Atomic-resolution studies of the structure, dynamics and interactions in chaperone assemblies : deciphering mitochondrial membrane protein import

Orateur : Soutenance de Thèse de Katharina WEINHÄUPL (IBS/Groupe de RMN biomoléculaire)

Molecular chaperones accompany, fold and protect other proteins until they reach their final conformationand location inside the cell. To this end, molecular chaperones need to be specialized in performing specific tasks, like folding, transport or disaggregation, and versatile in their recognition pattern to engage many different client proteins. Interactions between the different partners in this network and between the substrate and the chaperone are often dynamic processes, which are especially diffcult to study using standard structural biology techniques. Consequently, structural data on chaperone/substrate complexes are sparse, and the mechanisms of chaperone action are poorly understood. I present investigations of the structure, dynamics and substrate- interactions of two molecular chaperones, using various biophysical and in vivo methods. The main part of the presented work is devoted to the mitochondrial membrane protein chaperone TIM910, which binds its substrates - membrane proteins of the inner and outer mitochondrial membranes - in a ;highly dynamic manner. Not only is the TIM910 complex in constant exchange between monomeric and hexameric species, but the bound membrane preprotein also samples multiple conformations. Based on nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), analytical ultracentrifugation (AUC) and in vivo mutational experiments and available crystal structures, I propose a functional and structural model of the chaperone/membrane protein complex, and discuss the implication for membrane protein import in mitochondria. TIM910 binds its substrates in a hydrophobic pocket on the exterior of the chaperone in a modular fashion, where the number of TIM910 complexes bound depends on the length of the substrate. The second part focuses on the behavior of the N-terminal receptor domain of the ClpC1 unfoldase from M.tuberculosis in the presence of different antibiotics and ligands. The N-terminal domain of ClpC1 is the binding site for various new antibiotics against M.tuberculosis. The antibiotic cyclomarin completely abolishes dynamics induced by the ligand arginine-phosphate. We propose that this suppression of dynamics is the underlying principle for the mechanism of action of this antibiotic. This work highlights examples where only an integrated structural approach including various structural, dynamical and in vivo methods were allow the understanding of these dynamic assemblies at a molecular level.

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