 57(20), pp. 5921–5924. Available at: https://doi.org/10.1002/anie.201713064.

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Hiller, Sebastian and Burmann, Björn M. (2018) ‘Chaperone-client complexes: A dynamic liaison’, Journal of Magnetic Resonance, 289, pp. 142–155. Available at: https://doi.org/10.1016/j.jmr.2017.12.008.

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Mas, Guillaume and Hiller, Sebastian (2018) ‘Conformational plasticity of molecular chaperones involved in periplasmic and outer membrane protein folding’, FEMS Microbiology Letters, 365(13), p. fny121. Available at: https://doi.org/10.1093/femsle/fny121.

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Holdbrook, Daniel A. et al. (2017) ‘A Spring-Loaded Mechanism Governs the Clamp-like Dynamics of the Skp Chaperone’, Structure, 25(7), p. 1079–+. Available at: https://doi.org/10.1016/j.str.2017.05.018.

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Morgado, Leonor et al. (2017) ‘The dynamic dimer structure of the chaperone Trigger Factor’, Nature Communications, 8(1), p. 1992. Available at: https://doi.org/10.1038/s41467-017-02196-7.

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He, Lichun et al. (2016) ‘A molecular mechanism of chaperone-client recognition’, Science Advances, 2(11), p. e1601625. Available at: https://doi.org/10.1126/sciadv.1601625.

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Members (4)

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Sebastian Hiller

Principal Investigator
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Guillaume Mas

Project Member
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Bastian Kohl

Project Member
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Lichun He

Project Member