Motor mechanism of native ATPase complexes
Zusammenfassung der Projektergebnisse
The goal of this project was to refine the mechanistic and functional model for molecular motor proteins of the AAA+ family and their interaction with their substrates. High-resolution cryo EM or negative stain EM has been used to determine structural snapshots of AAA+ assemblies and/or their substrates in different physiological states in order to deduce their conformational dynamics. The structural data obtained by single particle cryo EM was complemented by results from biological and other biophysical techniques in order to investigate the structure-function relationship of AAA+ ATPases. The project was subdivided into three parts. In the first sub-project, we solved the structure of the contracted VipA/B Vibrio cholera type VI secretion tubule at 5.8Å resolution using cryo EM and single particle reconstruction. We used secondary structure prediction and structural homology between the T4 tail sheath protein gp18 and VipB to segment the helical 3D reconstruction. We concluded that the protomers consist of a structurally rather conserved domain located on the inside of the tubule and a unique domain positioned on the outside of the tubule, which mediates recycling of the tubules by harbouring a recognition motif for the AAA+ ATPase ClpV. This project produced the first high-resolution 3D reconstruction of a contractile nanomachine and laid the groundwork for more refined models of the type VI secretion system tubule of pathogenic bacteria. The direction of the second sub-project, in which proteasomal ATPases should have been investigated, was changed in 2011 to focus on proteasomal biogenesis. Here, we examined the structures of the yeast 15S proteasomal precursor complex and the Pba1/2 bound late 20S precursor complex by negative stain EM in order to localise the proteasomal chaperones Ump1, Pbal and Pba2 as well as to study the subunit arrangement of the proteasomal subunits. Comparison of the structures shows that the Pba1/2 heterodimer is partially embedded in the alpha ring of the 15S complex, which undergoes conformational changes upon dimerization thereby tightening the alpha ring and expelling Pba1/2. Cross-linking and mass spec analysis localised Ump1 in the cavity of the 15S precursor. In contrast to Ump1, Pba1/2 is recycled for multiple rounds of proteasome biogenesis. Here, we are the first to report structural rearrangement of proteasomal subunits during proteasome biogenesis and to pinpoint the location of Ump1 in the 15S precursor complex. We also disprove claims that proteasomal chaperones Pbal and Pba2 are degraded by the nascent proteasome. In the third project we solved the structure of Pexl/6 in 7 different physiological states. We also explored the function of individual domains in the hexamer using point mutations and in vivo assays. We can conclude that only the Pex6D2 ATPase domain hydrolyses ATP in the complex, while both D2 domains contribute to substrate interaction. When ATP is hydrolysed, the D2 domains rotate downwards, suggesting an N- to C-terminal substrate translocation through the hexameric pore. The project produced the first structure of a heterohexameric AAA+ assembly and a thorough analysis of nucleotide dependent movements in type II AAA+ ATPase complexes. It sheds first light onto critical parts of the crucial protein import machinery of peroxisomes.
Projektbezogene Publikationen (Auswahl)
- (2009). Motor mechanism for protein threading through Hsp104. Mol Cell34:81-92
Wendler, P., J. Shorter, D. Snead, C. Plisson, D. K. Clare, S. Lindquist, H. Saibil
- (2010). Cryo electron microscopy structures of Hsp100 proteins- crowbars in or out? Biochem. Cell Biol. 88:89-96
Wendler, P., H. Saibil
- (2010). Hsp104- ein eiskaltes Hitzeschockprotein. BIOspektrum 6:648650
Wendler, P.
- (2011). Structure and function of the AAA+ protein CbbX, a red-type Rubisco activase. Nature 479(7372):194-9
Mueller-Cajar, O., M. Stotz, P. Wendler, F.U. Hartl, A. Bracher, M. Hayer-Hartl
- (2011). Structure and mechanism of the Swi2/Snf2 remodeller Mot1 in complex with its substrate TBP. Nature 475 (7356):403-7
Wollmann, P., S. Cui, R. Viswanathan, O. Berninghausen, M.N. Wells, M. Moldt, G. Witte, A. Butryn, P. Wendler, R. Beckmann, D.T. Auble, KP Hopfner
- (2012). Structure and function of the AAA+ nucleotide binding pocket. BBA Jan;1823 (1):2-14
Wendler, P., S. Ciniawsky, M. Kock, S. Kube
(Siehe online unter https://doi.org/10.1016/j.bbamcr.2011.06.014) - (2013). Conserved distal loop residues in the Hsp104 and ClpB middle domain contact nucleotide-binding domain 2 and enable Hsp70-dependent protein disaggregation. J. Biol. Chem. 289(2):848-67
Desantis, M., E., E.A. Sweeny, D. Snead, E.H. Leung, M.S. Go, K Gupta, P. Wendler, J. Shorter
(Siehe online unter https://doi.org/10.1074/jbc.M113.520759) - (2014). 6A cryo EM structure of the contractile VipA/B nanomachine in type VI effector secretion. Cell Reports 8, 20-30
Kube, S., N. Kapitein, T. Zimniak, F. Herzog, A. Mogk, P. Wendler
(Siehe online unter https://doi.org/10.1016/j.celrep.2014.05.034) - (2014). Head-to-tail interactions of the coiled-coil domains regulate ClpB cooperation with Hsp70 in protein disaggregation. eLife 2014;10.7554/eLife.02481
Carroni, M., E. Kummer, Y. Oguchi, P. Wendler, D. K. Clare, I. Sinning, J. Kopp, A. Mogk, B. Bukau, H. Saibil
(Siehe online unter https://doi.org/10.7554/eLife.02481) - (2015). Molecular snapshots of the Pex1/6 AAA+ complex in action. Nat Commun. 6:7331
Ciniawsky, S., I. Grimm, D. Saffian, W. Girzalsky, R. Erdmann, P. Wendler
(Siehe online unter https://doi.org/10.1038/ncomms8331) - (2015). Proteasome assembly from 15S precursors involves major conformational changes and recycling of the Pba1-Pba2 chaperone. Nat. Commun. 6:6123
Kock, M., M. N. Nunes, M. Hemann, S. Kube, R. J. Dohmen, F. Herzog, P.C. Ramos, P. Wendler
(Siehe online unter https://doi.org/10.1038/ncomms7123)
