VCP

BBAPAP-39214; No. of pages: 7; 4C: 2, 3, 5, 6 Biochimica et Biophysica Acta xxx (2013) xxx–xxx

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap

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Shuai Liu 1, Qing-Shan Fu 1, Jian Zhao, Hong-Yu Hu ⁎

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State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

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Structural and mechanistic insights into the arginine/lysine-rich peptide motifs that interact with P97/VCP

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Article history: Received 2 July 2013 Received in revised form 5 September 2013 Accepted 26 September 2013 Available online xxxx

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P97 protein, also referred to as valosin-containing protein (VCP), is an AAA-ATPase (ATPase associated with a variety of cellular activities) that mediates vital cellular activities with the cooperation of many cofactors. A group of cofactors interact with the N-terminal domain of P97 (P97N) through their Arg/Lys-rich peptide motifs. We investigated the interactions between P97 and these motifs, including VCP-binding motif (VBM) and VCPinteracting motif (VIM). The solution structures of the VBM motif from HRD1 and the VIM motif from SVIP are both comprised mainly of a single α-helix. The VIM motifs generally have stronger P97N-binding affinities than the VBMs, and SVIP (VIM) can compete with HRD1–VBM for the interaction, providing a possibility that VIM-containing proteins (such as SVIP) act as competitors against VBM-containing proteins (such as HRD1) for interacting with P97. Based on biochemical features of the VBM motifs, we also identified NUB1L (NEDD8 ultimate buster-1 long) as a novel VBM-containing protein, which is involved in proteasomal degradation of NEDD8 through the P97 pathway. © 2013 Published by Elsevier B.V.

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Keywords: P97/VCP VCP-binding motif VCP-interacting motif Arginine/lysine-rich Structure Interaction

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1. Introduction

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The P97 (Cdc48 in yeast) protein, also known as valosin-containing protein (VCP), is a type-II AAA-ATPase (ATPase associated with a variety of cellular activities) [1]. It normally forms a homo-hexamer; each subunit is composed of an N-terminal domain, two stacked hexameric ATPase domains referred to as D1 and D2, and a C-terminal flexible tail [2–4]. The N-terminal domain and the C-terminus are mainly responsible for the interactions with various proteins [5,6]. By cooperating with a variety of cofactors, P97 is involved in a lot of cellular pathways, including protein degradation, membrane fusion, cell cycle regulation, and so on [7,8]. The best known examples are that P97 participates in endoplasmic reticulum-associated degradation (ERAD) by forming a complex with UFD1 and NPL4 cofactors [9–12], and regulates membrane fusion by interacting with P47 [12,13]. Mutations in P97 cause the inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD) [14,15], and autosomal dominantly inherited amyotrophic lateral sclerosis (ALS), a fetal neurodegenerative disease [16].

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Abbreviations: AAA, ATPase associated with a variety of cellular activities; BS1, binding site 1; ERAD, endoplasmic reticulum-associated degradation; ITC, isothermal titration calorimetry; P97N, the N-terminal domain of P97 (residues 1–213); P97-ND1, the N and D1 domains of P97 (residues 1–458); VBM, VCP-binding motif; VCP, valosin-containing protein; VIM, VCP-interacting motif; NMR, nuclear magnetic resonance ⁎ Corresponding author. Tel./fax: +86 21 54921121. E-mail address: [email protected] (H.-Y. Hu). 1 These authors contributed equally to this work.

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Based on versatile binding sites on P97 molecule, the interacting cofactors of P97 can be divided into two groups, the N-terminal domain binding and the C-terminus binding proteins [5,6,8]. The C-terminus of P97 interacts with the PUB domain of PNGase and the PUL domain of PLAA [17,18], while the N-terminal domain interacts with the majority of the UBX domain-containing protein cofactors [5,6]. Other P97 cofactors interact with the N-terminal domain of P97 (P97N) by small peptide motifs including binding site 1 (BS1 motif), VCP-binding motif (VBM), and VCP-interacting motif (VIM) [5,19]. The BS1 motif is a short hydrophobic peptide stretch which resided in P47 and UFD1 that interact with P97 [20]. The VBM motif was first identified in three ubiquitin-related enzymes, including deubiquitinase ataxin-3 (Atx3) [21], and two ubiquitin ligases UbE4B [22] and HRD1 [23]. UbE4B is involved in multi-ubiquitination and ERAD [24,25], while HRD1 is an RING-type E3 ligase functioning in ERAD [26]. The VIM motif was identified in a small VCP/P97-interacting protein (SVIP) [27], GP78 [28] and ANKZF 1 (Vms1 in yeast) [29]. Analogous with HRD1, GP78 is also an E3 ubiquitin ligase involved in ERAD by cooperating with P97 [30]. SVIP is an inhibitor of the ERAD pathway [31], while ANKZF 1 is proposed to be responsible for mitochondrial protein degradation [29]. Generally, the VBM and VIM motifs both contain conserved basic arginine and/or lysine residues in their sequences; and almost all the proteins containing these motifs are involved in ubiquitin-related protein degradation. The interactions between P97 and VIM/VBM-containing proteins are indispensable for their proper functions in the P97-related pathways [21,28,30]. Thus, it is of great significance to investigate the molecular mechanisms of these specific interactions based on structural analysis.

1570-9639/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.bbapap.2013.09.021

Please cite this article as: S. Liu, et al., Structural and mechanistic insights into the arginine/lysine-rich peptide motifs that interact with P97/VCP, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbapap.2013.09.021

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2.2. Protein expression and purification

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Fig. 1. Sequence alignment of the Arg/Lys-rich peptide motifs that interact with P97. The sequences of some currently known VBM and VIM motifs are compared. HRD1, HMGCoA reductase degradation 1, also known as synoviolin; E4B, ubiquitination factor E4B; Atx3, ataxin-3; SVIP, small VCP/P97-interacting protein; GP78, glycoprotein precursor 78 kD, also autocrine motility factor receptor (AMFR); ANKZF1, ankyrin repeat and zinc finger domain-containing 1.

All of the proteins were expressed in Escherichia coli BL21 (DE3). The His-tagged proteins were purified using Ni2+-NTA columns (Qiagen), and the GST-fused proteins were purified using glutathione Sepharose-4B columns (Amersham Biosciences). They were further purified by gel filtration chromatography with respective columns (GE Healthcare).

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2.3. Cell culture, transfection, Western blotting, and immunoprecipitation

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In this paper, we elucidated the molecular mechanism of the specific interactions between the Arg/Lys-rich motifs and P97 by biochemical and NMR structural approaches. We solved the structures of the VBM and VIM motifs from HRD1 and SVIP respectively, and compared their positive charges on the helical surfaces potentially for interacting with P97. Based on the knowledge of the VBM motifs, we identified an Arg/ Lys-rich VBM motif that interacts with P97 from the NEDD8 ultimate buster-1 long (NUB1L) protein.

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2. Materials and methods

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2.1. Plasmids and antibodies

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All plasmids were constructed using standard molecular biology techniques. For HA-tagged HRD1-Cyto and HRD1-CytoΔVBM, the cDNAs were subcloned into HA-pcDNA3.0 (Invitrogen); while for

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HEK 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (Gibco) and penicillin–streptomycin at 37 °C in 5% CO2. Transient transfection of cells with various expression vectors were carried out using PolyJet™ reagent (SignaGen) according to the manufacturer's instructions. Cells were harvested and lysed in a RIPA buffer (50 mM Tris– HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, cocktail protease inhibitor (Roche)). The cell lysates were incubated with FLAG antibody conjugated agarose for 1 h at 4 °C. The beads were washed with the RIPA buffer for three times, and subjected to immunoblotting analysis. The samples were loaded for SDS-PAGE with 12% acrylamide gels and transferred onto PVDF membranes (PerkinElmer). The indicated proteins were probed with respective primary and secondary antibodies, and visualized by using an ECL detection kit (Thermo-scientific).

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FLAG-tagged P97, the cDNA was subcloned into pCMV-tag 2B (Agilent). pGEX-4T3 (GE Healthcare) was used for GST-fused proteins, pHGB was for GB1-fused proteins [32], while pET-22b (Novagen) was for Histagged proteins. The antibodies against HA and FLAG were from Sigma.

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Fig. 2. HRD1 interacts with P97 through its C-terminal VBM motif. (A) Co-IP experiment for the interaction between HRD1 and P97. HA-tagged HRD1-Cyto (K236–H616) or HRD1CytoΔVBM (K236–G687) was co-transfected with FLAG-tagged P97 into HEK 293T cells, and 48 h after transfection, the cell lysates were subjected to immunoprecipitation with an anti-FLAG antibody. (B) GST pull-down experiment showing the interaction between HRD1–VBM and P97N. GST-fused HRD1–VBM (T588-H616) was applied to pull-down P97-ND1 (M1-Q458) and P97N (M1-L213). (C) NMR titration verifying the interaction between HRD1–VBM and P97N. Shown is the overlay of the HSQC spectra of 15N-labeled GB1–HRD1– VBM (100 μM) (red) and titration with P97N at a molar ratio of 1:1 (blue). The labeled peaks are from VBM. (D) Plot of the relative peak intensities of GB1–HRD1–VBM against the residue number in the presence of P97N at a molar ratio of 1:1.

Please cite this article as: S. Liu, et al., Structural and mechanistic insights into the arginine/lysine-rich peptide motifs that interact with P97/VCP, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbapap.2013.09.021

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Fig. 3. SVIP interacts with P97 through the VIM motif. (A) GST pull-down experiment demonstrating the interaction between SVIP and P97. GST-fused SVIP was applied to pull-down P97ND1 and P97N. (B) NMR titration showing the interaction between SVIP and P97. 15N-labeled SVIP was titrated with unlabeled P97N. The molar ratios of SVIP to P97N are 1:0 (red), 1:0.5 (green) and 1:1 (blue), respectively. (D) Plot of the relative peak intensities of SVIP against the residue number in the presence of P97N at a molar ratio of 1:1.

2.4. GST pull-down experiment

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The pull-down experiments were carried out in a PBS buffer (10 mM Na2HPO4, 140 mM NaCl, 2.7 mM KCl, 1.8 mM KH2PO4, pH 7.3). GST and GST-fused proteins were incubated with glutathione Sepharose-4B beads at 4 °C for 0.5 h. Other proteins were then incubated with immobilized GST or GST-fused proteins at 4 °C for 1 h. The beads were collected by centrifugation and washed for three times, then eluted by a GSH buffer (50 mM Tris–HCl, 10 mM GSH, pH 8.0).

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2.5. Isothermal titration calorimetry (ITC)

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ITC experiments were carried out using VP-ITC Microcalorimeter (Microcal). The sample cell (2 mL in volume) was filled with P97N (residues 1 213) with a concentration of 100 μM. The injection syringe (300 μL) was filled with GB1-fused VIM- or VBM-motif peptides with a concentration of 1 mM. The experiments were performed at 25 °C in 20 mM Tris–Cl, 50 mM NaCl (pH 7.5). The experimental parameters are: 25–30 injections; 5 μL and 5 s per injection; interval of 180 s; and stirring speed of 100 rpm.

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2.6. NMR titration and structure determination [33]

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N-labeled SVIP and GB1–HRD1–VBM were dissolved in a buffer containing 20 mM phosphate, and 50 mM NaCl, pH 6.5. All spectra

were recorded at 25 °C on a 600-MHz NMR spectrometer (Bruker Biospin). P97N was titrated to GB1–HRD1–VBM or SVIP at different molar ratios, and the 1H–15N HSQC spectra were acquired to monitor the peak-intensity changes upon titration. 15N/13C-labeled SVIP, GB1– HRD1–VBM and GB1–NUB1L–VBM were dissolved in 20 mM phosphate, and 50 mM NaCl, pH 6.5. For the backbone and side-chain assignments, the spectra of HSQC, HNCACB, CBCA(CO)NH, HNCO, C(CO) NH, H(CCO)NH, HCCH–TOCSY and HNHA were acquired. NOE restraints were obtained from 15N- and 13C-edited NOESY spectra. The NMR data was processed with NMRPipe and analyzed with Sparky. The structures were calculated using ARIA2.0, assessed with PROCHECK, and displayed with MOLMOL.

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3. Results

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3.1. Interaction of the VBM motif from HRD1 with P97

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There are a large group of P97/VCP-interacting proteins that harbor special peptide motifs consisting of several basic Arg or Lys residues in their sequences (Fig. 1). Based on sequence alignment, these Arg/Lys-rich peptide motifs can be further grouped into two types, VBM and VIM. HRD1 is an ER protein with a transmembrane domain in its Nterminus and a cytoplasmic part (HRD1-Cyto, residues 236–616), while a VBM motif resided in its C-terminal end [34]. We examined the interaction between HRD1 and P97 by biochemical approaches. The result

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Please cite this article as: S. Liu, et al., Structural and mechanistic insights into the arginine/lysine-rich peptide motifs that interact with P97/VCP, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbapap.2013.09.021

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Fig. 4. Binding affinities of various VBM/VIM-motif peptides with the N-terminal domain of P97. (A) ITC experiment for the GB1-fused VBMs from HRD1, E4B and Atx3 binding with P97N. (B) ITC for GB1-fused SVIP-VIM binding with P97N. (C) As in (B), GB1-fused GP78-VIM. (D) The dissociation constants (KD) from ITC experiments. The ITC data were fitted with one-site binding model to calculate the KD values as listed in the diagram. (E) Competitive GST pull-down experiment. SVIP was included at a molar ratio (over GST–HRD1–VBM) of 0.1, 0.2, 0.5, 1.0 and 1.5 respectively. The upper panel shows the GSH bead-bound proteins, while the lower panel shows the unbound proteins.

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3.2. Interaction of the VIM motif from SVIP with P97

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SVIP is a small protein functioning as an inhibitor of ERAD pathway [27,31]. Sequence alignment suggests that it contains a VIM

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showed that HRD1-Cyto could co-immunoprecipitate with P97 (Fig. 2A, lane 3), but deletion of the VBM motif abolished its association, indicating that HRD1 associated with P97 through the C-terminal VBM motif. GST pull-down experiment further indicated that the VBM motif of HRD1 directly interacted with the N-terminal part of P97, P97-ND1 (residues 1–458) or P97N (1–213) (Fig. 2B). Then, we prepared a GB1 fusion of HRD1–VBM (T588–H616) and performed an NMR titration experiment [33]. With the increasing amount of P97N, the peak intensities of some residues in the VBM region decreased dramatically as compared with those in GB1 tag (Fig. 2C and D). Collectively, these results demonstrate that HRD1 interacts with the N-terminal domain of P97 through its Cterminal VBM motif.

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motif with two Arg/Lys-rich regions (Fig. 1). GST pull-down indicated that SVIP interacted with P97-ND1 and P97N (Fig. 3A, lanes 4 & 7), as in the case of previous reports [27,35]. NMR titration showed that some peak intensities decreased with the addition of P97N. The most significantly changed region was in the residues within its VIM motif region (A12–L42) (Fig. 3B and C). Thus, these results corroborate the previous finding that SVIP interacts with P97 through its VIM motif [35].

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3.3. Comparison of the binding affinities of these Arg/Lys-rich motifs

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To quantify the binding affinities, we measured the dissociation constants (KD) of these VBM or VIM motifs from HRD1, E4B, Atx3, SVIP and GP78 respectively with P97N by ITC experiments (Fig. 4A–C). For the sakes of purification and stability, all the peptides were attached to a GB1 tag [32]. The ITC profiles were simulated to one-site binding model. As shown, the KD values of VBMs (from HRD1, E4B or Atx3)

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Please cite this article as: S. Liu, et al., Structural and mechanistic insights into the arginine/lysine-rich peptide motifs that interact with P97/VCP, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbapap.2013.09.021

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Fig. 5. Solution structures of HRD1–VBM and SVIP. (A, D) Backbone superposition of the 15 lowest-energy structures of HRD1–VBM (A) or SVIP (D). (B, E) Ribbon representation of HRD1– VBM (B) or SVIP (E). (C, F) Surface representation of HRD1–VBM (C) or SVIP (F). The conserved basic residues are marked in blue on the helical structures. The structure models were generated by MOLMOL.

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3.4. Solution structures of the Arg/Lys-rich motifs from HRD1 and SVIP

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To gain insights into the interactions with P97 in detail, we prepared SVIP and GB1–HRD1–VBM proteins, and solved their solution structures using NMR techniques (Table S1) [33]. The solution structure of HRD1– VBM is comprised mainly of an α-helix (A599–L607) (Fig. 5A and B). The four basic Arg residues are dispersedly located on the surface of the helix (Fig. 5C). As for SVIP, circular dichroism spectroscopy exhibited a negative peak at 202 nm and a shoulder at ~222 nm (Suppl. Fig. S1), indicating that SVIP contains both helical and random-coil structures in solution. The solution structure of SVIP is composed mainly of a long α-helix from Leu18 to Ala37 and two random-coil termini (Fig. 5D and E). The VIM motif is just localized on this α-helix, and those conserved basic residues form a positively charged patch on the

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with P97N are roughly 10-fold larger than those of VIMs (from SVIP or GP78) (Fig. 4D), indicating that the binding affinities of VBMs with P97N are weaker than those of VIMs. Both VBM and VIM motifs interact with the N-terminal domain of P97 but with different affinities. To examine whether there are mutual competition between these interactions with P97, we performed a competitive GST pull-down experiment. In the absence of SVIP, GST–HRD1– VBM pulled down P97-ND1 effectively (Fig. 4E, upper panel). With the increasing amount of SVIP, this interaction was gradually disrupted, suggesting that SVIP competed with HRD1–VBM for interacting with P97-ND1. This implies that VBM and VIM bind P97N in a mutually exclusive manner [35].

helical surface (Fig. 5F). Compared with HRD1–VBM (Fig. 5C), the VIM 220 motif of SVIP has more positive charges concentrated on an area of the 221 helical surface, which may be beneficial to P97 binding. 222 3.5. Both Arg/Lys-rich regions of SVIP are essential for P97 binding

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From sequence alignment, two Arg/Lys-rich regions are highlighted in SVIP as well as in GP78 (Fig. 6A), indicating that VIM is different from VBM in sequence (Fig. 1). We asked whether these two Arg/Lys regions interacted with P97 independently. GST pull-down showed that the individual peptides were incapable of binding to P97-ND1 (Fig. 6B), demonstrating that both Arg/Lys regions of SVIP are indispensable for the interaction with P97. To identify which residues of SVIP are crucial for its interaction with P97, we constructed several GST-fused SVIP mutants and performed GST pull-down analysis. The data showed that the K21A/R22A double mutant had lost the ability to bind with P97-ND1 (Fig. 6C, lane 7), while R31A/R32A gave a decreased affinity for P97-ND1. However, all the single-residue mutations had no considerable influence on the interaction (Fig. 6C). This suggests that the Arg/Lys pairs or clusters in VIM motif of SVIP are crucial for interacting with P97.

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3.6. Identification of a novel VBM motif from NUB1L

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It is likely that the P97-associated proteins containing VBM/VIM 240 motifs are mostly involved in the ubiquitin–proteasome pathway [5]. 241 By sequence alignment, we proposed that the VBM motif also existed 242

Please cite this article as: S. Liu, et al., Structural and mechanistic insights into the arginine/lysine-rich peptide motifs that interact with P97/VCP, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbapap.2013.09.021

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P97/VCP is involved in a variety of cellular functions with the assistance of many cofactor proteins [5,6,37]. Among these cofactors, the VBM- or VIM-containing proteins directly interact with the N-terminal domain of P97 via the Arg/Lys-rich peptide motifs. We investigated the interactions between P97N and VBM/VIM motifs by biochemical and structural approaches. We solved the solution structures of these peptide motifs from HRD1 and SVIP, both of which are comprised mainly of an α-helix with a positively charged surface. The key interface residues (K21–Q33) in SVIP are highly conserved in GP78-VIM (R625L637) that are critical for interacting with P97N [35], suggesting that SVIP interacts with P97 in a manner probably similar with GP78-VIM. Based on the biochemical features of the VBM motifs, we discovered NUB1L as a new VBM-containing protein involved in the P97mediated pathway. On the P97 side, a large hydrophobic groove between the two lobes of the N-terminal domain provides a special site for binding with the αhelical VIM motif [35]. Our data demonstrate that, (1) both VBM and VIM are mainly comprised of a single α-helix; (2) VBM exhibits a weaker binding affinity with P97N than VIM; and (3) SVIP (VIM) binds to

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in RAP80, CHIP, NUB1L, and another region of GP78 (Fig. 7A), all of which are related to the ubiquitin–proteasome system. We examined their interactions with P97-ND1 by GST pull-down, and found that the peptide motifs from CHIP and NUB1L were capable of binding with P97-ND1 (Fig. 7B). This means that P97 is potential to associate with CHIP and NUB1L, and is probably involved in the respective pathways [36]. Further studies demonstrated that NUB1L directly interacted with P97 via its VBM motif and promoted transfer of NEDD8 for proteasomal degradation (Liu et al., to be published elsewhere). We also solved the solution structure of NUB1L-VBM (Fig. 7C and D). As expected, the structure of NUB1L-VBM is comprised mainly of a long α-helix (E416–K438), and its positively charged patch on the helical surface (Fig. 7E) is much larger than that of HRD1–VBM (Fig. 5C).

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Fig. 6. Mutation and GST pull-down analysis for the interaction between SVIP and P97. (A) Sequence alignment indicating that the VIM motif of SVIP has two Arg/Lys-rich regions, 21KRAK24, and 31RRQR34. (B) The individual Arg/Lys-rich peptides of VIM from SVIP are not able to bind with P97. GST-fused SVIP-P12 (E10–Q45), SVIP-P1 (E10–E30) and SVIP-P2 (L25–Q45) were applied to pull-down P97-ND1. (C) Effects of the Arg/Lys mutations on the SVIP binding with P97-ND1. A variety of GST-fused SVIP-VIM mutants were applied to pull-down P97-ND1.

Fig. 7. Identification of a VBM motif in NUB1L interacting with P97. (A) Sequence alignment of the potential VBM motifs from RAP80, CHIP, GP78 and NUB1L. Besides the VIM motif yet identified, there is a VBM-like sequence in GP78. (B) GST pull-down examining the interactions of P97-ND1 with these peptides. A VBM motif (A414–G443) has been identified in NUB1L to interact with P97. (C) Backbone superposition of the 15 lowestenergy structures of NUB1L-VBM. (D) Ribbon representation of the NUB1L-VBM structure. (E) Surface representation of the HRD1–VBM structure showing the positive charges on the helix (blue).

P97N competing with HRD1–VBM. Therefore, we propose that VBM binds to the P97N domain on the helical surface site similar to the hydrophobic inter-domain cleft of VIM [35]. So, as a VIM-containing protein, SVIP potentially acts as an effective competitor against HRD1 for interacting with P97. This is consistent with the previous observation that SVIP functions as a negative regulator of ERAD [31]. Both VBM and VIM have at least four conserved basic residues that are vital for the interactions with P97. However, in VIM, the Arg/Lys residues are separated into two regions by several residues in sequence [38]. These separated conserved residues stand for a more extended binding

Please cite this article as: S. Liu, et al., Structural and mechanistic insights into the arginine/lysine-rich peptide motifs that interact with P97/VCP, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbapap.2013.09.021

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Acknowledgements

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The authors thank Dr. Donghai Lin, Chenjie Zhou and Meng Wu for NMR technical help, and Dr. Xu Shen for assistance in ITC assay. This work was supported by grants from the National Basic Research Program of China (2011CB911104, 2012CB911003), and the National Natural Science Foundation of China (31270773).

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Appendix A. Supplementary data

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Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbapap.2013.09.021.

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In summary, P97 specifically interacts with a group of cofactors on the VBM or VIM motifs consisting of basic Arg or Lys residues in sequences. The structures of these motifs are comprised mainly of a long α-helix. In general, the VIM motifs interact with P97 stronger than the VBMs. Based on the Arg/Lys-rich motifs, more related motifs or proteins will be identified to interact with P97 involved in P97-mediated cellular pathways.

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interface, which probably provides an explanation for the stronger binding affinity of VIM. Because the biochemical features of VIM and VBM are well obtained, based on these, new VIM- or VBM-containing proteins will probably be identified and well characterized in the future, which may lead to the discovery of some novel functions of P97 and P97-mediated pathway. Moreover, investigating the cooperation of these P97 cofactors containing VBM or VIM motif will provide better understanding of such issues as why P97 takes part in so many biological pathways and how they are regulated in cells.

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Please cite this article as: S. Liu, et al., Structural and mechanistic insights into the arginine/lysine-rich peptide motifs that interact with P97/VCP, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbapap.2013.09.021

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