p97 N-terminal domain
Created by Preeya Fozdar
The p97 N-terminal domain (pdb id: 3QQ7) is a structurally essential component of the AAA (ATPases associated with various cellular activities) ATPase p97 protein as it is the main binding site for the over 40 possible cofactors1. The p97 protein is a vital component of the protein degradation and membrane fusion pathways which are necessary for proper cellular function. The mechanisms are dependent upon different proteins or protein complexes that target p97 to a specific function. There are two main protein classes that bind to the p97 N-terminal domain, the ubuiquitin regulatory X domain proteins such as p47, and the ubiquitin like domain proteins such as the heterodimer Npl4-Ufd1. The breakdown of these interactions can lead to many conditions and diseases. Focusing specifically on the p97 N-terminal domain, which is responsible for the complementary interactions, it is possible to understand the process and significance of protein degradation.
The N-terminal domain of AAA ATPase p97 is a short sequence of 186 residues with a molecular weight of 21208.85 Daltons. The p97 N-terminal domain consists of two subdomains linked by six amino acids, an n-terminal with a double phi beta barrel and a c-terminal with antiparallel beta sheets. The cleft between the two subdomains is negatively charged and has many hydrophobic residues which interact with ligand residues to create a tight binding pocket. The hydrophobic interactions of Val38, Phe52, Ile70, Leu72, Tyr110, and Tyr143 are most significant although there is also hydrogen bonding observed with Val108 and Arg53. The initial interaction begins often when a highly conserved phenylalanine residue with a nonpolar benzyl group slides into the p97 N-terminal cleft2. These residues and interactions are the basis of many proteins which associate with p97 and target it towards a specific function. The N-terminal domain is crucial for targeting, however, for function, p97 is bound to two ATPase domains, D1 and D2, to form the p97 monomer. Six monomers come together to form a hexameric protein. The beta barrel of the N-terminal domain and the alpha helix of the D1 domain are the main site of interaction. The highly conserved glycines in the linker between the two allows for movement because the two D domains need flexibility as they perform their ATPase function. This interaction with the two D domains only occupies 8% of the solvent accessible surface area of the N-terminal domain, much of the rest is used to bind cofactors3. The structure of the N-terminal domain facilitates cofactor binding, which mediates targeting, and ATPase function, which supplies energy, so the whole protein complex can carry out the function of removing misfolded proteins or mediate membrane fusion.
The p97 N-terminal domain has several homologs across species with related functions. One main homolog is the mouse homolog, murine p97 (PDB ID: 1R7R), which contains the same p97 N-terminal domain. The E-value for homology is 2.11x10-129. The primary structure is the same for both4. The tertiary structure differs very slightly but important features are consistent. The subdomains are similar and the ligand binding sites are there in both although there are less ligands in mice. The function of both is also the same. Mouse p97 N-terminal domain has the same complex with p47 and will facilitate membrane fusion. There is also a clear protein degradation pathway involving many p97 protein complexes in the ER. There are homologous proteins in Xenopus laevis, Saccharomyces cerevisiae, and, interestingly, Thermoplasma acidophilum, VAT (PDB ID: 1CZ4). Archaea are not known to need membrane fusion, therefore the functionality is different for these proteins despite somewhat conserved primary and tertiary structure5. The tertiary structure contains a similar beta barrel and the protein has been implicated in protein folding and unfolding, which are the early stages of the p97 mediated degradation process seen in mammals6.
The largest class of p97 cofactors is ubquitin regulatory x domain (UBX) containing proteins including FAF1. FAF1 is involved in cell apoptosis, ubiquitylation, and degradation of proteins which is also mediated by p97. FAF1, and other UBX proteins, contain a highly conserved R…FPR amino acid sequence that binds to the p97 n-terminal domain at the previously mentioned cleft. The p97-FAF1UBX interaction is mostly polar with 46% polar residues, 29% charged residues, and 25% nonpolar residues. The UBX protein’s Leu616, Phe619, and Phe645 residues are involved in hydrophobic interactions while the Arg621 residue is involved in hydrogen bonding. Most of these residues lie on the S3/S4 loop of the UBX protein which is allowed to turn due to conserved proline residues3. P47 is another UBX protein that has significant structural similarity to FAF1 despite having few conserved residues. The conserved residues, Arg301 and Phe343 specifically, are critical to p97 N-terminal domain binding.
The ubiquitin like domain (UBD) containing protein Npl4 in conjuction with Ufd1 is an important cofactor for p97 in the ERAD pathway. The binding at the p97 N-terminal domain is very similar to that of UBX containing proteins but with a few important differences. The charged nonpolar cleft still bonds to the S3/S4 loop however this loop is embellished with a 310 helix which contains the residues that directly contact the p97 N-terminal domain. The Val-15, Leu-74, and Phe-76 of the Npl4-Ufd1 bind to the Lys-109, Gly-54, and Tyr-143 of the N-terminal domain. It is because of these residues on the 310 helix that Npl4 can bind to p97 despite its short S3/S4 loop. Ubuiquitin has a similar loop without the 310 helix and has very low affinity binding to the p97 N-terminal domain. The Ufd1 part of the dimer also binds to the p97 N-terminal domain in the same manner as p47. However, the Npl4-Ufd1 complex binds in a 1:1 ratio with the p97 N-terminal domain, while p47 binds in a trimer to p97. Importantly, since both classes of proteins bind to the cleft of the N-terminal domain, there is an inherent exclusivity and p97 will only be targeted to one function at a time7. Overall, the structure of the p97 N-terminal domain is important for cellular function as improper cofactor binding or loss of ATPase activity from the other p97 domains can lead to misfolded protein build up and improper membrane fusion.
The role of p97 ATPase, specifically the capacity of the p97 N-terminal domain to bind to different proteins creating unique complexes, is necessary for proper function and the lack of this protein can lead to a variety of diseases. Defects in p97 have been implicated in many neurodegenerative diseases which are often the result of protein aggregates. Huntington’s and Parkinson’s have both been linked to p97 due to its role in protein degradation. It has even been linked to cancer, however the mechanism of these disease models is still unclear. The processes that p97 ATPase mediates are important for cellular function.