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Structural basis of the interaction between the AAA ATPase p97/VCP and its adaptor protein p47

2004; Springer Nature; Volume: 23; Issue: 5 Linguagem: Inglês

10.1038/sj.emboj.7600139

1460-2075

Ingrid Dreveny, Hisao Kondo, Keiji Uchiyama, Anthony W. Shaw, Xiaodong Zhang, Paul S. Freemont,

Cellular transport and secretion

Article26 February 2004free access Structural basis of the interaction between the AAA ATPase p97/VCP and its adaptor protein p47 Ingrid Dreveny Ingrid Dreveny Centre for Structural Biology, Department of Biological Sciences, Imperial College London, South Kensington Campus, London, UK Search for more papers by this author Hisao Kondo Hisao Kondo Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Keiji Uchiyama Keiji Uchiyama Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Anthony Shaw Anthony Shaw Centre for Structural Biology, Department of Biological Sciences, Imperial College London, South Kensington Campus, London, UKPresent address: Department of Cardiological Sciences, St Georges Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK Search for more papers by this author Xiaodong Zhang Corresponding Author Xiaodong Zhang Centre for Structural Biology, Department of Biological Sciences, Imperial College London, South Kensington Campus, London, UK Search for more papers by this author Paul S Freemont Corresponding Author Paul S Freemont Centre for Structural Biology, Department of Biological Sciences, Imperial College London, South Kensington Campus, London, UK Search for more papers by this author Ingrid Dreveny Ingrid Dreveny Centre for Structural Biology, Department of Biological Sciences, Imperial College London, South Kensington Campus, London, UK Search for more papers by this author Hisao Kondo Hisao Kondo Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Keiji Uchiyama Keiji Uchiyama Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Anthony Shaw Anthony Shaw Centre for Structural Biology, Department of Biological Sciences, Imperial College London, South Kensington Campus, London, UKPresent address: Department of Cardiological Sciences, St Georges Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK Search for more papers by this author Xiaodong Zhang Corresponding Author Xiaodong Zhang Centre for Structural Biology, Department of Biological Sciences, Imperial College London, South Kensington Campus, London, UK Search for more papers by this author Paul S Freemont Corresponding Author Paul S Freemont Centre for Structural Biology, Department of Biological Sciences, Imperial College London, South Kensington Campus, London, UK Search for more papers by this author Author Information Ingrid Dreveny1, Hisao Kondo2, Keiji Uchiyama2, Anthony Shaw1, Xiaodong Zhang 1 and Paul S Freemont 1 1Centre for Structural Biology, Department of Biological Sciences, Imperial College London, South Kensington Campus, London, UK 2Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK *Corresponding authors. Centre for Structural Biology, Department of Biological Sciences, Imperial College London, South Kensington Campus, SW7 2AZ London, UK. Tel.: +44 20 75945 327; Fax: +44 20 75 94 3057; E-mail: [email protected] Centre for Structural Biology, Department of Biological Sciences, Imperial College London, South Kensington Campus, SW7 2AZ London, UK. Tel.: +44 20 75945 327; Fax: +44 20 75 94 3057; E-mail: [email protected] The EMBO Journal (2004)23:1030-1039https://doi.org/10.1038/sj.emboj.7600139 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The AAA ATPase p97/VCP is involved in many cellular events including ubiquitin-dependent processes and membrane fusion. In the latter, the p97 adaptor protein p47 is of central importance. In order to provide insight into the molecular basis of p97 adaptor binding, we have determined the crystal structure of p97 ND1 domains complexed with p47 C-terminal domain at 2.9 Å resolution. The structure reveals that the p47 ubiquitin regulatory X domain (UBX) domain interacts with the p97 N domain via a loop (S3/S4) that is highly conserved in UBX domains, but is absent in ubiquitin, which inserts into a hydrophobic pocket between the two p97 N subdomains. Deletion of this loop and point mutations in the loop significantly reduce p97 binding. This hydrophobic binding site is distinct from the predicted adaptor-binding site for the p97/VCP homologue N-ethylmaleimide sensitive factor (NSF). Together, our data suggest that UBX domains may act as general p97/VCP/CDC48 binding modules and that adaptor binding for NSF and p97 might involve different binding sites. We also propose a classification for ubiquitin-like domains containing or lacking a longer S3/S4 loop. Introduction p97/VCP is a member of the AAA+ family (ATPases associated with various cellular activities), comprising enzymes that are involved in a wide range of different cellular processes including proteolysis, DNA repair and membrane fusion (for reviews, see Ogura and Wilkinson, 2001; Lupas and Martin, 2002). One possible common feature of AAA+ function is a folding or unfolding step usually catalysed in an ATP-dependent manner. The ATPase domain is characterised by a conserved sequence of 200–250 residues that includes the Walker A and B motifs. Classical AAA proteins contain an additional 'second region of homology' or SRH. AAA+ proteins can be further subdivided into type II containing two ATPase domains, referred to as D1 and D2 (for example, p97, N-ethylmaleimide sensitive factor (NSF), ClpA, ClpB, ClpC) or type I (e.g. ClpX, HslU) containing only one ATPase domain, termed D2. Additional less conserved domains are often found at the N-terminus (N domain) or the C-terminus and are often implicated in adaptor binding (for a review, see Dougan et al, 2002). Over the last few years, significant insight into the structures of AAA+ proteins has been obtained both by cryo-electron microscopy (cryo-EM) and crystallography (for a review, see Ogura and Wilkinson, 2001). Recently determined crystal structures include FtsH (Krzywda et al, 2002; Niwa et al, 2002) and ClpA (Guo et al, 2002b). However, much less is known about the nature of the interaction between AAA+ proteins and their adaptor molecules. To date, the only structure of such a complex is that of the ClpA N domain and its adaptor protein ClpS (Guo et al, 2002a; Zeth et al, 2002), although both structures are unrelated to p97 N domain and p47 C, respectively. The hexameric mammalian p97/VCP interacts with various adaptor proteins, which are proposed to function in different cellular pathways (Kondo et al, 1997; Meyer et al, 2000; Hetzer et al, 2001). The adaptor complex of UFD1 and NPL4 is believed to be crucial for p97-mediated ubiquitin-dependent processes (Meyer et al, 2000; and for a review, see Bays and Hampton, 2002). For p97-mediated membrane fusion required for organelle biogenesis, the p47 and VCIP135 (valosin-containing protein [VCP][p97]/p47 complex-interacting protein, p135) proteins have been shown to be essential (Uchiyama et al, 2002). In addition, p97 also interacts with a large number of seemingly unrelated proteins, including SVIP (small VCP/p97-interacting protein) (Nagahama et al, 2003), clathrin (Pleasure et al, 1993), the breast/ovarian cancer susceptibility gene product, BRCA1 (Zhang et al, 2000a), a DNA unwinding factor (Yamada et al, 2000) and the ubiquitination factor UFD2 (Kaneko et al, 2003). The yeast homologue of p97, CDC48, also binds ubiquitin (Dai and Li, 2001; Rape et al, 2001) and can distinguish between native and non-native proteins in the absence of cofactors (Thoms, 2002). No adaptor proteins have been described for the archaeal homologue VAT, which has been suggested to act as a chaperone utilising its N domains for substrate binding (Golbik et al, 1999). The p97 membrane fusion adaptor p47 is a conserved 370 residue eukaryotic protein that forms a tight complex with p97 (Kondo et al, 1997) and mediates the binding of p97 to its t-SNARE (soluble NSF attachment protein receptor) syntaxin5 (Rabouille et al, 1998). VCIP135 binds to the p97/p47/syntaxin5 complex dissociating it via p97-catalysed ATP hydrolysis, possibly preparing SNAREs for another round of membrane fusion (Uchiyama et al, 2002). The AAA protein NSF is essential for heterotypic membrane fusion by interacting with α-SNAP (soluble NSF attachment protein) during the process. Interestingly, α-SNAP competes with p47 for syntaxin5 binding (Rabouille et al, 1998); however, while p47 forms a stable complex with p97 inhibiting its ATPase activity (Meyer et al, 1998), α-SNAP has to recruit syntaxin5 first before it can bind to NSF, thereby stimulating its ATPase activity (for a review, see Whiteheart et al, 2001). p47 contains two binding sites for p97 with the C-terminal region sufficient for tight p97 binding, while p97 N domain is essential for p47 binding (Uchiyama et al, 2002). The C-terminal domain of p47 (282–370) adopts a ubiquitin-like β-grasp fold (Yuan et al, 2001), generally referred to as a ubiquitin regulatory X domain (UBX) domain (Hofmann and Bucher, 1996; Buchberger et al, 2001). UBX domains are suggested to be involved in ubiquitin-related processes (Buchberger et al, 2001; Buchberger, 2002), but as yet no common function has been described. In order to characterise the structural basis of one p97–adaptor complex, we have carried out a crystallographic analysis of hexameric p97 ND1 domains and the C-terminal region of p47 comprising the UBX domain (residues 244–370; p47 C). Our 2.9 Å structure represents the first structure of a UBX–protein complex, as well as AAA protein in its natural oligomeric state bound to an adaptor protein. Additional mutagenesis studies based on the structure reveal that a highly conserved loop specific to UBX domains mediates the p97 interaction. This interaction could represent a general mode of UBX domain recognition by p97/VCP/CDC48 or p97-like N domains. Results Overall structure The structure of p97 ND1 in complex with p47 C was determined from crystals of space group P65, grown in low salt conditions. The structure was solved by the molecular replacement method employing ND1 as a search model. Within the asymmetric unit, the crystals contain an ND1 hexamer and two p47 C molecules bound to adjacent ND1 protomers. In our density maps, we also observe ill-defined density for a third p47 C molecule bound to another p97 protomer. The first 17–22 residues of the p97 N domain are not visible in our density maps. A ribbon diagram of the complex structure is shown in Figure 1A and a representative sample of electron density in Figure 1D. In the crystal packing, a plane of hexamers interlock in a 'cog-like' manner, with the p47 C molecules serving as 'bridges' between the different hexamers (Figure 1B). The p47 C-bound hexamers also spiral around the molecular axis, to obey the P65 symmetry. The p47 C fragment interacts with the N domain of one ND1 protomer and is positioned slightly below the plane of the hexameric ring (Figure 1A), pointing towards the D2 domain of full-length p97 (Huyton et al, 2003). The same p47 C molecule also makes contacts with an adjacent hexamer in the crystal lattice (Figure 1C). In the next packing layer below the plane, there are no major contacts. Figure 1.Overview of the p97 ND1–p47 C structure. (A) Ribbon representation of the p97 ND1 (blue)–p47 C (red/orange) crystal structure (top and side views). The radius of the complex structure is indicated. The disconnected secondary structure at the bottom (and right) of the p97 hexamer corresponds to parts of a third p47 C molecule we observe in the density maps. (B) Crystal packing arrangement of the p97 ND1–p47 C complex in one plane as viewed down the crystallographic 65 screw axis. (C) Stereo close-up view of one p47 C molecule (in red/gold) in the crystal lattice. The N domains from two ND1 hexamers (blue) are labelled. The ND1 secondary structure elements from the two hexamers that form the p47 C interface are depicted in light blue ('binary' complex of p47 UBX with p97 Nn and p97 Nc; additional lattice contact between the N-terminal extended chain of p47 C with the p97' hexamer). p47 C secondary structure elements that interact with ND1 are depicted in gold. (D) Electron density (2Fo-Fc map contoured at 1.2σ) of the p47 C S3/S4 loop region. Download figure Download PowerPoint In contrast to the crystal structure of ND1 (Zhang et al, 2000b), the six protomers of the ND1 hexamer in the complex structure are crystallographically independent copies. However, a comparison between both ND1 crystal structures shows no major domain movements, although both structures display totally different packing arrangements. The rmsd for different protomers superimposed on the P622 ND1 crystal structure ranges from 0.5 to 0.7 Å. Interestingly, the hydrogen-bonding interactions between the N domain and D1 are maintained, indicating that there is a preferred binding mode between the two domains. Differences between protomers in the complex are mainly observed in surface side-chain conformations and in the loop region 427–437, which is flexible and has poor-quality electron density. Overall, the Cα atoms can be superimposed with an rmsd ranging from 0.45 to 0.62 Å. In the active site of D1, bound ADP is observed, further confirming the preference of ND1 for ADP (Zhang et al, 2000b). The two copies of p47 C observed in the complex superimpose with an rmsd of ∼0.9 Å, although there are slight differences at the end of β-strand 2 (S2). Both make nearly identical contacts with p97 ND1 covering a buried surface area of ∼1700 Å2. A comparison of p47 C bound to p97 ND1 with the solution structure of p47 UBX (Figure 2A, Table II) shows differences in the N-terminal part, which may be due to the shorter fragment used for the solution structure determination (282–370). The loop between β-strands 1 and 2 (S1 and S2) is shorter and in consequence results in a different hydrogen interaction pairing between the two strands. The loop region between strands 4 and 5 (S4 and S5) is flexible in solution and residues 349–352 are also not well defined in the crystal structure. Interestingly, residues N-terminal of S1 are ordered in the crystal complex, forming an additional helix (H1) that folds back onto the UBX domain between S2 and the main helix H2 of the UBX domain (Figure 2A). Pro294 facilitates this loop formation and there are hydrophobic contacts (Ala283 and Phe324) and a salt bridge (Glu280-Arg307) that stabilise the orientation of H1 with respect to the UBX core. At the N-terminal end of H1, Pro273 allows the extended chain to adopt a sharp bend, although no side-chain density can be observed for residues in this region, which in the final model are poly-Ala. The first ∼10 residues of the p47 C sequence as well as the His-tag are disordered and could not be observed. In summary, the secondary structure organisation for p47 C in complex with ND1 is αββαββαβ with an additional 310-type helix (labelled H3 in Figure 2A). Figure 2.Comparison of p47 UBX with other UBX domains/ubiquitin and p97 N domain with NSF. (A) Superpositions of p47 C (red) with p47 UBX solution structure (grey, pdb-code 1JRU), FAF1 UBX (blue, pdb-code 1H8C) and ubiquitin (yellow, pdb-code 1UBI). Note the shorter turn between S3 and S4 for ubiquitin and the additional helix H1 in p47 C. (B) Top view of a superposition of p97 ND1 and NSF N domain. The p97 N domain has 9% sequence identity with NSF (1.9 Å rmsd over 117 residues). The p97 N domain is coloured blue, p97 D1 grey and NSF N domain gold. Residues at the p97 N–p47 C complex interface are shown in ball-and-stick representation (red). Residues implicated in α-SNAP binding by NSF N are coloured magenta and are located on the opposite side of the p47-binding site. Download figure Download PowerPoint Table 1. Data collection and refinement statistics Data collection statistics Cell parameters 157.7 Å; 157.7 Å; 243.2 Å; 90°; 90°; 120° Space group P65 Wavelength 0.9394 Å Resolution max. 2.9 Å Completeness (last shell) 96.3% (99.3%) Rmerge (last shell) 9.6% (58.0%) Redundancy 6.9 〈I/σI〉 10.5 (3.1) Refinement Resolution range 40–2.9 Å No. of reflections 72860 (6732 in test set) R-factor 24.8% Rfree 29.5% No. of residues 2871 No. of ADP 6 Solvent 63 Ramachandran plot Most favoured regions 81.7% Add. allowed regions 16.8% Disallowed regions 0.2% Rmerge=∑hkl∑i∣I(hkl)i−〈I(hkl)〉∣/∑hkl∑iI(hkl)i. R-factor=∑hkl∣Fo(hkl)−Fc(hkl)∣/∑hkl∣Fo(hkl)∣, where Fo and Fc are observed and calculated structure factors, respectively. Table 2. Superpositions of p47 C with ubiquitin and ubiquitin-like domains Structure aligned with p47 C (PDB code) No. of aligned residues at 3.5 Å distance cutoff (identical residues) rmsd (Å) p47 residue range aligned (identical residues) Residue range of structure aligned p47 (282–370) sol (1JRU) 52 (39)a 2.1 297–369 295–369 (319–369)a FAF1 UBX (1H8C) 68 (12) 1.8 295–370 5–81 UBI (1UBI) 65 (12) 1.5 296–370 1–72 GATE-16 (1EO6) 62 (4) 1.8 294–369 27–111 a Differences in the N-terminal part due to a shorter loop between β-strands 1 and 2 in the crystal structure. Alignment of all residues gives rise to an rmsd of 2.7 Å. Interactions between p47 C and p97 ND1 In our crystal structure, we observe a complex between the UBX domain of p47 C and a p97 N domain within the p97 hexamer. Additional interactions are mainly observed between the N-terminal extended chain of p47 C (modelled as poly-Ala) and an adjacent p97 hexamer (Figure 1C), which forms a lattice contact. The main β-sheet of the p47 UBX domain contacts the N domain of p97, resulting in a buried surface area of ∼1700 Å2, which is typical for a protein–protein interaction surface (Lo Conte et al, 1999). The most striking feature of the interaction is the insertion of a loop from p47 C (S3/S4 loop; residues 342–345) into a hydrophobic pocket formed between the two p97 N subdomains (p97 Nn and p97 Nc; see Figures 1C and 3). The aromatic ring of Phe343 inserts into the N-domain cleft and makes contacts with a hydrophobic patch comprising residues from the first ψ-loop (Asp35CG, Ser37CB, Val38) and Ile70 (Figure 3B). Pro344 allows for the sharp turn observed in the S3/S4 loop. In addition, the side chain of Asn345 forms a hydrogen bond with the main-chain carbonyl of residue Glu141 in the N domain. A number of other hydrophobic interactions between p47 C and ND1 are observed, which may contribute to the overall stability of the complex, including p47 C residues Leu308, Met340, Ala363, Val364 and Val366, and p97 N residues Phe52, Arg53, Gly54, Pro106, Tyr110 and Tyr143. Hydrogen-bonding interactions are also clearly seen between p47 C Arg301NH2 and p97 N Val108O, as well as p47 C Asn345ND2 and p97 N Glu141O (Figure 3B). p47 homologues from yeast to humans typically share 14–30% sequence identity. The majority of residues at the interface are conserved, although for Drosophila and yeast there are a number of exceptions (see Figure 4A). However, Phe343 and Pro344 within the S3/S4 loop are absolutely conserved, as is Arg301. The extended chain N-terminal of H1 is modelled as poly-Ala and forms a lattice contact. Part of this interaction consists of a short intermolecular β-sheet with a p97 Nc domain from a different hexamer in the plane of the crystal packing (p97 Nc′ in Figure 1C). Figure 3.Binding of p47 C to p97 N domain. (A) Electrostatic surface representation of p97 N domain interacting with p47 C (ribbon coloured red). Key residues of p47 C at the N-domain interface are shown in ball-and-stick representation. D1 is depicted in blue. (B) Detailed view of specific interactions at the p47 C–p97 N interface. p47 C is shown as a ribbon representation (red) with key residues in ball-and-stick, p97 N is depicted in blue. Hydrogen-bonding interactions (p47 C Arg301NH2 and p97 N Val108O as well as p47 C Asn345ND2 and p97 N Glu141O) are indicated by a dotted line. Key residues conserved within UBX domains are labelled in red. Download figure Download PowerPoint Figure 4.Sequence alignments showing p97–p47 interacting residues. (A) Sequence alignment of p47 homologues (rat p47: accession code tr∣O35987∣, human p47: accession code tr∣Q9UNZ2∣, Drosophila EYC: accession code tr∣Q9U9C9∣ and yeast SHP1: accession code sp∣P34223∣) starting at residue 245 of rat p47. (B) Structure-based sequence alignment of p47 and FAF1 UBX domains and ubiquitin. Residues located at the interface with p97 N in the 'binary' complex are marked with a red arrow. Secondary structure elements of p47 C are shown above the alignment. Conserved residues are highlighted in grey, 'h' stands for hydrophobic (yellow), 'b' for basic and 'a' for acidic residues (blue). Download figure Download PowerPoint Comparison with other UBX domain-containing proteins FAF1, a protein implicated in apoptosis, also contains a C-terminal UBX domain and represents the only other known UBX domain structure (Buchberger et al, 2001). Superposition of the p47 and FAF1 UBX domains reveals a close structural similarity (Figure 2A; Table II). Very few residues are conserved between both domains, as illustrated by a structure-based sequence alignment (Figure 4B), with the majority of these residues likely to contribute to the stability of the fold (Ile300, Gly306, Phe324, Leu339, Pro344, Leu355 and Leu360). Strikingly, residues that mediate the interaction between p97 N and p47 C are conserved in FAF1, namely Arg301 and Phe343 and two other hydrophobic residues (Leu308, Val366). The S3/S4 loop, although similar in sequence (Figure 4B), shows small differences in conformation compared to p47 (Figure 2A), which may be due to FAF1 being unbound. Nearly all UBX domains contain an arginine or less frequently a lysine at equivalent positions to Arg301 in p47 and a hydrophobic residue followed by a proline in the putative S3/S4 loop, indicating the functional importance of these residues (Buchberger et al, 2001). It is notable that VCIP135, which binds to p97 in a mutually exclusive manner with p47, also contains a predicted UBX domain (Uchiyama et al, 2002), and the main p97-interacting residues (Arg301 and Phe343) are conserved. The S3/S4 loop is essential for binding to full-length p97 Despite a lack of sequence similarity, the UBX domain of FAF1 and ubiquitin were suggested to be evolutionary related (Buchberger et al, 2001; Buchberger, 2002). From our crystal structure, the p47 UBX domain is in fact more similar to ubiquitin (Ramage et al, 1994) than FAF1 (Buchberger et al, 2001), despite both having low respective sequence identities (see Figure 2A and Table II). This supports the notion that ubiquitin is the closest structural relative to the UBX domain known to date. In a structure-based sequence alignment (Figure 4B), the equivalent S3/S4 loop in ubiquitin is shortened by two residues, which interestingly correspond to Phe343 and Pro344. In ubiquitin, a glycine at the same position as Asn345 allows a sharp turn, which results in a shorter loop. To address the question as to whether the S3/S4 loop is important in mediating a full-length p97–p47 C interaction, we designed several mutants, including one that mimicked the shorter loop observed in ubiquitin (Thr342, Phe343, Pro344, Asn345 to Ala, Gly) and three single-point mutants of p97-interacting residues (Phe343Ser, Asn345Ala, Arg301Ala). In vitro binding studies clearly show that p47 C binding to full-length p97 is almost abolished for the loop mutant (Figure 5A) and significantly reduced for the single-point mutations (Figure 5B). Together, these data provide direct evidence for the importance of the S3/S4 loop in mediating p47 C–p97 binding. Figure 5.The S3/S4 loop is essential for binding to p97. (A) Binding studies of p47 C and mutant p47 C harbouring a shortened S3/S4 loop (Thr342, Phe343, Pro344, Asn345 to Ala, Gly) with full-length p97. The binding of p97 to the p47 C mutant (lane 4) is very much reduced compared to p47 C wild type (lane 2). (B) Effect of single-point mutations in p47 C upon p97 binding. S3/S4 loop mutants Phe343Ser (lane 6) and Asn345Ala (lane 8) as well as the hydrogen bonding interrupting mutant Arg301Ala (lane 4) show reduced binding to full-length p97 compared to wild-type p47 C (lane 2). Download figure Download PowerPoint Comparison of N domains between p97 and its homologues p97 homologues are found in species as diverse as archaebacteria and humans, whereas p47 homologues are only present in eukaryotes. The residues at the p97 N–p47 C binding interface are located around and within the cleft formed between the two p97 N subdomains Nn and Nc (Figure 2B). Residues that form part of the buried interaction surface in p97 Nn are located between strands S1 and S2 (referred to as one of the two ψ-loops), within H1, between H1 and S3, and on S4. The linker region between p97 Nn and p97 Nc is also involved as well as residues on S7, between H4 and S9, on S11 and on H5. Interestingly, all these residues are not conserved between p97, VAT and NSF. Moreover, a comparison of the p47 interaction regions in p97, VAT and NSF shows significant differences in secondary structure/and or chain length. Differences include an inserted long helix between strands S1 and S2, a lack of H1, a longer linker between the two subdomains and slightly different orientations of H4, S11 and H5 in NSF (Figure 2B). The loop between S10 and S11 in NSF is longer (16 residues) and is partly disordered in the crystal structure (May et al, 1999; Yu et al, 1999). In contrast, VAT N (27% sequence identity with p97 N, rmsd of 1.9 Å over 127 residues) has a shorter linker region between the two subdomains and the region between H4 and S9 is also shortened (Coles et al, 1999). However, the p47 contact residues are mostly conserved within eukaryotic p97 homologues, such as humans and rats (99% sequence identity), both of which are known to interact with p47, as well as the yeast homologue CDC48 (66% sequence identity). Discussion The p47 UBX domain binds to p97 N The crystal structure of the p97 ND1–p47 C complex provides the first detailed description of the structural organisation of an AAA protein hexamer in complex with its adaptor molecule, as well as the mode of interaction between a UBX domain and a target protein. The p47 UBX domain predominantly binds to a single p97 N domain ('binary complex') forming a large buried surface area, which is consistent with our previous NMR chemical shift perturbation studies on p47–p97 (Yuan et al, 2001) that implicated the p47 UBX β-sheet as forming part of the p97 N-domain interaction region. Peptide-mapping studies also show that residues from both p97 N subdomains are required for p47 (271–370) binding, which agrees with our crystal structure where residues from both subdomains contact p47 (Uchiyama et al, 2002). Furthermore, cryo-EM studies of the full-length p97–p47 complex show p47 at the periphery of the p97 hexamer (Rouiller et al, 2000; see Figure 6 and later discussion). The mainly hydrophobic nature of the p97 N–p47 UBX interface is also consistent with the observation that full-length p97–p47 complex is stable in the presence of high salt concentrations (up to 0.5 M KCl). Direct evidence, however, for the observed complex is provided by our mutagenesis studies of the p47 S3/S4 loop, which protrudes into the cleft formed between the p97 N subdomains. Mutant p47 C with a shortened loop shows significantly reduced full-length p97 binding in vitro, as do p47 C single-point mutants within the loop region. A further point mutant on β-strand S1 that disrupts a hydrogen-bonding interaction at the interface also shows reduced p97 binding in vitro. In conclusion, the binding of p47 to p97 is mediated primarily via the S3/S4 loop of the p47 UBX domain. This observed interaction is supported by mutagenesis data and in vitro full-length p97-binding studies. Figure 6.Model of p47–p97 as a physiological complex. Fitting of a model based on our crystal structure with six p47 C molecules bound to the periphery of the p97 ND1 hexamer onto a cryo-EM projection map of the full-length p97–p47 complex (figure adapted from Rouiller et al, 2000). Note the remarkable agreement in both dimensions and overall fit of the ND1–p47 C structure. An arrow indicates the position of the p47 C domain bound to the ND1 hexamer, which fits well in density attributed to p47 by Rouiller et al (2000). Download figure Download PowerPoint There is at present no evidence to support the binding of the N-terminal extended chain to p97, modelled as poly-Ala in our crystal structure. This interaction, which forms a lattice contact with a different p97 ND1 hexamer, could mimic an additional p47–p97 interaction, positioning the remaining p47 chain at the top of the hexamer. Interestingly, in the related ubiquitin-like structure, GABARAP (γ-aminobutyric acid receptor type A receptor-associated protein), a proline (Pro10) located at the N-terminal end of the equivalent H1 helix, mediates two conformations, which are likely to be biologically relevant (Coyle et al, 2002). In p47, this proline (Pro273) and/or Pro265 and three glycines (Gly255, Gly257, Gly261) could be responsible for different conformations in this region. Parts of this sequence motif are also found in UFD1 (Meyer, personal communication), a known p97-binding protein, although t