VCP at 3.6 Å

Journal of

Structural Biology Journal of Structural Biology 144 (2003) 337–348 www.elsevier.com/locate/yjsbi

 The crystal structure of murine p97/VCP at 3.6 A Trevor Huyton,a,1,2 Valerie E. Pye,a,2 Louise C. Briggs,a Terence C. Flynn,b Fabienne Beuron,a Hisao Kondo,c Jianpeng Ma,b,d,e Xiaodong Zhang,a and Paul S. Freemonta,* a

e

Centre for Structural Biology, Department of Biological Sciences, Imperial College London, South Kensington SW7 2AZ, UK b Department of Bioengineering, Rice University, 6100 Main Street, Houston, TX 77005, USA c Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge CB2 2XY, UK d Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, BCM-125, Houston, TX 77030, USA Graduate Program of Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, TX 77030, USA Received 15 August 2003, and in revised form 1 October 2003

Abstract p97/VCP is a member of the AAA ATPase family and has roles in both membrane fusion and ubiquitin dependent protein  crystal structure of murine p97 in which D2 domain has been modelled as poly-alanine and degradation. Here, we present a 3.6 A the remaining
100 residues are absent. The resulting structure illustrates a head-to-tail packing arrangement of the two p97 AAA domains in a natural hexameric state with D1 ADP bound and D2 nucleotide free. The head-to-tail packing arrangement observed in this structure is in contrast to our previously predicted tail-to-tail packing model. The linker between the D1 and D2 domains is partially disordered, suggesting a flexible nature. Normal mode analysis of the crystal structure suggests anti-correlated motions and distinct conformational states of the two AAA domains. Ó 2003 Published by Elsevier Inc. Keywords: AAA; p97/VCP/CDC48; ATPase; Crystal structure; Mechanism; Normal mode analysis

1. Introduction p97 belongs to the AAA family of ATPases (ATPases associated with diverse cellular activities) occurring in eubacteria, archaebacteria, and eukaryotes (Confalonieri and Duguet, 1995). The AAA proteins are characterised by the presence of one (type I) or two (type II) conserved AAA domains of around 230–250 residues (typically termed D1 and D2), which contain the Walker A (P-loop) and B (DExx box) motifs. The presence of an additional region of high sequence conservation, termed the second region of homology (SRH), distinguishes AAA ATPases from other Walker type ATPases (Neu* Corresponding author. Fax: +44-20-7594-3057. E-mail address: [email protected] (P.S. Freemont). 1 Present address: Structural Biology, Walter and Eliza Hall Research Institute, Melbourne, 3050 Victoria, Australia. 2 These authors contributed equally to this work.

1047-8477/$ - see front matter Ó 2003 Published by Elsevier Inc. doi:10.1016/j.jsb.2003.10.007

wald et al., 1999). Members of the AAA ATPase family are involved in many cellular processes with AAA modules often being linked to other domains that promote their diverse cellular functions including membrane fusion (p97 and NSF), organelle biosynthesis (PAS1p), motor proteins (TorsinA), DNA helicases (RuvB), proteolysis (Lon, Clp, and FtsH), and transcriptional regulation (SUG1 and TBP1) (Neuwald et al., 1999; Ogura and Wilkinson, 2001). Mammalian p97 (first termed VCP, valosin-containing protein) was originally described as a precursor protein containing a biologically active peptide valosin (Koller and Brownstein, 1987). Subsequently, in Xenopus laevis, an oligomeric ATPase with a sedimentation coefficient of 14.5S was identified as the homologue of mammalian VCP (Peters et al., 1992; Peters et al., 1990). Highly conserved p97 homologues have also been identified in a diverse range of organisms, including Saccharomyces cerevisiae (CDC48) (Frohlich et al., 1991;

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Moir et al., 1982) and Thermoplasma acidophilum (VAT) (Pamnani et al., 1997). The presence of archaeal homologues of p97 is consistent with an involvement in processes other than membrane fusion, since there is no evidence that membrane fusion events occur in archaeal cells apart from cytokinesis. p97 is one of the most abundant mammalian cytosolic proteins comprising as much as 1% of the cell cytosol. The p47 and Ufd1–Npl4 adaptors compete for p97 and direct its activity into Golgi membrane fusion or ubiquitindependent protein degradation (Meyer et al., 2000); in membrane fusion, p97 is proposed to mediate homotypic fusion process which reconstitutes endoplasmic reticulum and Golgi apparatus membranes after mitosis (Acharya et al., 1995; Latterich et al., 1995), whilst the highly homologous NSF controls fusion of various heterotypic membranes, including vesicle-mediated transport and neurotransmission (Acharya et al., 1995; Misura et al., 2000; Rabouille et al., 1995). The selectivity of the various membrane fusion processes catalysed by p97 and NSF is presumably due to the requirement of additional specific cofactors. p97 mediated membrane fusion events requires p47 for interactions with the Golgi t-SNARE (targetsoluble NSF attachment protein receptor) syntaxin 5 (Kondo et al., 1997; Rabouille et al., 1998). In an analogous manner, NSF mediated membrane fusion requires SNAP soluble NSF attachment proteins) such as a-SNAP for the interaction and disassembly of vesicletarget SNARE complexes. The membrane fusion pathway catalysed by p97 and NSF, are however distinctly different. The NSF/SNAP/SNARE complex can be readily dissociated by ATP hydrolysis (Sollner et al., 1993), unlike the p97/p47/SNARE complex. Recently the novel protein VCIP135 (valosin-containing protein [VCP] p97/p47 complex-interacting protein, p135) has been identified. VCIP135 binds to the p97/p47/syntaxin5 complex and promotes its dissociation via p97 catalysed ATP hydrolysis (Uchiyama et al., 2002). Biochemically, p97 also mediates ubiquitin conjugate association through its cofactors such as p47, or the Ufd1–Npl4 complex (Meyer et al., 2002). Surprisingly both p47 and Ufd1–Npl4 bind ubiquitin and thereby link p97 to ubiquitin tagged substrates. In the case of p47, mono-ubiquitin is bound preferentially over poly-ubiquitin. Conversely Ufd1–Npl4 binds both poly-ubiquinated and mono-ubiquinated substrates. Furthermore, p97 copurifies with IjBa and the 26S proteasome (Dai et al., 1998), and is involved in the degradation of many other ubiquitin–proteasome degradation pathway substrates including short lived cyclin molecules (Dai and Li, 2001). The specificity therefore of p97 function could be mediated by differential ubiquitin conjugation. Electron microscopy has been prominent in structural studies of p97 and related AAA proteins NSF, CDC48, and VAT, showing that they form stacked hexameric ring complexes (Fleming et al., 1998; Frohlich et al.,

1995; Hanson et al., 1997; Peters et al., 1990; Rockel et al., 1999; Rouiller et al., 2000; Zhang et al., 2000). More recently three-dimensional cryo-EM reconstruc resolution have provided more tions of p97 at 20–25 A accurate representations of the flexibility and conformational changes that occur upon nucleotide binding and hydrolysis (Beuron et al., 2003; Rouiller et al., 2002). Normal mode analyses have also been used to investigate the intrinsic flexibility of the various domains of p97 and their correlated motions (Beuron et al., 2003). The inherent flexibility required to transmit conformational changes throughout these types of molecules has meant that despite the extensive structural studies of NSF and p97, the only crystal structure of a Ôfull-lengthÕ duplicate AAA domain containing protein to be determined is the Escherichia coli ClpA in an inactive conformational state (Guo et al., 2002). Here we  describe the crystal structure of murine p97 at 3.6 A resolution, which represents the first crystal structure of a duplicate AAA protein in its biological hexameric state. We discuss the mechanistic implications of our model in terms of p97 function.

2. Materials and methods 2.1. Cloning of recombinant p97 Full-length murine p97 (residues 1–806) was amplified by PCR using gene specific primers containing unique NdeI and NotI restriction endonuclease sites. The PCR product was then cloned using these sites into pET22b (Novagen) to generate a construct (p97#60) in frame with a C-terminal hexahistidine tag. 2.2. Expression and purification of recombinant p97 Rosetta (DE3) cells (Novagen) were transformed with p97#60 and cells were grown at 37 °C to a mid log phase (OD600 nm
0.5–0.6) before induction of protein expression by the addition of 1 mM IPTG. Cells were grown for a further 3 h before harvesting. Cell pellets were resuspended in buffer A (25 mM Hepes, 500 mM KCl, 2 mM bMe, pH 8.0) containing 0.1 mM PMSF and 100 lg/ml of leupeptin, pepstatin, aprotinin, antipain, and benzamidine. The cells were lysed by sonication and clarified by centrifugation for 30 min at 20 000g 4 °C, before loading onto a 5 ml Hi-Trap Chelating column (Amersham–Pharmacia) precharged with nickel sulphate and equilibrated with buffer A. The column was washed extensively with buffer A containing 50 and 100 mM imidazole before elution of recombinant p97 over a linear gradient to 250 mM imidazole. Fractions containing p97 were dialysed into gel filtration buffer (25 mm Hepes, 250 mM KCl, 2 mM bMe, and 1 mM MgCl2 ) before loading onto a superdex 200 16/60 gel

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filtration column (Amersham–Pharmacia) where p97 eluted corresponding to a hexamer of MW
600 kDa. The purity of p97 at this stage was
95% as judged from 10% SDS–PAGE gels and contained some low level contaminants which could not be removed by further purification steps. 2.3. Crystallisation, data collection and processing Purified recombinant p97 was concentrated to 7 mg/ ml in 25 mM Hepes, 250 mM KCl, 2 mM bMe, and 1 mM MgCl2 before addition of 10 mM AMP–PNP or 2 mM 20 -BrAMP-PNP (Jena Biosciences). The ‘‘fulllength’’ recombinant p97 was crystallised by the hanging drop vapour diffusion method. Protein was mixed with an equal volume of reservoir solution containing 1.0– 1.4 M ammonium phosphate (monobasic), 1–5% PEG 400, 2 mM bMe and 0.1 M sodium citrate, pH 5.6. Crystals appeared after 5–7 days and grew to maximum dimensions of
50  50  75 lm over a period of 2–3 weeks at 21 °C. Data were collected at beamlines ID29 and ID14-4 of the ESRF. Crystals were soaked sequentially in mother liquor containing gradual increments of ethylene glycol up to a final concentration of 30%, before being mounted in a nylon loop and flash frozen in liquid nitrogen. Diffraction data show anisotropic tendencies mainly in the c-direction. All diffraction data were collected at )173 °C and processed using the HKL suite of programs (Otwinowski and Minor, 1997). Data collection and final refinement statistics are given in Table 1. Crystals belong to hexagonal space, group P622 with unit-cell dimensions a ¼ 146 A , c ¼ 166 A , a ¼ 90°, b ¼ 90°, and c ¼ 120° b ¼ 146 A and one molecule in the asymmetric unit.

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2.4. Structure solution and refinement The position of ND1 domains was obtained by molecular replacement with AMoRe (Navaza, 1994)  processed in spacegroup using data from 25 to 3.6 A P622 and ND1 coordinates (1E32). Rigid body refinement was carried out using CNS (Brunger et al., 1998), the partial model of ND1 was used to calculate model phases, the 2Fo-Fc map clearly indicated the location of the central b-sheet of D2 (a=b sub-domain, Fig. 1A). Using the D1 a=b sub-domain as a model, only two possible arrangements could fit the observed density; a direct translation of D1 along the hexamer axis or the same translation followed by a 180° flip along the bsheet. However the direct translation model, when positioned into the density with appropriate adjustment for secondary structure, allowed additional densities to be observed which correspond to the a-helical subdomain of D2. A translation model of D2 followed by a 180° flip, however did not allow the observation of any additional density corresponding to the a-helical sub-domain and was deemed incorrect. Therefore the direct translation model was used for subsequent rebuilding and refinement. Although data were collected from crystals of p97 in the presence of 20 -BrAMP-PNP close to the Br-edge, no corresponding peaks were observed in fo-fc anomalous density maps and hence no experimental phases were obtained from the dataset. However, this dataset was of higher quality than previous native datasets and was subsequently used for refinement. Each rebuilding was followed by rigid body refinement followed by group B-factor refinement using data from . Refinement and rebuilding were carried out 20 to 3.6 A

Table 1 Data collection and refinement statistics Crystal

Refinement

Wavelength Resolution (highest shell) Spacegroup

 0.9202 A  (3.66–3.6 A ) 25–3.6 A P622

Unit cell dimensions

Measured reflections

 a ¼ 145:3 A  b ¼ 145:3 A  c ¼ 165:5 A 133 872

Unique reflections Completeness (highest shell)

12 461 99.8% (100%)

Resolution (highest shell) Reflections (work/free) Rwork (%)b (highest shell) Rfree (%) (highest shell) ) r.m.s.d. bond lengths (A

 (3.8–3.6 A ) 25–3.6 A 11046/1266 32.9 (34.1) 35.8 (36.8) 0.01

r.m.s.d. bond angles (°) Number of protein atoms Number of non-protein atoms 2 ) Average B factors (A Ramachandran plot % core, % allowed, % generous, % dissallowed

1.6 4677 27 104.4 80.3, 17.4, 1.8, 0.5

Overall Rmerge a (highest shell) 8.5% (23.8%) Overall I=r (highest shell) 17.4 (5.3) P P P P a Rmerge ¼ hkl i jIhkl  hIhkl ij= hkl i jIhkl;i j, where Ihkl;i is the intensity of an individual measurement of the reflection with indices hkl and hIhkl i is the Pmean intensity of that reflection. P b R ¼ h jjFobs ðhÞj  jFcalc ðhÞjj= h jFobs ðhÞj Rwork and Rfree were calculated from the working and test reflection sets, respectively.

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Fig. 1. Electron density maps of full-length p97 before and after crystallographic refinement. All maps are contoured at 0.8r level unless otherwise noted. (A) Experimental map calculated with N–D1 molecular replacement phases showing the location of the D2 a=b sub-domain. (B) 2Fo-Fc maps of b sheet within D2 showing the variation in quality of the final maps. (C) Refined 2Fo-Fc map of the D1 P-loop and bound ADP (left, 1.2r) compared to the equivalent region of D2 where no extra density is observed for bound nucleotide (right). (D) 2Fo–Fc maps of the linker regions between N and D1 (left) and D1 and D2 (right).

Fig. 2. Overall structure of the p97. (A) Ribbon representation of the p97 protomer showing the Ôhead-to-tailÕ packing arrangement of D1 and D2. Colour coding are as follows: N–yellow, D1 a=b-blue, D1 a-cyan, N–D1 linker–magenta, D2 a=b–red, D2 a–orange, D1–D2 linker, mauve. ADP bound to D1 is shown in white surface. (B) Ribbon representations (top and side views) of the p97 hexamer with each protomer coloured differently, wider ring is the N–D1. (C) Molecular surface representations of the p97 hexamer showing the overall shape and dimensions of the hexamer body (D) Docking of the p97 hexamer into cryo-EM density maps of endogenous p97 in the presence of AMP-PNP. Note that the current p97 model consists of residues 17–700 with poly-alanine for residues 461–700. The remaining D2 and C-terminal regions were not visible in our density maps. Top: top view with the model showing as Ca trace. Bottom: side view with the model as molecular surface and the cryo-EM reconstructions as grey transparent volume. Short arrows indicate the N domains. All the figures were produced using PREPI ([email protected]).

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Fig. 3. Comparison of p97 D1 and D2 and with ClpA. (A) Superposition of p97 D1 and D2 as ribbon representations showing the overall similarity between domains and the different linker position. D1 is coloured blue while D2 is coloured red. The N–D1 linker is coloured magenta while the D1– D2 linker is coloured yellow. Note that the N–D1 linker is located closer to the P-loop. (B) superposition of p97 D1, ClpA D1, and ClpA D2 showing the similar a-helical sub-domain orientation and the linker position (especially the conserved hydrophobic residues which are shown as magenta sticks). (C) Superposition of p97 protomer with ClpA protomer on their respective D1 domains showing a significantly different position of D2. (D) A ClpA D2 hexamer was constructed based on p97 D1, which results in a looser D1 packing. Interestingly, this hypothetical hexamer fit well into the endogenous p97 ‘‘ADP’’ EM map which we interpreted as D2–ADP, D1–empty and N domains flexible.

Fig. 4. (A): Ellipsoidal representation of anisotropic atomic displacement tensor calculated for p97 hexamer. Side view of the complete hexamer (N, crimson; D1, green; and D2, orchid). Each ellipsoid represents one Ca residue of the protein backbone. Size and orientation of the ellipsoids indicate magnitude and direction, respectively of linear combination of Ca atomic fluctuations for lowest frequency normal modes (first 203 modes). An arbitrary temperature was used to calculate the magnitudes of the displacements. Figure was made using the RASTEP functionality (Burnett and Johnson, 1996; Trueblood et al., 1996; Merritt, 1999) of Raster3D (Bacon and Anderson, 1988; Merritt and Bacon, 1997). (B) Anti-correlated motions between D1 and D2 domains as revealed in mode 9 of ANM calculation of the truncated hexamer. Orientation of the hexamer is identical to (A), note that the N domains were not used in the calculation. Arrows indicate the direction of corresponding motions of D1 and D2 domains—as D1 moves inwards towards the D1 pore, D2 moves outwards opening the D2 pore; or vice versa. Figure was made using MOLSCRIPT (Kraulis, 1991) and rendered by Povray (www.povray.org).

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using CNS (Brunger et al., 1998) and XtalView (McRee, 1999) in space group P622, with one p97 monomer per asymmetric unit. The D2 part of the model appears highly disordered, as reflected by the poor density and high 2 ). The final model consists of residues B-factor (>100 A 17–464, and a poly-alanine model for residues 465–700 (excluding residues 610–615 and 665–668). Additional density in the middle of the molecule between D1 and D2 and in the outer rim of the hexamer could be observed but were not interpretable. The final model has a Rfree of 2 , with 35.8% (Rcryst ¼ 32:9%), average B factor of 104.4 A reasonable geometry for a partial poly-alanine model at this resolution (see Table 1; r.m.s.d. bond length , r.m.s.d. bond angle ¼ 1.6°, 97.8% of residues ¼ 0.01 A have dihedral angles in the most favoured or additional allowed region, with 0.5% in the disallowed region). We acknowledge that the phasing technique employed in this structure determination has its limitations, and that the less well-defined density for the D2 domain could be ascribed to either poor phasing or the inherent flexibility of D2. However rigid body refinement of the recently solved p97-ADP-AlFx structure (DeLaBarre and Brunger,  data also showed large differences in 2003) using our 3.6 A 2 ) and D2 (170.1 A 2 ) the B-factors between ND1 (74.2 A (data not shown). We conclude therefore, that the poor quality electron density and higher B-factors for D2 is due to the inherent flexibility of D2 in our p97 crystals and not a lack of phasing. Coordinates have been deposited with the RCSB structural database (http://www.rcsb.org) under accession code 1R7R. 2.5. EM docking Fitting the crystal hexamer structure into EM reconstruction densities was performed using Situs v2.1 (Chacon and Wriggers, 2002). The previously published cryo-EM density maps of endogenous p97 in ‘‘ADP’’ and ‘‘AMP–PNP’’ bound states (Beuron et al., 2003; Zhang et al., 2000) were converted to Situs format using CONFORMAT and normalised using HISTOVOX, retaining the number and order of the x,y,z increments and the grid spacing. The hexameric p97 structure was used as a rigid-body to search against the cryo-EM maps using CoLoRes (Contour-based Low Resolution docking) and a 20° angular sampling of the rotational search . For the p97 ‘‘AMP–PNP’’ map a Situs unnorat 24 A malised correlation coefficient of 0.046 and a good visual fit was produced for the one solution. However for the p97 ‘‘ADP’’ map a Situs unnormalised correlation coefficient of 0.029 and a poor visual fit was yielded. 2.6. Normal mode analysis The magnitude and directionality of the positional fluctuation of a given atom i in a protein structure can

be described by an anisotropic thermal ellipsoid determined by the atomic displacement tensor U i (GrosseKunstleve and Adams, 2002; Trueblood et al., 1996),


hDxi Dxi i hDxi Dy i i hDxi Dzi i


U i ¼

hDy i Dxi i hDy i Dy i i hDy i Dzi i

:
hDzi Dxi i hDzi Dy i i hDzi Dzi i
The matrix elements of U i can be expressed in terms of normal modes (1 to 3N )6) in the form of hDrki Drpi i ¼ a

3N 6 X

aikm aipm =km

ðk; p ¼ x; y; zÞ;

m¼1

here h  i stands for ensemble average, N is the number of atoms, a is a parameter related to temperature that affects the magnitude of displacement, km is the eigenvalue of the mth mode (the force constant), and aikm is the corresponding Cartesian component for the ith atom in the eigenvector of the mth mode (Brooks III et al., 1988). In X-ray crystallography, the atomic displacement tensor is directly related to the Debye–Waller temperature factor by a relation of   1 T i T i hexpðiq Dr Þi ¼ exp  q U q ; 2 here q is a reciprocal lattice vector and Dri is the atomic displacement vector (Grosse-Kunstleve and Adams, 2002). When there are enough independent X-ray diffractions, the anisotropic atomic displacement tensors can be determined experimentally; however, for the resolution of the system we are dealing with, we are only able to determine the tensors theoretically in terms of normal modes. By examining the distribution of the principal axes of the thermal ellipsoids, one can not only determine the relative mobility of different regions of protein structure such as functional domains, but also the overall directionality of the motions of that region relative to the other parts of the molecule.

3. Results 3.1. Overall p97 structure Our crystal structure of murine p97 comprises residues 17–700 and consists of: complete models for the N terminal substrate binding domain and the D1 AAA domain, and a poly-alanine partial model for the D2 AAA domain, in which the a-helical and C-terminal regions are highly disordered (see representative densities in Fig. 1). This partial model however, still contains valuable information in terms of intra- and inter-domain arrangements and linkage between the D1 and D2 domains. Overall, the p97 hexamer is Ômushroom shapedÕ , and height
80 A , and a central with diameter
160 A  (Fig. 2). The hexamer consists of two hole of
10 A

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ring-like layers with the N–D1 domains forming the wider layer and the D2 domains forming the narrower layer (Fig. 2). The N–D1 hexamer, in terms of height and diameter, is identical to that of our previously published N–D1 crystal structure (1E32). The D2 layer is similar in . The D2 height to D1, with a diameter of
120 A monomer adapts a typical AAA domain structure containing an a=b sub-domain followed by an a-helical subdomain. In our p97 structure, we observe density in the active sites of D1 protomers, which is consistent with the presence of ADP, same as in our ND1 structure (Fig. 1C left). In contrast, we do not observe similar densities in the D2 protomers (Fig. 1C right) although our crystallisation conditions contain 5 mM AMP–PNP. We therefore interpret D2 protomers as being nucleotide free or Ôempty.Õ It is plausible that the conformation of the Ndomain and linkers could inhibit nucleotide binding, or alternatively, the crystallisation environment, including 1.0–1.4 M Phosphate and low overall pH (4.5), could affect nucleotide binding to D2. 3.2. D1–D2 head-to-tail packing In our p97 crystal structure, the D1 and D2 domains form a head-to-tail packing arrangement contrary to our previous predictions based on a low-resolution cryo-EM reconstruction (Zhang et al., 2000). In our current p97 model, the position of D2 corresponds to a D1 protomer  and along translated along the hexamer axis by
34 A  followed by a the radius of the hexamer plane by
3 A small rotation (Fig. 2). The density for the D1–D2 linker region (residues 459–481, Fig. 1D right) is weaker than that for the N–D1 linker (Fig. 1D left) consistent with it being significantly more flexible. However we were able  to model the D1–D2 linker region, which travels >48 A from the bottom of D1 all the way to the bottom of D2, before going into the first a-helix of the D2 a=b sub-domain, and is largely exposed, consistent with proteolytic results (our unpublished results). The interface between the D1 (atomic models) and D2 protomers 2 buried surface (poly-alanine model) is small (
460 A area) with few apparent interactions. The linker region (residues 459–481) contributes to interactions with both D1 and D2. The observed head-to-tail packing is also in agreement with that observed in the ClpA structure (Guo et al., 2002 and discussion below). While the manuscript was in review, another structure of p97/VCP was published (DeLaBarre and Brun), that ger, 2003) albeit to a lower resolution (4.7 A exhibits the same relative domain orientations. This structure was solved using a combination of molecular replacement and selenomethionine MAD phasing and has nucleotide bound in both of the AAA domains, ADP in D1 and ADP-AlFx in D2. Overall the two  for 617 aligned Ca structures are similar (r.m.s.d. 2.67 A atoms), although differences in the relative positions of

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the D2 domains with respect to the hexamer axis are observed. These differences could be due to the different packing arrangements in the crystal and/or the different nucleotide states of D2 and/or the flexible nature of D2. 3.3. Protomer–protomer interactions and D1–D2 comparison In the N–D1 hexamer ring (Fig. 2B, bottom, upper ring), a large protomer–protomer interface (total buried 2 ) is observed. This interface is parsurface of 5816 A tially mediated through the N domain, as the D1 pro2 . tomer–protomer interface alone is only 3782 A Furthermore, the nucleotide ADP is bound in between 2 to the adjacent proprotomers and contributes 246 A tomer interface. Within the D2 domain, the interface is 2 (based on our poly-alsignificantly reduced to 1887 A anine model) though this could be due to the incomplete model of D2. Nevertheless, this interface is still somewhat looser, mainly due to the loss of N domain and/or nucleotide. This is reflected by the poor electron density and high B-factors we observe, and indicates the intrinsic flexible nature of the D2 domain and hexamer ring in this conformation. This is also reflected by a low number of contacts between the D1 and D2 hexamers. Superposition of the D1 and D2 domain by their respective a=b sub-domains show a reasonable fit (r.m.s.d.  on 212 aligned Ca atoms). However the position of 1.4 A the a-helical sub-domain in both protomers is slightly different (Fig. 3A), with that in the D2 domain located further away from the a=b sub-domain than in D1. This results in a less compact AAA domain which is perhaps not surprising, given that nucleotide binds between the a=b sub-domain and the a-helical sub-domain. In D1 we observe bound ADP, whereas in D2 we observe no bound nucleotide (Fig. 1C). More strikingly, the linker between D1 and D2 is positioned further away from the D2 P-loop when compared to the linker between N and D1 (Fig. 3A, yellow vs. mauve). 3.4. Comparison with ClpA structure The only other available high-resolution structure of type II AAA proteins is the bacterial protease ClpA (Guo et al., 2002). Comparison with the ClpA structure (1KSF) reveals that the D1 domains of p97 and ClpA  for 86 can be superposed reasonably well (r.m.s.d. 1.9 A aligned Ca atoms). The positions of the N domains of the p97 with respect to the AAA domains are analogous with the positions of the N-domains in the ClpA structure, as illustrated in Fig. 3. The N domains are on the periphery of the D1 AAA domains and have a hexagonal star-like arrangement. It is likely that the N domains protrude from the double AAA core in order to be more accessible to adaptor proteins such as p47, Ufd1–Npl4, ClpS, and SspB.

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However, the relative position of D2 is dramatically  apart from each other differently, more than 40 A (Fig. 3C). In the p97 hexamer, D2 is almost a direct translation of D1 along the hexamer axis, while in ClpA, when D1 is superposed with p97 D1, the D2 protomers clash at the hexamer axis, preventing a proper hexameric packing. It is notable that in the ClpA structure, both D1 and D2 have bound ADP and the protomers form a hexameric spiral instead of a ring structure. The ClpA D2 a=b sub-domain aligns reasonably well with p97 D2, though the a-helical sub-domain is significantly different in terms of secondary structure (in ClpA, this domain contains a b-sheet). Overall, the relative orientation of the a-helical sub-domain and a=b sub-domain (p97 D1, ClpA D1, and ClpA D2) are similar, all of which are ADP bound (Fig. 3B). Like p97, the ClpA N– D1 linker interacts with bound nucleotide (Leu188 and the adenine ring in ClpA; Val206 and adenine ring in p97), similarly the linker between D1–D2 domains also interact with ADP in ClpA (Val460 interacts with the adenine ring). Interestingly, the P-loops between ClpA D1, ClpA D2, and p97 D1 (all ADP bound) align ex r.m.s.d. Ca respectively). tremely well (0.27 and 0.39 A And the distance between equivalent hydrophobic residues in the linker region (Val206 in p97, Leu188 and Val460 in ClpA) and the conserved P-loop Threonine is , almost identical in all three AAA domains (
10.5 A Fig. 3B). In contrast, the linker region between p97 D1– D2 domain is further away from the P-loop. 3.5. Fitting into EM density The p97 hexamer was rigid-body fitted into the endogenous p97 ‘‘AMP–PNP’’ cryo-EM density (Beuron et al., 2003) using the program Situs v2.1 (Chacon and Wriggers, 2002). The p97 hexamer and the EM reconstruction suitably concur, as illustrated in Fig. 2D except the N domains. The N domains in the crystal structure would have to be rotated upward to fit into the EM density (see arrows in Fig. 2D). This could reflect the multiple conformations N domains could adapt and/or the different p97 conformational states the crystal structure and the EM reconstruction represent. Not surprisingly, the hexamer structure does not fit well into the p97 ‘‘ADP’’ cryo-EM density (Zhang et al., 2000) (data not shown) as the two EM reconstructions show distinct conformations. In a recent cryo-electron microscopy study, Rouiller and colleagues (2002) determined the three-dimensional structures of full length recombinant p97 as well as DN p97 mutants purified in different nucleotide states at
. Our studies have been carried out on endogenous 24 A rat liver p97 and the nucleotide (ADP or AMP–PNP) added prior to sample preparation for cryo-electron microscopy (Beuron et al., 2003). Both studies however have led to similar conclusions, namely that the D1 and

D2 rings rotate with respect to each other and that this is accompanied by concomitant changes in their overall diameter and central pore size. The flexibility of N domains are also revealed by both studies depending on the nucleotide state considered. However there are significant differences between the reconstructions and their subsequent interpretation. Our EM maps exhibit Ôside holesÕ whereas those from Rouiller and colleagues show significant side protrusions. Our AMP–PNP map also reveals more densities for N domains than Rouiller et al. (2002), which results in a very different aspect ratio for our reconstruction (compare Fig. 2E, Beuron et al., 2003; to Fig. 2B in Rouiller et al., 2002). Overall our AMP–PNP map appears more similar in shape and dimensions to the ADP-AlFx from Rouiller et al. A reconstruction of recombinant p97 incubated with 5 mM AMP–PNP prior to the observation by cryo-EM is in general agreement with the AMP–PNP map of the endogenous protein (our unpublished results) showing that the side holes are not specific to the endogenous protein but probably depend on the actual nucleotide state captured in the different reconstructions. A plausible explanation of the observed differences between the different p97 reconstructions could be in the reconstruction method. It is notable that the euler assignment in all the nucleotide states by Rouiller and colleagues (2002) involved the use of the same starting model consisting of two stacked NSF D2 rings. In our work the first three-dimensional models were computed for each dataset independently using an angular reconstitution approach (Van Heel, 1987) which limits possible model bias from the reconstructions. Another major difference between the reconstructions of Rouiller et al and our own lies in the interpretation of the resulting EM maps. In our reconstructions, we assign the D1 domain to be positioned in the wider part of the maps based in particular on antibody labelling (Beuron et al., 2003). Rouiller and colleagues (2002) however provide a surprising interpretation of their DN p97 mutant and propose the opposite D1–D2 assignment, namely D1 is positioned in the narrower domain. The cryo-EM reconstruction of endogenous p97 in complex with the adaptor protein p47 bound to the N domain of the hexameric ATPase further validate our interpretation (our unpublished results) as does the recent cryo-EM reconstruction of NSF (Furst et al., 2003). 3.6. Normal mode analysis The normal modes of the refined p97 crystal structure were calculated by an anisotropic network model  and a (ANM), which employed a cut-off distance of 13 A force constant of 1.0 (Atilgan et al., 2001; Beuron et al., 2003; Tirion, 1996). The low-frequency modes (the first 203 modes) were used to compute the anisotropic atomic displacement tensor for each Ca atom and to

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subsequently construct the thermal ellipsoid. The results reveal that, overall, the most mobile portions of p97 lie at its outer edges (the N domain, the a helical domain of D1, and the D2 domain), where most of the functionality of the protein resides (Fig. 4A). The N domain is able to move predominantly up and down about a pivot/hinge point near the connection between the N and D1 domains. Such a motion can change the relative orientation of the N domains with respect to the hexameric ring. The D2 domain appears to move primarily towards and away from the inner pore/cavity. Such a motion could potentially alter the size of the cavity. There are relatively small motions at the central pore of the hexamer, near the position of the a=b sub-domain of D1 (Fig. 4A). In our previous work (Beuron et al., 2003), we described the motions of the lowest frequency normal modes derived from cryo-EM density maps of p97 using a quantized elastic deformational model (QEDM) (Kong et al., 2003; Ming et al., 2002a; Ming et al., 2002b; Tama et al., 2002). The most interesting modes from QEDM demonstrate flexibility at the N–D1 interface, negative co-operativity between D1 and D2, and radial swelling of the D1 and D2 inner pores. The N–D1 flexibility is manifested as the N domains move as two separate sets of three (three-up and three-down), with each N domain moving the opposite direction as its two nearest neighbours. The current work verifies this hingemotion at N–D1, and in addition reveals flexion of the D2 domains. The high degree of motion exhibited by D2 in the present study indicates that D2 may play a larger role in the negative cooperative process between D1 and D2, as revealed by the previous QEDM analysis (Beuron et al., 2003), which results in axial twisting of the two rings. The anisotropic atomic displacement tensors also substantiate the radial swelling/contracting of the D2 pore indicated in the QEDM study (Beuron et al., 2003), however, much smaller motions are observed around the D1 pore. To more closely examine the nature of the motions between domains D1 and D2, ANM was performed on a truncated hexamer, completely lacking the N domains (Fig. 4B). Mode 9 revealed that D1 domain and D2 domain have the ability to move in an anti-correlated fashion. D1 domain moves inward toward the D1 pore, while D2 domain moves outward, thus expanding the D2 pore; and vice versa. Such an anti-correlated motion could lead to possible negative cooperativity between the nucleotide binding sites in the D1 and D2 domains.

4. Discussion 4.1. Structure of p97/VCP In our p97 structure, D2 is largely disordered, which 2 ), and poor is reflected by the high B factors (175 A

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quality electron density, reflecting an intrinsic flexibility of D2 rather than poor phasing information as there is good electron density for N–D1 domains, and the a=b sub-domain of D2 is clearly defined in our maps (Fig. 1A). Though the a-helical sub-domain of D2 is disordered in the 2Fo-Fc map, we could see densities corresponding to three a-helices that we were able to model. Together with the clear placement into our experimental maps of the a=b sub-domain, we unambiguously assigned the position of D2 relative to D1 as a head-to-tail packing arrangement. Due to the lack of a high-resolution model for D2 and the poor quality electron density, we modelled D2 as poly-alanine with residue assignments based on both predicted secondary structure information and a D2 homology model using D1 as a template (38% sequence identity). There is additional unconnected density seen on the periphery of the D2 ring, in close proximity to the N-domain, which could be the highly flexible and disordered C-terminal regions (
60–80 residues). In the D1 hexamer, ADP is bound in a deep pocket between the a=b and a-helical sub-domains, as well as between individual D1 protomers similar to our previous crystal structure for N–D1 (Zhang et al., 2000). Furthermore, the N–D1 linker region interacts with the adenine ring of ADP through Val206, and N domain interacts with D1 a-helical sub-domains, all of which contribute to stabilize the D1 hexamer packing arrangement. In comparison with D1, we observe no bound nucleotide in the D2 protomers, which results in fewer contacts between the a=b sub-domain and a-helical sub-domain and between individual D2 protomers. In addition, there is little interaction between D1 and D2 domains. Combined with the flexible nature of the D1– D2 linker, it is not surprising that D2 is partially disordered in our crystals. However our D2 model does allow a comparison between a nucleotide bound and nucleotide free hexamer state. In the absence of nucleotide, the a-helical sub-domain becomes flexible, and is positioned further away from the hexamer axis. Therefore, release of nucleotide would weaken the interactions between sub-domains and result in a looser hexamer structure. 4.2. Comparison with ClpA crystal structure A comparison with the ClpA structure reveals that the linker position (between N–D1 and D1–D2) is largely determined by bound nucleotide. Although, the linker sequence varies between different AAA proteins, both in terms of amino acid composition and length, a highly conserved Gly has been proposed to act as a pivot point that delineates the start of the AAA domain (Zhang et al., 2000; and manuscript in preparation). For the D1–D2 linker, by using the conserved Gly as the end point of the linker and the end of the a-helical

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sub-domain of D1 as the beginning, the D1–D2 linker in p97 comprises
23 residues (between 459 and 481) while the equivalent in ClpA is
25 residues (436–462). In our structure, the D1–D2 linker is flexible and largely in an extended conformation while in ClpA, the linker consists of a short non-structured region followed by an ahelix. For p97 the N–D1 linker is 22 residues long and is largely extended in conformation. Unfortunately, in the ClpA structure, part of the N–D1 linker is not present in the model, although the C-terminal part of the linker (residues 182–190) adapts an extended conformation. The differing secondary structure of linker regions probably reflects differences in amino acid sequences. Nevertheless, the distance between the conserved hydrophobic linker residue and the P-loop appears independent of sequence, but is largely determined by bound nucleotide. Given the conservation of the pivotal glycine and the adenine contacting hydrophobic residue, the dependence of the linker position (and by inference, the relative orientation of sub-domains) on bound nucleotide, could represent a general property for all type II AAA proteins. The ClpA structure is unlikely to reflect an active state as both D1 and D2 are ADP bound and since the monomers form a spiral arrangement within the crystal (Guo et al., 2002). Nevertheless, since the linker position appears to be dependent upon bound nucleotide, the relative orientation of ClpA D1 and D2 could indicate the orientation of p97 D1 relative to D2 when D2 has ADP bound. Therefore, constructing a ClpA D2 hexamer (ADP bound) based on our D1 hexamer here (ADP bound) should give an indication of p97 D1–D2 linker position upon p97 D2 nucleotide binding/hydrolysis. Formation of this hexamer positions the ClpA D1 a-helical sub-domain away from the molecular axis (Fig. 3D), forming a looser D1 ring. Interestingly, this arrangement fits surprisingly well into our previously published full-length cyro-EM reconstruction of endogenous p97 in ‘‘ADP’’ form (Beuron et al., 2003; Zhang et al., 2000), which we interpreted as D2 in an ‘‘ADP’’ form, D1 in an ‘‘empty’’ form and N domains being flexible. 4.3. Normal mode analysis The theoretical anisotropic B factors calculated for the hexameric p97 structure show that the most mobile portions of p97 lie at its outer edges (the N domain, the a-helical sub-domain of D1, and the D2 domain). This is particularly interesting as the N domains in the crystal structure are well defined and in a stable conformation and would only reflect the intrinsic mobile nature of the domain. The N domains collectively show potential motions along up-and-down direction and towards the hexamer axis. This supports the hypothesis that N domains could move upwards towards the hexamer axis as indicated by our cryo-EM studies and previous normal

mode studies (Beuron et al., 2003). The D1 a=b subdomain seems to be most stable, which supports the proposal that D1 could maintain the hexamer while conformational changes occur largely through the D1 a-helical sub-domain. In contrast, D2 seems flexible, implying larger conformational changes for D2 during the ATP binding/hydrolysis cycle. 4.4. Conclusions The first type II AAA structure in a biological hexamer allows for the first time, an unambiguous assignment of the domain arrangement within this class of AAA proteins. A comparison between the D1 and D2 domains and their respective conformational states within the hexamer and comparison with ClpA provide new insights into the mechanism of p97/VCP, in particular how nucleotide binding and hydrolysis could relocate the D1–D2 linker and therefore aid the release of N domains from the stably locked positions, priming interactions with other cofactors. Furthermore, normal mode analysis of the structure and previous structural studies indicate negative co-operativity between the two AAA domains and distinct conformational outcomes of the two AAA domains.

Acknowledgments We are grateful to Tony Shaw, Pawel Dokurno, and Richard Newman for their earlier contributions, Ingrid Dreveny, and Hajime Niwa for fruitful discussions, Suhail Islam for helping with the graphics. T.H. wishes to thank E. Dodson and G. Murshadov of University of York for helpful discussions. T.H. was funded by Cancer Research UK. J.M. acknowledges financial support from the Robert A. Welch Foundation (Q-1512), the American Heart Association (AHA-TX0160107Y), the National Institute of Health (R01-GM067801), and a National Science Foundation Career Award (MCB0237796). J.M. received the Award for Distinguished Young Scholars Abroad from the National Natural Science Foundation of China. X.Z. and P.F. wish to thank the Wellcome Trust for their generous funding.

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