Structural studies of the protein remodeller p97 and associated partners
p97 is a member of the AAA (ATPase associated with various cellular activities) ATPase family and is an essential protein, conserved throughout evolution from archaea to mammals. p97 may be thought of as a motor protein that binds to adaptor proteins and transfers energy from ATP binding and hydrolysis through the adaptor to substrate proteins, which tend to be ubiquitylated.– The subsequent remodelling, unfolding or degradation of substrate proteins regulates diverse processes such as Golgi reassembly, ubiquitin proteasome degradation and spindle disassembly at the end of mitosis. The mechanism by which p97 and its adaptors perform these tasks is poorly understood.
Figure 1: Ribbon representations of full-length p97 (deposited structure) and p97 ND1 complexed with p47 UBX (deposited structure). Top and side views (from Dreveny et al., 2004) are shown.
p97 is a homohexamer with subunits arranged in a ring with a pore in the centre (Peters et al., 1992, Zhang et al., 2000, Beuron et al., 2003, Huyton et al., 2003, DeLaBarre and Brunger, 2003, Rouiller et al., 2000, Figure 1). Each subunit is composed of an N-terminal domain (double ψ–β-barrel) involved in adaptor binding and two AAA domains (α/β-subunit and α-helical subunit) responsible for nucleotide binding and hydrolysis. The structure of its C-terminal extension remains elusive.
Our laboratory was the first to elucidate the hexameric arrangement of the N and D1 domains of p97 (Zhang et al., 2000). We have also determined the full-length structure of p97 by X-ray crystallography (Huyton et al., 2003) and showed nucleotide-dependent domain motions by cryo-EM (Beuron et al., 2003). Recently, we have quantitatively analyzed nucleotide binding to the two ATPase rings (Briggs et al., 2008).
Interacting Proteins
A plethora of proteins have been reported to associate with p97 (Figure 2). Much work is still required to reveal how p97 associates with different adaptors and functions in so many different activities. Our studies are aimed at understanding the mechanism and specificity of p97 and its adaptor complexes and involve using a combined structural biology approach comprising X-ray crystallography, cryo-EM and NMR. To date, we have determined the domain structure of the adaptor protein p47 by NMR (Yuan et al., 2001, Yuan et al., 2004), solved the p97 ND1–p47 UBX domain complex structure (Beuron et al., 2004) and studied its nucleotide-dependent conformational changes (Beuron et al., 2006).
Figure 2: Classification of p97 adaptor/interacting proteins according to structural (UBX) or general functional criteria (protein degradation, mitosis, membrane traffic and DNA/RNA repair). A non-exhaustive list of key interacting partners of these proteins has been added at the outer circle; where there is cross-interaction with one of the other p97 interactors the colour code has been maintained (note the frequency of ubiquitin in the scheme, adapted from Dreveny et al., 2004).
UBX domain proteins
The trimeric UBX domain-containing protein p47 associates with p97 and forms a complex involved in membrane fusion, which has been structurally characterized in our lab (Yuan et al., 2001, Yuan et al., 2004, Beuron et al., 2004). Since then, a large number of other UBX domain-containing proteins have been identified and are being studied in the lab.
Ufd1–Npl4 and endoplasmic reticulum-associated degradation
The Ufd1–Npl4 heterodimer (UN) in complex with p97 is involved in the retrotranslocation of misfolded or aggregated proteins from the endoplasmic reticulum and their subsequent transfer to the proteasome, which degrades them. Despite the crucial importance of ERAD for cell survival, few structures of the players involved are known, and it is poorly understood how they interact and work. The UN complex in particular has resisted most efforts of being characterized structurally. We are trying to address the scarcity of data with a comprehensive approach combining X-ray crystallography, cryo-EM and NMR. Read more.
Mutations in p97 associated with human diseases
A number of devastating human diseases (such as inclusion-body myopathy, Paget's disease of bone and fronto-temporal dementia) are linked to signel point mutations in p97. We are studying these mutants. In particular, we are interested in their effect on the structure and thermodynamic stability of the ATPase and on its interaction with adaptor proteins. Eventually, we want to understand how these mutations lead to disease. Read more.