AAA-ATPase unfoldase dynamics

Principal investigator: Frank Gabel

Group members involved: Emilie Mahieu, Jacques Covès, Bruno Franzetti

A healthy proteome is essential for the correct functioning at the molecular, cellular and organismal level of any living being. One important mechanism to achieve this is the specific recognition, unfolding and degradation of abnormally folded proteins which otherwise can lead to a number of diseases due to protein aggregation (e.g. some neurodegenerative diseases in humans). Unfolding is accomplished by different classes of AAA-ATPase machines (unfoldases), in concert with peptidase core particles where the unfolded proteins are catalyzed. While recent studies have provided structural models of the individual partners and their complexes, the dynamics and kinetics of the working unfoldases remains largely unknown.

In the present project we apply a combination of structural biology techniques in solution (SAXS/SANS/NMR) to address the molecular mechanism of the unfolding process of the proteasome-activating nucleotidase (PAN), a stable and thermo-activable 240 kDa complex from thermophilic archaea. We use PAN with ssrA-tagged GFP substrate as an in vitro model system to study the function, kinetics and structural dynamics by biophysical approaches of both the unfoldase and the substrate.

Figure 1: Left: archaeal protein degradation system PAN + 20S core particle + GFP substrate. Right: Conformational changes of PAN during the active unfolding process of GFP and GFP aggregation in the absence of the 20S core particle. The inset displays the special sample environment developed at ILL for this project, combining a neutron scattering and fluorescence.

Collaborations:

• Anne Martel, European Neutron Center, ILL Grenoble.

• Carlomagno NMR group at Birmingham University, United Kingdom.

Funding:

• ANR PRCI Grant "PROTstretch" (2015-2019)

Publications:

• Mahieu, E., Covès, J., Krüger, G., Martel, A., Moulin, M., Carl, N., Härtlein, M., Carlomagno, T., Franzetti, B. and Gabel, F. (2020) Observing Protein Degradation by the PAN-20S Proteasome by Time-Resolved Neutron Scattering. Biophys. J. 119(2), 375-388.

• Mahieu, E. and Gabel, F. (2018) Biological small-angle neutron scattering: recent results and development. Acta Crystallogr. D Struct. Biol. 74(Pt 8), 715-726.

• Ibrahim, Z., Martel, A., Moulin, M., Kim, H.S., Härtlein, M., Franzetti, B. and Gabel, F. (2017) Time-resolved neutron scattering provides new insight into protein substrate processing by a AAA+ unfoldase. Sci. Rep. 7, 40948.