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VCP-binding region
Biochemical and Biophysical Research Communications 407 (2011) 531–534
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Crystal structure of human FAF1 UBX domain reveals a novel FcisP touch-turn motif in p97/VCP-binding region Wonchull Kang, Jin Kuk Yang ⇑ Department of Chemistry, College of Natural Sciences, Soongsil University, Seoul 156-743, Republic of Korea
a r t i c l e
i n f o
Article history: Received 9 March 2011 Available online 15 March 2011 Keywords: Fas-associated factor 1 UBX domain Touch-turn motif p97 b-Grasp fold
a b s t r a c t UBX domain is a general p97/VCP-binding module found in an increasing number of proteins including FAF1, p47, SAKS1 and UBXD7. FAF1, a multi-functional tumor suppressor protein, binds to the N domain of p97/VCP through its C-terminal UBX domain and thereby inhibits the proteasomal protein degradation in which p97/VCP acts as a co-chaperone. Here we report the crystal structure of human FAF1 UBX domain at 2.9 Å resolution. It reveals that the conserved FP sequence in the p97/VCP-binding region adopts a rarely observed cis-Pro touch-turn structure. We call it an FcisP touch-turn motif and suggest that it is the conserved structural element of the UBX domain. Four FAF1 UBX molecules in an asymmetric unit of the crystal show two different conformations of the FcisP touch-turn motif. The phenyl ring of F619 in the motif stacks partly over cis-Pro620 in one conformation, whereas it is swung out from cis-P620, in the other conformation, and forms hydrophobic contacts with the residues of the neighboring molecule. In addition, the entire FcisP touch-turn motif is pulled out in the second conformation by about 2 Å in comparison to the first conformation. Those conformational differences observed in the p97/VCPbinding motif caused by the interaction with neighboring molecules presumably represent the conformational change of the UBX domain on its binding to the N domain of p97/VCP. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction p97/VCP, a multi-functional ATPase of the AAA+ family, is implicated in diverse cellular processes such as post-mitotic Golgi reassembly, endoplasmic reticulum associated degradation, nuclear envelope reconstruction, cell cycle, suppression of apoptosis and DNA damage response [1]. The diverse functions of p97/VCP are mediated by the interaction with various adaptor proteins in each different biological context [2,3]. The largest group of the adaptors comprises the proteins containing the UBX domain, such as FAF1, p47, SAKS1, and UBXD7, which directly binds to N domain of p97/VCP [4–7]. The UBX domain comprises about 80 amino acid residues and is currently considered as a general p97/VCP-binding module, with a growing number of UBX proteins being identified [8]. The structures of UBX domain were determined by NMR spectroscopy on FAF1 UBX and p47 UBX, revealing that it adopts a bgrasp fold similar to ubiquitin [5,9]. The first structural glimpse on its interaction with p97/VCP was obtained from the crystal structure of p47 UBX in complex with p97/VCP ND1 fragment at 2.9 Å resolution [10]. It showed that the conserved FP sequence
Abbreviations: FAF1, Fas-associated factor 1; VCP, valosin-containing protein; IKK, IjB kinase. ⇑ Corresponding author. Fax: +82 2 824 4383. E-mail address: [email protected] (J.K. Yang). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.03.052
motif in the loop connecting strands S3 and S4 is inserted into the cleft of the p97/VCP N domain and is essential for the binding activity [10]. FAF1 was initially identified as a Fas-interacting protein in the death inducing signaling complex and was shown to potentiate the apoptotic cell death [11–13]. Later, FAF1 was shown to intervene in the NF-jB signaling at two points of the pathway. First, FAF1 binds to the IKK b subunit, thereby disrupting the IKK complex assembly and inhibiting its activation [14]. Second, FAF1 also binds to p65 subunit of NF-jB and inhibits the translocation of NFjB into the nucleus [15]. Most recently, FAF1 was revealed to inhibit the proteasome-mediated protein degradation process either by interacting with p97/VCP which may serve as a molecular chaperone presenting the ubiquitinated client proteins to the proteasome or by interacting with ubiquitinated client proteins [4]. As a consequence of its involvement in these cellular processes, FAF1 acts as a pro-apoptotic protein. Human FAF1, composed of 650 amino acid residues, contains a UBX domain at the C-terminus through which it binds to p97/VCP N domain [4,16]. In an effort to elucidate the conserved structural features of the UBX domain for its binding to p97/VCP and also to provide a structural basis of the interaction between FAF1 and p97/ VCP, we initiated X-ray crystallographic analysis on FAF1 UBX domain, and here we present its crystal structure determined at 2.9 Å resolution.
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W. Kang, J.K. Yang / Biochemical and Biophysical Research Communications 407 (2011) 531–534
2. Materials and methods 2.1. Protein preparation, crystallization and data collection The human FAF1 UBX domain (residues 571–650) was overexpressed, purified and crystallized as described previously [17]. Briefly, FAF1 UBX protein was overexpressed in fusion with Histagged thioredoxin in Escherichia coli strain Rosseta2 (DE3). The fusion part was removed by TEV protease during the purification process. The FAF1 UBX alone was concentrated to 9.7 mg/ml for its crystallization. We collected X-ray diffraction data to 2.9 Å resolution, at Photon Factory, Japan on the beamlines AR-NW12. Data collection statistics are summarized in Table 1.
2.2. Structure solution and refinement The structure of the FAF1 UBX domain was solved by the molecular replacement using Phaser in CCP4 program suite [18,19]. The search model was its previously reported NMR structure (PDB code 1H8C) [9]. The crystal of FAF1 UBX was found to be twinned with the apparent space group of F4132 and its actual space group of F23. Its twin fraction is 0.426 and the twin operator is (k, -h, l). Initial trial for solving the structure was performed for the apparent space group F4132 without noticing the twining, and two FAF1 UBX molecules were positioned successfully with TFZ score 8.0. The R-factors resisted going down from around 50% during the refinement, and so the twining was suspected. We checked its twining and prepared the de-twinned data by Detwin in CCP4 program suite [19]. Its final structure determination and refinement were successfully performed with de-twinned data in the actual space group F23 of which the asymmetric unit contains four copies of FAF1UBX molecules. Subsequent manual model rebuilding was carried out using Coot [20] interspersed with rounds of automatic refinement by Refmac5 [21] and CNS [22]. An asymmetric unit of the crystal contains four FAF1 UBX molecules and the refined model covers residues from Glu571 to Glu650. The statistics for the data collection, structure determination and refinement are summarized in Table
1. The atomic coordinates and structure factors have been deposited with a code 3QCA in the Protein Data Bank (http:// www.pdb.org). 3. Results and discussion 3.1. Overall structure of FAF1 UBX FAF1 UBX domain comprises five b-strands (S1–S5), one a-helix (H1), and three 310 helices (G1–G3) (Fig. 1A). The five strands form a mixed b-sheet in the order of 2–1–5–3–4, and the sheet rolls around the a-helix H1 to form the well-known b-grasp fold, as previously shown in the NMR structure of FAF1 UBX [9]. Notably, among the three short 310 helices G1 to G3, only G3 was recognized in the previous NMR structure as a helix H2. FAF1 UBX domain contains three Phe-Pro sequence stretches: FP608-9, FP619-20, and FP639-40. In this study, FP608-9 has a usual trans peptide, and the other two, FP619-20 and FP639-40, have cis peptides. These features are contradictory to the previously reported NMR structure of FAF1 UBX in which the peptide conformations are exactly the opposite, that is, FP608-9 in cis and both FP619-20 and FP639-
Table 1 Data collection and structure refinement statistics for human FAF1 UBX. Data collection Space group Twin faction Twin operator Unit cell parameters Resolution No. of measured reflections No. of unique reflections Rmergea Completeness hI/r(I)i Redundancy
F23 0.426 k, -h, l 175.73 Å 30.0–2.90 Å 4,14,219 10,089 6.2%(32.2%) 99.9%(100.0%) 9.8(2.2) 41.1(29.7)
Refinement Rwork/Rfreeb r.m.s.d. bond lengths r.m.s.d. bond angles Subunits in asymmetric unit No. of atoms (protein/water) Average B factors (protein/water) Ramachandran favored region Ramachandran disallowed region
24.7%/29.3% 0.011 Å 1.28° 4 2546/31 65.8 Å2/42.8 Å2 97.4% 2.6%
Values in parentheses are for the highest resolution shell, 3.06–2.90 Å. a Rmerge = RhRi|I(h)ihI(h)i|/RiRiI(h)i, where I(h) is the intensity of reflection h, Rh is the sum over all reflections, and Ri is the sum over i measurements of reflection h. b R = Rh||Fobs(h)||Fcalc(h)||/Rh|Fobs(h)|, where F(h) is the structure factor of reflection h. Rfree was calculated using 5% data excluded from refinement, and Rwork using the other 95% data.
Fig. 1. Structure FAF1 UBX. (A) Overall structure of FAF1 UBX. (B) Four FAF1 UBX molecules in an asymmetric unit. The arrow indicates the S3/S4 loop.
W. Kang, J.K. Yang / Biochemical and Biophysical Research Communications 407 (2011) 531–534
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in trans. Among these three FP sequences, the second one, FP619, is conserved throughout VCP-binding UBX domains of various UBX-containing proteins such as FAF1, p47, Ubxd7, and SAKS1 [10]. Moreover, it was shown that the S3/S4 loop containing this FP of p47, another UBX-containing p97/VCP adaptor protein, is essential for UBX binding to p97/VCP N domain [10]. With this cis-Pro620, the S3/S4 loop takes a sharp turn connecting the two b-strands, S3 and S4 (Figs. 2 and 3). This structural feature and its implication will be further discussed below. 20
3.2. Two different conformations of p97/VCP-binding region The asymmetric unit of the FAF1 UBX crystal contains four copies of FAF1 UBX domain (designated a to d) (Fig. 1B). In the crystal packing, FAF1 UBX molecules a and c are positioned in the virtually identical contact environment, and molecules b and d are located in another kind of the contact environment, which is different from that of a and c (Fig. 1B). As a result, the molecules a and c adopt an almost identical conformation, and the molecules b and d commonly show another kind of conformation which is different from that of molecules a and c. Superposition of the four FAF1 UBX molecules reveals that a conformational difference is limited to the S3/ S4 loop region. For example, the root-mean-square (r.m.s.) deviation of Ca atoms in the S3/S4 loop region (residues 617–622) is 1.41 Å between molecules a and b, but is 0.70 Å between molecules a and c. In addition to the slightly different positions of the loop, the side-chain phenyl ring of F619 shows two distinct conformations. It stacks partly over the cis-P620 in molecules a and c, whereas it is completely swung out from cis-P620 in molecules b and d (Fig. 2). We refer to these conformations as type-ac and type-bd. The conformational difference can be accounted for the fact that the S3/S4 loops in molecules b and d are involved in the interaction with other molecules in the crystal lattice but those in molecules a and c are not (Fig. 1B). The S3/S4 loop region of molecules b and d is in contact with hydrophobic residues of neighboring molecules, whereas the corresponding region of molecules a and c is not. The side chain phenyl ring of F619 on the S3/S4 loop is swung out by the hydrophobic interactions with P609, W610, and D611 of the neighboring molecule. It is reminiscent of the observation in the crystal structure of p47 UBX in complex with p97/VCP ND1 domain: the corresponding F343 of p47 UBX forms hydrophobic contacts with D35, V38, and L72 of p97/VCP N domain [10]. So it is very tempting to suggest that the type-bd and type-ac conformations of FAF1 UBX may represent its ‘‘bound’’ state and the ‘‘unbound’’ state respectively.
Fig. 3. FcisP touch-turn. (A) FcisP touch-turn, a rare case of an open b-turn, formed by the four residues TFPR (residues 618–621) from the molecule a. The dashed line represents the distance between Cai and Cai+3.
3.3. FcisP touch-turn motif of UBX domain The most functionally important feature of UBX domain is the conserved FP sequence on the S3/S4 loop which was experimentally shown to be essential for p97/VCP-binding activity [10]. In the previously reported crystal structure of p47 UBX in complex with p97/ VCP N domain, the S3/S4 loop containing FP sequence was inserted into the cleft in the p97/VCP N domain [10]. The p47 UBX mutant with a shortened S3/S4 loop without the conserved FP residues lost most of the p97/VCP-binding activity [10] and UBXD1 UBX lacking those conserved residues does not bind to p97/VCP N domain [23]. So the conserved FP sequence on S3/S4 loop was of our special interest throughout this study. We observed in this crystal structure that the peptide bond between these two residues is clearly in a cis conformation, i.e., the proline residue is a cis-Pro (Fig. 2B). The FP sequence is strictly conserved among all known p97/VCPbinding UBX domains and is known to be essential for the binding of UBX domain to p97/VCP. Therefore, it is tempting to speculate that the observed structural feature of the cis peptide should also be conserved. In the crystal structure of p47 UBX in complex with p97/VCP ND1 [10], it is in an unusual partly extended conformation, intermediate between trans and cis. The discrepancy may be due to incomplete structure refinement in that region. In the previous NMR structures of FAF1 UBX and p47 UBX, the conserved FP sequence has a trans peptide between them [5,9]. This discrepancy
Fig. 2. Structure FAF1 UBX in complex with p97/VCP N domain. (A) The structural difference in the S3/S4 loop region between the type-ac and the type-bd conformations is illustrated for molecules a and b, respectively. (B) 2Fo-Fc electron density map for FP619-20 of molecules a and b at 1.2 r level.
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may be due to the NMR structure determination procedure, which usually assumes the peptide bond as trans. A geometric analysis also supports our proposal on the conservation of FcisP structural motif. The S3/S4 loop of FcisP clearly forms a b-turn with the four residues TFPR618-621 (Fig. 3). This b-turn can be classified as type VIb turn, whose structural features are as follows. First, the type VIb turn has no hydrogen bond among the four residues consisting of the turn and is called ‘open’ [24,25]. As a consequence, the distance between Cai and Cai+3 is about 6.0 Å, which is significantly longer than the one for the ‘normal’ hydrogenbonded b-turns. Second, the (i + 2)-th residue of the type VIb turn must be cis-Pro. Otherwise, the consecutive four residues would adopt an extended loop and cannot form a b-turn at all [26]. The FcisP b-turn of FAF1 UBX displays these two features of the type VIb turn, except the distance between Cai and Cai+3 is significantly shorter (4.58–4.91 Å) (Fig. 3). Recently, an even tighter and rarely observed form of the type VIb turn was described and termed as the cis-Pro touch-turn, in which the distance between Cai and Cai+3 is typically 4–5 Å as in the case of FAF1 UBX FcisP motif [27]. This analysis indicates that the S3/S4 loop of FcisP in FAF1 UBX is actually a rarely observed cis-Pro touch-turn and the corresponding proline residue in other UBX proteins such as p47 must be also cis-Pro. Therefore, we call it an FcisP touch-turn motif and propose that it is a conserved structural element of UBX domains for binding to p97/VCP. Interestingly, Videau et al. [27] found that all the 15 examples of cis-Pro touch-turn motif collected from 500 high resolution crystal structures are commonly located in functionally important sites, that is, the catalytic sites in the case of enzymes and the binding sites in the case of nucleic acid binding proteins. The FcisP touch-turn motif in UBX domains also plays a critical role in binding to another molecule, p97/VCP. To the best of our knowledge, it is the first example of a cis-Pro touch-turn described in the protein–protein binding interface. In summary, we determined the crystal structure of FAF1 UBX domain at 2.9 Å resolution. It revealed two previously unrecognized structural features of the UBX domain in its p97/VCP-binding region. First, the S3/S4 loop containing the conserved FP sequence, which is critical for the p97/VCP binding activity, adopts a rare cisPro touch-turn structure. We suggest that this FcisP touch-turn structural motif should be the conserved structural element of the UBX domain determining its p97/VCP binding activity. Second, the two different conformations observed in the current crystal structure may represent the unbound and the bound state of the UBX domain. Interestingly, the side chain phenyl ring of F619 in the motif partly stacks over P620 in the unbound state, whereas it is completely swung out in the bound state. Acknowledgments This work was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A080930). The authors thank the staff of the Pohang Light Source beamlines 4A in Korea, and the Photon Factory beamlines AR-NW12 in Japan. References [1] N. Vij, AAA ATPase p97/VCP: cellular functions, disease and therapeutic potential, J. Cell. Mol. Med. 12 (2008) 2511–2518.
[2] I. Dreveny, V.E. Pye, F. Beuron, L.C. Briggs, R.L. Isaacson, S.J. Matthews, C. McKeown, X. Yuan, X. Zhang, P.S. Freemont, P97 and close encounters of every kind: a brief review, Biochem. Soc. Trans. 32 (2004) 715–720. [3] H.O. Yeung, P. Kloppsteck, H. Niwa, R.L. Isaacson, S. Matthews, X. Zhang, P.S. Freemont, Insights into adaptor binding to the AAA protein p97, Biochem. Soc. Trans. 36 (2008) 62–67. [4] E.J. Song, S.H. Yim, E. Kim, N.S. Kim, K.J. Lee, Human Fas-associated factor 1, interacting with ubiquitinated proteins and valosin-containing protein, is involved in the ubiquitin-proteasome pathway, Mol. Cell. Biol. 25 (2005) 2511–2524. [5] X. Yuan, A. Shaw, X. Zhang, H. Kondo, J. Lally, P.S. Freemont, S. Matthews, Solution structure and interaction surface of the C-terminal domain from p47: a major p97-cofactor involved in SNARE disassembly, J. Mol. Biol. 311 (2001) 255–263. [6] H. McNeill, A. Knebel, J.S. Arthur, A. Cuenda, P. Cohen, A novel UBA and UBX domain protein that binds polyubiquitin and VCP and is a substrate for SAPKs, Biochem. J. 384 (2004) 391–400. [7] G. Alexandru, J. Graumann, G.T. Smith, N.J. Kolawa, R. Fang, R.J. Deshaies, UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1alpha turnover, Cell 134 (2008) 804–816. [8] C. Schuberth, A. Buchberger, UBX domain proteins: major regulators of the AAA ATPase Cdc48/p97, Cell. Mol. Life Sci. 65 (2008) 2360–2371. [9] A. Buchberger, M.J. Howard, M. Proctor, M. Bycroft, The UBX domain: a widespread ubiquitin-like module, J. Mol. Biol. 307 (2001) 17–24. [10] I. Dreveny, H. Kondo, K. Uchiyama, A. Shaw, X. Zhang, P.S. Freemont, Structural basis of the interaction between the AAA ATPase p97/VCP and its adaptor protein p47, EMBO J. 23 (2004) 1030–1039. [11] K. Chu, X. Niu, L.T. Williams, A Fas-associated protein factor, FAF1, potentiates Fas-mediated apoptosis, Proc. Natl. Acad. Sci. USA 92 (1995) 11894–11898. [12] S.W. Ryu, S.K. Chae, K.J. Lee, E. Kim, Identification and characterization of human Fas associated factor 1, hFAF1, Biochem. Biophys. Res. Commun. 262 (1999) 388–394. [13] S.W. Ryu, S.J. Lee, M.Y. Park, J.I. Jun, Y.K. Jung, E. Kim, Fas-associated factor 1, FAF1, is a member of Fas death-inducing signaling complex, J. Biol. Chem. 278 (2003) 24003–24010. [14] M.Y. Park, J.H. Moon, K.S. Lee, H.I. Choi, J. Chung, H.J. Hong, E. Kim, FAF1 suppresses IkappaB kinase (IKK) activation by disrupting the IKK complex assembly, J. Biol. Chem. 282 (2007) 27572–27577. [15] M.Y. Park, H.D. Jang, S.Y. Lee, K.J. Lee, E. Kim, Fas-associated factor-1 inhibits nuclear factor-kappaB (NF-kappaB) activity by interfering with nuclear translocation of the RelA (p65) subunit of NF-kappaB, J. Biol. Chem. 279 (2004) 2544–2549. [16] C.W. Menges, D.A. Altomare, J.R. Testa, FAS-associated factor 1 (FAF1): diverse functions and implications for oncogenesis, Cell Cycle 8 (2009) 2528–2534. [17] W. Kang, H.Y. Shin, J.K. Yang, Crystallization and preliminary X-ray crystallographic analysis of human FAF1 UBX domain, Acta Cryst. F66 (2010) 211–213. [18] A.J. McCoy, R.W. Grosse-Kunstleve, P.D. Adams, M.D. Winn, L.C. Storoni, R.J. Read, Phaser crystallographic software, J. Appl. Cryst. 40 (2007) 658–674. [19] Collaborative Computational Project Number 4, The CCP4 suite: programs for protein crystallography, Acta Cryst. D50 (1994) 760–763. [20] P. Emsley, K. Cowtan, Coot: model-building tools for molecular graphics, Acta Cryst. D60 (2004) 2126–2132. [21] A.A. Vagin, R.A. Steiner, A.A. Lebedev, L. Potterton, S. McNicholas, F. Long, G.N. Murshudov, REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use, Acta Cryst. D60 (2004) 2184–2195. [22] A.T. Brunger, P.D. Adams, G.M. Clore, W.L. DeLano, P. Gros, R.W. GrosseKunstleve, J.S. Jiang, J. Kuszewski, M. Nilges, N.S. Pannu, R.J. Read, L.M. Rice, T. Simonson, G.L. Warren, Crystallography & NMR system: A new software suite for macromolecular structure determination, Acta Cryst. D54 (1998) 905–921. [23] L. Madsen, K.M. Andersen, S. Prag, T. Moos, C.A. Semple, M. Seeger, R. Hartmann-Petersen, Ubxd1 is a novel co-factor of the human p97 ATPase, Int. J. Biochem. Cell Biol. 40 (2008) 2927–2942. [24] J.S. Richardson, The anatomy and taxonomy of protein structure, Adv. Protein Chem. 34 (1981) 167–339. [25] O. Koch, G. Klebe, Turns revisited: a uniform and comprehensive classification of normal, open, and reverse turn families minimizing unassigned random chain portions, Proteins 74 (2009) 353–367. [26] K.C. Chou, J.R. Blinn, Classification and prediction of beta-turn types, J. Protein Chem. 16 (1997) 575–595. [27] L.L. Videau, W.B. Arendall 3rd, J.S. Richardson, The cis-Pro touch-turn: a rare motif preferred at functional sites, Proteins 56 (2004) 298–309.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Crystal structure of human FAF1 UBX domain reveals a novel FcisP touch-turn motif in p97/VCP-binding region Wonchull Kang, Jin Kuk Yang ⇑ Department of Chemistry, College of Natural Sciences, Soongsil University, Seoul 156-743, Republic of Korea
a r t i c l e
i n f o
Article history: Received 9 March 2011 Available online 15 March 2011 Keywords: Fas-associated factor 1 UBX domain Touch-turn motif p97 b-Grasp fold
a b s t r a c t UBX domain is a general p97/VCP-binding module found in an increasing number of proteins including FAF1, p47, SAKS1 and UBXD7. FAF1, a multi-functional tumor suppressor protein, binds to the N domain of p97/VCP through its C-terminal UBX domain and thereby inhibits the proteasomal protein degradation in which p97/VCP acts as a co-chaperone. Here we report the crystal structure of human FAF1 UBX domain at 2.9 Å resolution. It reveals that the conserved FP sequence in the p97/VCP-binding region adopts a rarely observed cis-Pro touch-turn structure. We call it an FcisP touch-turn motif and suggest that it is the conserved structural element of the UBX domain. Four FAF1 UBX molecules in an asymmetric unit of the crystal show two different conformations of the FcisP touch-turn motif. The phenyl ring of F619 in the motif stacks partly over cis-Pro620 in one conformation, whereas it is swung out from cis-P620, in the other conformation, and forms hydrophobic contacts with the residues of the neighboring molecule. In addition, the entire FcisP touch-turn motif is pulled out in the second conformation by about 2 Å in comparison to the first conformation. Those conformational differences observed in the p97/VCPbinding motif caused by the interaction with neighboring molecules presumably represent the conformational change of the UBX domain on its binding to the N domain of p97/VCP. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction p97/VCP, a multi-functional ATPase of the AAA+ family, is implicated in diverse cellular processes such as post-mitotic Golgi reassembly, endoplasmic reticulum associated degradation, nuclear envelope reconstruction, cell cycle, suppression of apoptosis and DNA damage response [1]. The diverse functions of p97/VCP are mediated by the interaction with various adaptor proteins in each different biological context [2,3]. The largest group of the adaptors comprises the proteins containing the UBX domain, such as FAF1, p47, SAKS1, and UBXD7, which directly binds to N domain of p97/VCP [4–7]. The UBX domain comprises about 80 amino acid residues and is currently considered as a general p97/VCP-binding module, with a growing number of UBX proteins being identified [8]. The structures of UBX domain were determined by NMR spectroscopy on FAF1 UBX and p47 UBX, revealing that it adopts a bgrasp fold similar to ubiquitin [5,9]. The first structural glimpse on its interaction with p97/VCP was obtained from the crystal structure of p47 UBX in complex with p97/VCP ND1 fragment at 2.9 Å resolution [10]. It showed that the conserved FP sequence
Abbreviations: FAF1, Fas-associated factor 1; VCP, valosin-containing protein; IKK, IjB kinase. ⇑ Corresponding author. Fax: +82 2 824 4383. E-mail address: [email protected] (J.K. Yang). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.03.052
motif in the loop connecting strands S3 and S4 is inserted into the cleft of the p97/VCP N domain and is essential for the binding activity [10]. FAF1 was initially identified as a Fas-interacting protein in the death inducing signaling complex and was shown to potentiate the apoptotic cell death [11–13]. Later, FAF1 was shown to intervene in the NF-jB signaling at two points of the pathway. First, FAF1 binds to the IKK b subunit, thereby disrupting the IKK complex assembly and inhibiting its activation [14]. Second, FAF1 also binds to p65 subunit of NF-jB and inhibits the translocation of NFjB into the nucleus [15]. Most recently, FAF1 was revealed to inhibit the proteasome-mediated protein degradation process either by interacting with p97/VCP which may serve as a molecular chaperone presenting the ubiquitinated client proteins to the proteasome or by interacting with ubiquitinated client proteins [4]. As a consequence of its involvement in these cellular processes, FAF1 acts as a pro-apoptotic protein. Human FAF1, composed of 650 amino acid residues, contains a UBX domain at the C-terminus through which it binds to p97/VCP N domain [4,16]. In an effort to elucidate the conserved structural features of the UBX domain for its binding to p97/VCP and also to provide a structural basis of the interaction between FAF1 and p97/ VCP, we initiated X-ray crystallographic analysis on FAF1 UBX domain, and here we present its crystal structure determined at 2.9 Å resolution.
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2. Materials and methods 2.1. Protein preparation, crystallization and data collection The human FAF1 UBX domain (residues 571–650) was overexpressed, purified and crystallized as described previously [17]. Briefly, FAF1 UBX protein was overexpressed in fusion with Histagged thioredoxin in Escherichia coli strain Rosseta2 (DE3). The fusion part was removed by TEV protease during the purification process. The FAF1 UBX alone was concentrated to 9.7 mg/ml for its crystallization. We collected X-ray diffraction data to 2.9 Å resolution, at Photon Factory, Japan on the beamlines AR-NW12. Data collection statistics are summarized in Table 1.
2.2. Structure solution and refinement The structure of the FAF1 UBX domain was solved by the molecular replacement using Phaser in CCP4 program suite [18,19]. The search model was its previously reported NMR structure (PDB code 1H8C) [9]. The crystal of FAF1 UBX was found to be twinned with the apparent space group of F4132 and its actual space group of F23. Its twin fraction is 0.426 and the twin operator is (k, -h, l). Initial trial for solving the structure was performed for the apparent space group F4132 without noticing the twining, and two FAF1 UBX molecules were positioned successfully with TFZ score 8.0. The R-factors resisted going down from around 50% during the refinement, and so the twining was suspected. We checked its twining and prepared the de-twinned data by Detwin in CCP4 program suite [19]. Its final structure determination and refinement were successfully performed with de-twinned data in the actual space group F23 of which the asymmetric unit contains four copies of FAF1UBX molecules. Subsequent manual model rebuilding was carried out using Coot [20] interspersed with rounds of automatic refinement by Refmac5 [21] and CNS [22]. An asymmetric unit of the crystal contains four FAF1 UBX molecules and the refined model covers residues from Glu571 to Glu650. The statistics for the data collection, structure determination and refinement are summarized in Table
1. The atomic coordinates and structure factors have been deposited with a code 3QCA in the Protein Data Bank (http:// www.pdb.org). 3. Results and discussion 3.1. Overall structure of FAF1 UBX FAF1 UBX domain comprises five b-strands (S1–S5), one a-helix (H1), and three 310 helices (G1–G3) (Fig. 1A). The five strands form a mixed b-sheet in the order of 2–1–5–3–4, and the sheet rolls around the a-helix H1 to form the well-known b-grasp fold, as previously shown in the NMR structure of FAF1 UBX [9]. Notably, among the three short 310 helices G1 to G3, only G3 was recognized in the previous NMR structure as a helix H2. FAF1 UBX domain contains three Phe-Pro sequence stretches: FP608-9, FP619-20, and FP639-40. In this study, FP608-9 has a usual trans peptide, and the other two, FP619-20 and FP639-40, have cis peptides. These features are contradictory to the previously reported NMR structure of FAF1 UBX in which the peptide conformations are exactly the opposite, that is, FP608-9 in cis and both FP619-20 and FP639-
Table 1 Data collection and structure refinement statistics for human FAF1 UBX. Data collection Space group Twin faction Twin operator Unit cell parameters Resolution No. of measured reflections No. of unique reflections Rmergea Completeness hI/r(I)i Redundancy
F23 0.426 k, -h, l 175.73 Å 30.0–2.90 Å 4,14,219 10,089 6.2%(32.2%) 99.9%(100.0%) 9.8(2.2) 41.1(29.7)
Refinement Rwork/Rfreeb r.m.s.d. bond lengths r.m.s.d. bond angles Subunits in asymmetric unit No. of atoms (protein/water) Average B factors (protein/water) Ramachandran favored region Ramachandran disallowed region
24.7%/29.3% 0.011 Å 1.28° 4 2546/31 65.8 Å2/42.8 Å2 97.4% 2.6%
Values in parentheses are for the highest resolution shell, 3.06–2.90 Å. a Rmerge = RhRi|I(h)ihI(h)i|/RiRiI(h)i, where I(h) is the intensity of reflection h, Rh is the sum over all reflections, and Ri is the sum over i measurements of reflection h. b R = Rh||Fobs(h)||Fcalc(h)||/Rh|Fobs(h)|, where F(h) is the structure factor of reflection h. Rfree was calculated using 5% data excluded from refinement, and Rwork using the other 95% data.
Fig. 1. Structure FAF1 UBX. (A) Overall structure of FAF1 UBX. (B) Four FAF1 UBX molecules in an asymmetric unit. The arrow indicates the S3/S4 loop.
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40
in trans. Among these three FP sequences, the second one, FP619, is conserved throughout VCP-binding UBX domains of various UBX-containing proteins such as FAF1, p47, Ubxd7, and SAKS1 [10]. Moreover, it was shown that the S3/S4 loop containing this FP of p47, another UBX-containing p97/VCP adaptor protein, is essential for UBX binding to p97/VCP N domain [10]. With this cis-Pro620, the S3/S4 loop takes a sharp turn connecting the two b-strands, S3 and S4 (Figs. 2 and 3). This structural feature and its implication will be further discussed below. 20
3.2. Two different conformations of p97/VCP-binding region The asymmetric unit of the FAF1 UBX crystal contains four copies of FAF1 UBX domain (designated a to d) (Fig. 1B). In the crystal packing, FAF1 UBX molecules a and c are positioned in the virtually identical contact environment, and molecules b and d are located in another kind of the contact environment, which is different from that of a and c (Fig. 1B). As a result, the molecules a and c adopt an almost identical conformation, and the molecules b and d commonly show another kind of conformation which is different from that of molecules a and c. Superposition of the four FAF1 UBX molecules reveals that a conformational difference is limited to the S3/ S4 loop region. For example, the root-mean-square (r.m.s.) deviation of Ca atoms in the S3/S4 loop region (residues 617–622) is 1.41 Å between molecules a and b, but is 0.70 Å between molecules a and c. In addition to the slightly different positions of the loop, the side-chain phenyl ring of F619 shows two distinct conformations. It stacks partly over the cis-P620 in molecules a and c, whereas it is completely swung out from cis-P620 in molecules b and d (Fig. 2). We refer to these conformations as type-ac and type-bd. The conformational difference can be accounted for the fact that the S3/S4 loops in molecules b and d are involved in the interaction with other molecules in the crystal lattice but those in molecules a and c are not (Fig. 1B). The S3/S4 loop region of molecules b and d is in contact with hydrophobic residues of neighboring molecules, whereas the corresponding region of molecules a and c is not. The side chain phenyl ring of F619 on the S3/S4 loop is swung out by the hydrophobic interactions with P609, W610, and D611 of the neighboring molecule. It is reminiscent of the observation in the crystal structure of p47 UBX in complex with p97/VCP ND1 domain: the corresponding F343 of p47 UBX forms hydrophobic contacts with D35, V38, and L72 of p97/VCP N domain [10]. So it is very tempting to suggest that the type-bd and type-ac conformations of FAF1 UBX may represent its ‘‘bound’’ state and the ‘‘unbound’’ state respectively.
Fig. 3. FcisP touch-turn. (A) FcisP touch-turn, a rare case of an open b-turn, formed by the four residues TFPR (residues 618–621) from the molecule a. The dashed line represents the distance between Cai and Cai+3.
3.3. FcisP touch-turn motif of UBX domain The most functionally important feature of UBX domain is the conserved FP sequence on the S3/S4 loop which was experimentally shown to be essential for p97/VCP-binding activity [10]. In the previously reported crystal structure of p47 UBX in complex with p97/ VCP N domain, the S3/S4 loop containing FP sequence was inserted into the cleft in the p97/VCP N domain [10]. The p47 UBX mutant with a shortened S3/S4 loop without the conserved FP residues lost most of the p97/VCP-binding activity [10] and UBXD1 UBX lacking those conserved residues does not bind to p97/VCP N domain [23]. So the conserved FP sequence on S3/S4 loop was of our special interest throughout this study. We observed in this crystal structure that the peptide bond between these two residues is clearly in a cis conformation, i.e., the proline residue is a cis-Pro (Fig. 2B). The FP sequence is strictly conserved among all known p97/VCPbinding UBX domains and is known to be essential for the binding of UBX domain to p97/VCP. Therefore, it is tempting to speculate that the observed structural feature of the cis peptide should also be conserved. In the crystal structure of p47 UBX in complex with p97/VCP ND1 [10], it is in an unusual partly extended conformation, intermediate between trans and cis. The discrepancy may be due to incomplete structure refinement in that region. In the previous NMR structures of FAF1 UBX and p47 UBX, the conserved FP sequence has a trans peptide between them [5,9]. This discrepancy
Fig. 2. Structure FAF1 UBX in complex with p97/VCP N domain. (A) The structural difference in the S3/S4 loop region between the type-ac and the type-bd conformations is illustrated for molecules a and b, respectively. (B) 2Fo-Fc electron density map for FP619-20 of molecules a and b at 1.2 r level.
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may be due to the NMR structure determination procedure, which usually assumes the peptide bond as trans. A geometric analysis also supports our proposal on the conservation of FcisP structural motif. The S3/S4 loop of FcisP clearly forms a b-turn with the four residues TFPR618-621 (Fig. 3). This b-turn can be classified as type VIb turn, whose structural features are as follows. First, the type VIb turn has no hydrogen bond among the four residues consisting of the turn and is called ‘open’ [24,25]. As a consequence, the distance between Cai and Cai+3 is about 6.0 Å, which is significantly longer than the one for the ‘normal’ hydrogenbonded b-turns. Second, the (i + 2)-th residue of the type VIb turn must be cis-Pro. Otherwise, the consecutive four residues would adopt an extended loop and cannot form a b-turn at all [26]. The FcisP b-turn of FAF1 UBX displays these two features of the type VIb turn, except the distance between Cai and Cai+3 is significantly shorter (4.58–4.91 Å) (Fig. 3). Recently, an even tighter and rarely observed form of the type VIb turn was described and termed as the cis-Pro touch-turn, in which the distance between Cai and Cai+3 is typically 4–5 Å as in the case of FAF1 UBX FcisP motif [27]. This analysis indicates that the S3/S4 loop of FcisP in FAF1 UBX is actually a rarely observed cis-Pro touch-turn and the corresponding proline residue in other UBX proteins such as p47 must be also cis-Pro. Therefore, we call it an FcisP touch-turn motif and propose that it is a conserved structural element of UBX domains for binding to p97/VCP. Interestingly, Videau et al. [27] found that all the 15 examples of cis-Pro touch-turn motif collected from 500 high resolution crystal structures are commonly located in functionally important sites, that is, the catalytic sites in the case of enzymes and the binding sites in the case of nucleic acid binding proteins. The FcisP touch-turn motif in UBX domains also plays a critical role in binding to another molecule, p97/VCP. To the best of our knowledge, it is the first example of a cis-Pro touch-turn described in the protein–protein binding interface. In summary, we determined the crystal structure of FAF1 UBX domain at 2.9 Å resolution. It revealed two previously unrecognized structural features of the UBX domain in its p97/VCP-binding region. First, the S3/S4 loop containing the conserved FP sequence, which is critical for the p97/VCP binding activity, adopts a rare cisPro touch-turn structure. We suggest that this FcisP touch-turn structural motif should be the conserved structural element of the UBX domain determining its p97/VCP binding activity. Second, the two different conformations observed in the current crystal structure may represent the unbound and the bound state of the UBX domain. Interestingly, the side chain phenyl ring of F619 in the motif partly stacks over P620 in the unbound state, whereas it is completely swung out in the bound state. Acknowledgments This work was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A080930). The authors thank the staff of the Pohang Light Source beamlines 4A in Korea, and the Photon Factory beamlines AR-NW12 in Japan. References [1] N. Vij, AAA ATPase p97/VCP: cellular functions, disease and therapeutic potential, J. Cell. Mol. Med. 12 (2008) 2511–2518.
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