Crystal Structure of p97 N-D1 Hexamer in Complex with p47 UBX Domain

Abstract

The p97 adenosine triphosphatase is a key player in protein homeostasis, responsible for unfolding ubiquitylated substrates. It engages with various adaptor proteins through its N-terminal domain, with the p97-p47 complex attracting particular attention for its involvement in membrane remodeling. Although the structures of p97 in complex with the Ubiquitin regulatory X (UBX) domain from various adaptors have been reported, the stoichiometry is conflicting. Here, we report the crystal structure of the p97 N-D1 hexamer in complex with the p47 UBX domain at a resolution of 2.7 Å. The structure reveals a stoichiometry of 6:6 between the p97 N-D1 and the p47 UBX domain. These findings provide valuable insights into the binding stoichiometry of p97 N-D1 and p47 UBX domain, which are crucial for understanding the role of p97 and adaptor proteins in cellular processes such as the ubiquitin-proteasome pathway, membrane fusion, and cell cycle regulation.

INTRODUCTION

The p97 adenosine triphosphatase (also called valosincontaining protein) in metazoans and cell division control protein 48 (Cdc48) ortholog in Saccharomyces cerevisiae play a crucial role as a core element within the ubiquitinproteasome system (UPS). Its primary function involves the extraction and disassembly of substrates across different cellular sites, encompassing a wide array of cellular functions such as proteasomal and lysosomal degradation, membrane fusion, regulation of the cell cycle, and the process of apoptosis.¹

The p97 consists of an N-terminal domain (NTD), two tandem ATPase domains (D1 and D2), and a flexible Cterminal tail domain.² The structural studies revealed that two hexameric rings formed by the D1 and D2 domains are coaxially stacked and NTD exists in the periphery of the D1 ring.² After nucleotide hydrolysis, the NTD of the up-conformation in the ATP-bound state rotates downwards by 12.5 Å, and this conformation is referred to as the down-conformation in the adenosine diphosphate (ADP)-bound state.^2b,3 Many adaptor proteins primarily bind to p97 through the NTD, and the first identified one was NSFL1 cofactor (p47).⁴ The p97-p47 complex plays an essential role in the remodeling of cellular membranes through an interaction with the monoubiquitinated soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins.^4-5 The conformational equilibrium of the NTD regulates the interactions of p97 with various adaptor proteins.⁶ A degenerative diseases in humans such as inclusion body myopathy (IBM), frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), and Paget’s disease (PD) of bone contain point mutations in p97, which are located at the interface between the N and D1 domains.^1b,7 Most adaptor proteins bind to the NTD of p97 using conserved domain or motif including ubiquitin regulatory X domain (UBX), ubiquitin-binding domain (UBD), UBX-like domain (UBL), p97-binding motif (VBM), p97-interacting motif (VIM).⁸ Interestingly, crystallographic studies have revealed that the suppressor of high-copy PP1 (SHP) box binds to the far-most side of the Nc lobe of p97. This finding provides an explanation for the dual binding mode, involving either the UBX domain or the UBL domain, in conjunction with the SHP box.^8-9

p47 has three independent motifs including ubiquitinassociated (UBA), Saccharomyces cerevisiae Shp1, Drosophila melanogaster eyes closed gene, and vertebrate p47 (SEP), and ubiquitin regulatory X (UBX).¹⁰ The 11-residuelong Suppressor of high-copy PP1 (SHP) box between SEP and UBX of p47 has been identified as a binding motif of p97-NTD.^8-9,11 Although the oligomeric status of p47 was suggested as a trimer mediated by the SEP domain, recent biophysical studies using NMR spectroscopy and analytical ultracentrifugation revealed a monomeric p47.^5a Initial crystallographic studies showed that the p97-NTD binds to UBX domain with a stoichiometry of 1:1,^10a,12 but there are conflicted reports showing that p47 binds to p97 N-D1 hexamer in a molar ratio of either 3:6 or 6:6.^4,13 The p97 hexamer and Ufd1-Npl4 (UN) complex, a representative adaptor protein of p97, forms a complex with a stoichiometry of 1:6, which extract client proteins from the cellular membranes.^11a,14 On the other hands, Fas-associated Factor 1 (FAF1) binds to p97 in a stoichiometry of 3:6.¹⁵

In this study, we determined the crystal structure of p97 N-D1 hexamer in complex with p47-UBX domain at a resolution of 2.7 Å, providing valuable insights into the spatial arrangement and interactions between these key components. Our investigation yielded compelling evidence that underscores the pivotal role of the p47 SHP box in modulating the binding stoichiometry of the p97-p47 complex. Additionally, through a comparative analysis of the p97-p47 complex structures, incorporating different lengths of the p47 protein, we explored structural variations and dynamics within the complex. These findings pave the way for future structural studies of the physiological complex using full-length p97 and full-length p47, allowing for a more comprehensive understanding of the functional implications and regulatory mechanisms underlying the p97-p47 interaction.

EXPERIMENTAL

Cloning and Protein Purification

The human p97-N/D1 domain (residues 21–458) was expressed and purified as described previously.¹⁶ The gene for human p47-UBX domain (residues 287–370) amplified by PCR from its cDNA (KRIBB, Republic of Korea; clone ID: BKU007063) was cloned into a pET-26b (+) vector using two restriction endonucleases of NdeI and XhoI. The constructed expression vector is designed to produce the UBX domain of human p47 with a hexahistidine tag at C-terminus. The human p47-UBX domain was purified as the same procedures for p97-N/D1 domain.¹⁶ Individual protein was concentrated to 1 mM by centrifugal ultrafiltration (Amicon Ultra, Millipore, USA).

Crystallization and X-ray Data Collection

The protein solution of a complex composed of p97-N/ D1 domain and p47-UBX domain was prepared by mixing at a molar ratio of 1:1. Initial crystallization screening was performed using the sitting drop vapor diffusion method at 295 K with crystallization reagent kits supplied by Hampton Research (USA) and Qiagen (Germany), respectively. The best crystal of the complex was grown with the hanging drop vapor diffusion method by mixing 2.0 µl of protein solution with an equal amount of reservoir solution containing 14% (w/v) PEG 3350, 0.2M ammonium citrate tribasic (pH 7.0), and 0.01M hexamine cobalt (III) chloride and equilibration against 0.5 ml of the reservoir solution. For data collection, crystals were soaked for a few seconds in a 10 µl droplet of a cryoprotective solution, which had the composition of the reservoir solution with an additional 30% (v/v) glycerol, and directly flash-cooled in liquid nitrogen before data collection. X-ray diffraction data were collected at 100 K using an ADSC Quantum 270 CCD detector on beamline BL-17A at the Photon Factory (PF) in Japan. X-ray diffraction data were indexed, integrated, and scaled using the Collaborative Computational Project Number 4 (CCP4) suite.¹⁷

Structure Determination and Refinement

The initial model of the heterodimer comprising p97-N/ D1 domain and p47-UBX domain was determined by molecular replacement with Phaser in the CCP4 suite using p97 N/D1 hexamer (RCSB Protein Data Bank ID: 1S3S) as a search model.^10a,17 Subsequent manual model rebuilding and refinement were performed iteratively using Coot and Buster of the CCP4 Cloud.¹⁸ The refinement statistics are presented in Table S1. Coordinates and structure factors have been deposited in the Protein Data Bank under accession code 8HRZ and are publicly available as of the date of publication. Figures were prepared using PyMol (PyMOL Molecular Graphics System, Version 2.5.0 Schrödinger, LLC).

RESULTS AND DISCUSSION

Overall Structure of p97 N-D1 Hexamer in Complex With p47 UBX Domain

The p97 is composed of an NTD, two hexamers of ATPase domains (D1 and D2), and a flexible C-terminal tail domain (Fig. 1). On the other hands, p47 possesses three domains, including UBA, SEP, SHP box, and UBX (Fig. 1).

Figure 1. Schematic representation of p97 and p47 structure organization. Black dashed lines indicate the range studied in crystallography. The green line with a double arrow represents the interaction between p97-NTD and p47-UBX, while the magenta dashed line signifies the interaction between p97-Nc and p47- SHP box.

The formation of the p97-p47 complex is known to occur through the interaction of p97 with SHP box and UBX domains. However, there has been controversy regarding the binding stoichiometry of this complex.^4,13 Since the UBX domain binds to the p97 NTD at a 1:1 ratio, it is likely to interact with the p97 N-D1 hexamer in the same ratio. However, a previous crystal structure revealed the p97 ND1 hexamer in complex with three UBX domains, which was unexpected.^10a Recent cryogenic-electron microscopy (cryo-EM) studies have suggested that full-length p97 can engage with six p47 molecules in the presence of ADP.¹⁹ However, the electron density maps for the six p47-UBX were not clear due to the resolution limit (5.9 Å) and the instability of p47 in the cryo-EM studies.¹⁹ Furthermore, structural studies of the p97-p47 complex using NMR and biophysical methods have revealed that a monomeric p47 binds to the p97 hexamer, and the conformation of the complex is dependent on the presence of ATP or ADP.^5a To address the conflicting views regarding the p97-p47 complex, we designed a construct expressing the p47 UBX domain alone including the residues I287 to T370, without the SHP box, as a second binding motif to p97. The crystal structure of the p97 N-D1 hexamer in complex with p47 UBX domain was determined at a resolution of 2.7 Å. The structure has been refined to 21.2% for R_work and 25.5% for R_free (Table S1). In the asymmetric unit of a monoclinic crystal system, two p97 N-D1 hexamers along with twelve p47 UBX domains were modeled. A composite omit maps for p47 UBX domain, covering the entire p47 model, revealing that six p47 UBX domains bound to the p97-NTD (Fig. 2). This highlights the presence of six p47 UBX domains, in contrast to the findings of cryo-EM studies.¹⁹ The p97 D1 domain containing an ADP molecule, adopt down-conformation of NTD (Fig. 2). Once structure refinement reached convergence, 2Fo-Fc electron density maps were generated covering the entire model of the p47 UBX domain (Fig. S1). Thus, our crystal structure clearly demonstrated that p47 UBX domain was complex with the p97 N-D1 in a stoichiometry of 6:6.

Figure 2. Overall structure of the p97-p47 complex. (a) Side view of the complex. The color codes are as follow: p47 UBX, orange; p97 NTD, purple; p97 D1, blue. The ribbon diagrams are overlaid with omit Fo-Fc electron density maps for UBX domains contoured at 3.0 times root-mean-square deviation (green). The omit Fo-Fc electron density maps were calculated by Polder Maps in the Phenix suite.²⁰ (b) Top view of the complex, highlighting the spatial arrangement of p47 UBX and p97 N-D1 hexamer.

Influence of p47 SHP Box on Binding Stoichiometry

The crystal structure obtained in our study reveals the presence of two heterododecamers in the asymmetric unit, consisting of two p97 N-D1 hexamers complexed with twelve p47 UBX domains (Fig. S2(a)). Additionally, crystallographic dimers of p47 UBX domains were observed at the periphery of the p97 hexamer, exhibiting D2 symmetry (Fig. S2(b)). Expanding the crystal structure beyond the asymmetric unit revealed highly packed dimers of the heterododecamers. Furthermore, employing crystallographic symmetry operations allowed the generation of a crystallographic dimer of dimer composed of p47 UBX domains at the edge of the p97 hexamer (Fig. S2). As a result, the lattice interactions within the crystal structure were contributed by four p47 UBX domains.

To understand the position of the p47 SHP box within the complex, we superimposed our structure with the crystal structure of the p97 NTD complexed with Ufd1 SHP box (PDB ID: 5B6C) (Fig. 3).⁹ Previous crystal structure revealed that the sequences of the SHP box exhibited the characteristic features of hxxFxGxGxxh, where h represents a hydrophobic amino acid and x denotes any amino acid.⁹ Specifically, the residue range of the p47 SHP box spans from F250 to L260. However, due to intrinsic flexibility, the region between the UBX domain and the SHP box (from F250 to S286) was excluded to ensure structural stability and to provide a clearer understanding of the stoichiometry between the p47 UBX domain and the p97 N-D1 hexamer. Assuming the presence of the SHP box in p47, the crystallographic contacts between p47 UBX domains will be disrupted (Fig. 3 and S2). Further details Consequently, our crystal structure accurately represents a 1:1 stoichiometry between the p97 N-D1 hexamer and the p47 UBX domain. These findings contribute to our understanding of the influence of the p47 SHP box on the binding stoichiometry of the p97-p47 complex, shedding light on the structural dynamics and functional implications of this important interactions. Additionally, our structure supports the previously reported crystal structures of p97-NTD complexed with the UBX domain at 1:1 ratio.^10a,12

Figure 3. Crystal packing arrangement of the complex. Comparing to Figure S2, Figure 3 contains a single p97 N-D1 hexamer. (a) Top view of the complex. The ribbon diagrams of the crystallographic pairs were shown in transparently. The color codes were the same as those in Figure 2. (b) Closed-up view of the lattice interaction between two p97-p47 complexes. The crystal structure of p97 NTD in complex with the Ufd1 SHP box (PDB ID: 5B6C) was superimposed with the p97 NTD from the p97-p47 complex, and the ribbon diagram of Ufd1 SHP box was represented using a magenta-colored ribbon.

Comparative Analysis of p97-p47 Complex Structures

The previous crystal structure of the p97-p47 complex (PDB ID: 1S3S) consists of the p97 N-D1 hexamer in complex with three p47 UBX domains.^10a It is worth noting that one of the p47 UBX domains in this structure only contains a partial structure, potentially limiting the comprehensive understanding of the complex.^10a To identify the differences between the previous structure (PDB ID: 1S3S) and the crystal structure obtained in our study, we superimposed our structure onto the prior one (Fig. 4). Interestingly, our analysis revealed significant distinctions between the two structures. Consistent with the findings reported in their publication, the SHP box from the complete p47 model was observed to interact with the p97 NTD in the crystallographic pair, despite the SHP box model being represented as poly-Ala (Fig. 4).^10a This interaction highlights the importance of the SHP box in the formation of the p97-p47 complex. In summary, the superimposition and comparison of our crystal structure with the previous crystal structure (PDB ID: 1S3S) offer valuable insights into the differences and the stoichiometry related to the SHP box and the UBX domain. These findings enhance our understanding of the structural characteristics and potential functional implications of the p97-p47 complex.

Figure 4. Crystal packing arrangement of the p97-p47 complex (PDB ID: 1S3S). (a) Top view of the complex. The ribbon diagrams of the crystallographic pairs were shown in transparently. Three p47 UBX domains were represented as green-colored ribbon diagrams. 6:6 model of p97-p47 complex in our study was superimposed to their structure and p47 model was represented as orange-colored ribbon diagrams. The color codes were the same as those in Figure 2. (b,c) Closed-up view of the lattice interactions between the p97-p47 complexes in the previous structure (PDB ID: 1S3S).

CONCLUSION

In conclusion, this study elucidated the overall structure of the p97 N-D1 hexamer in complex with the p47 UBX domain, confirming a binding stoichiometry of 1:1. The crystal structure revealed the presence of two heterododecamers in the asymmetric unit, consisting of two p97 N-D1 hexamers complexed with twelve p47 UBX domains. This provides insights into lattice interactions within the crystal structure. Our structure also emphasized the significance of the p47 SHP box in the formation and dynamics of the p97-p47 complex. Comparative analysis with the previous crystal structure (PDB ID: 1S3S) highlights significant differences, emphasizing the importance of a comprehensive structure for a thorough understanding. The interaction of the SHP box with the p97-NTD in the prior structure further supports its role in complex formation. These findings enhance our understanding of the structural characteristics and functional implications of the p97-p47 complex, paving the way for future investigations into its cellular functions and potential therapeutic interventions. The intrinsically disordered motif of the SHP box may regulate the number of the ubiquitinated proteins by enhancing the interaction between p97-NTD and p47 SHP-UBX, consequently influencing the binding stoichiometry between them. Furthermore, our structure provides evidence that the UBX domains interact with the p97 N-D1 hexamer at a 6:6 ratio. Given that UBX domain proteins constitute the largest family of p97 cofactors, determining the exact stoichiometry is crucial for understanding the regulation and recognition of p97. Additional structural studies, including those involving the extended p47 region beyond the UBX domain, will help elucidate the physiological interactions and dynamics between p97 and p47.