Structural and functional insights into the cofactor diversity of the human AAA ATPase p97
Final Report Abstract
The hexameric type II AAA ATPase (ATPase associated with various activities) p97 participates in a variety of cellular processes including pathways such as DNA replication and repair involving chromatin remodeling, and is a key player in multiple protein quality control pathways mediated by the ubiquitin proteasome system (UPS) and autophagy. Correspondingly, p97 has been linked to pathophysiological states including cancer, neurodegenerative disorders and premature aging. p97 is characterized by an N- terminal domain, the presence of two highly conserved ATPase domains (D1 and D2) and an unstructured C-terminal tail. p97 uses energy from ATP hydrolysis to extract or segregate ubiquitylated target proteins from stable protein assemblies, membranes and chromatin. Since p97 is involved in highly diverse cellular processes, its activity must be tightly controlled. This is achieved by a large number of regulatory cofactors, which either associate with the N-terminal domain or interact with the extreme C-terminus and target p97 to specific pathways, which sometimes involves the simultaneous association with more than one cofactor. As part of this project we investigated the interaction of cofactors with the N domain and determined the first crystal structure of a SHP binding motif in complex with p97. Unlike UBX/UBXL domains and the two helical binding motifs VIM/VBM, which bind into a hydrophobic binding pocket formed between the two subdomains of the N domain, the SHP motif binds to a so far uncharacterized hydrophobic site on the lateral surface of the N domain. The observed binding mode is conserved between structurally unrelated proteins. The SHP motif is often found in combination with UBX or UBXL domains The association of the SHP-binding motif on the lateral surface of the N domain and of UBX/UBXL domains in the hydrophobic interdomain cleft on top of the N domain allows for a bipartite binding of both binding domains/motifs to either the same N domain or to two adjacent N domains. This bipartite interaction mode not only increases the affinity but also restricts the conformational flexibility of p97. These data provided new insights into the assembly of regulatory proteins, which is regulated by mechanisms including mutually exclusive binding, conformational changes induced by nucleotide binding/hydrolysis, different oligomeric assemblies and hierarchical binding. Furthermore, we determined crystal structures of the physiologically relevant p97 hexamer in the apo and ATPγS bound state. The different nucleotide states are reflected in significant conformational changes. A rotation of about 14° of the D2 and ND1 modules with respect to each other, which is mediated by the long flexible linker between the D1 and D2 module was observed. These structures also revealed: (i) p97 contains a functional important sensor helix, which is proposed to be involved in sensing the nucleotide status in the cis-subunit, which is then transmitted to the C-terminus of p97. (ii) The C- terminal α-helix undergoes a significant conformational change. The helix is kinked and inserts between two adjacent monomers into the ATP-binding pocket of the trans-monomer and an arginine directly coordinates the γ-phosphate of the ATP, leading to a closure of the D2 nucleotide-binding pocket. (iii) The central putative substrate-binding pore in the D2 subunit formed by the hexameric p97 arrangement switches between a locked and a dynamically released conformation in the apo and the ATPγS bound states. (iv) An intersubunit signaling network (ISS) couples the conformation of the putative substratetranslocating pore to the nucleotide state of the cis-subunit, which is then transmitted to the trans-subunit and coordinates in this way ATP-hydrolysis in adjacent monomers. In addition, the ISS is involved in signal transmission from the D2 domain via the D1D2 linker to the D1 domain. These structures provided mechanistic insights into interdomain communication mediated by conformational changes of the C terminus as well as an intersubunit signaling network, which couples the nucleotide state to the conformation of the central putative substrate-binding pore.
Publications
- Control of p97 function by cofactor binding. FEBS Lett 589: 2578-2589, 2015
Buchberger A., Schindelin H. and Hänzelmann P.
(See online at https://doi.org/10.1016/j.febslet.2015.08.028) - Characterization of an Additional Binding Surface on the p97 N-Terminal Domain Involved in Bipartite Cofactor Interactions. Structure 24: 140-147, 2016
Hänzelmann P. and Schindelin H.
(See online at https://doi.org/10.1016/j.str.2015.10.027) - Structural Basis of ATP Hydrolysis and Intersubunit Signaling in the AAA+ ATPase p97, Structure 24:127-139, 2016
Hänzelmann P. and Schindelin H.
(See online at https://doi.org/10.1016/j.str.2015.10.026)
