Unusual bipartite mode of interaction between the nonsense-mediated decay factors, UPF1 and UPF2
2009; Springer Nature; Volume: 28; Issue: 15 Linguagem: Inglês
10.1038/emboj.2009.175
ISSN1460-2075
AutoresMarcello Clerici, André Mourão, Irina Gutsche, Niels H. Gehring, Matthias W. Hentze, Andreas E. Kulozik, Jan Kadlec, Michael Sattler, S. Cusack,
Tópico(s)RNA modifications and cancer
ResumoArticle25 June 2009free access Unusual bipartite mode of interaction between the nonsense-mediated decay factors, UPF1 and UPF2 Marcello Clerici Marcello Clerici European Molecular Biology Laboratory, Grenoble Outstation, Grenoble Cedex 9, France Unit of Virus Host-Cell Interactions, UJF-EMBL-CNRS, UMI3265, Grenoble Cedex 9, France Search for more papers by this author André Mourão André Mourão Munich Center for Integrated Protein Science, Department Chemie, Technische Universität München, Garching, Germany Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Irina Gutsche Irina Gutsche Unit of Virus Host-Cell Interactions, UJF-EMBL-CNRS, UMI3265, Grenoble Cedex 9, France Search for more papers by this author Niels H Gehring Niels H Gehring Molecular Medicine Partnership Unit, European Molecular Biology Laboratory and University of Heidelberg, Heidelberg, Germany Department of Pediatric Oncology, Hematology and Immunology, Children's Hospital, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Matthias W Hentze Matthias W Hentze Molecular Medicine Partnership Unit, European Molecular Biology Laboratory and University of Heidelberg, Heidelberg, Germany Search for more papers by this author Andreas Kulozik Andreas Kulozik Molecular Medicine Partnership Unit, European Molecular Biology Laboratory and University of Heidelberg, Heidelberg, Germany Department of Pediatric Oncology, Hematology and Immunology, Children's Hospital, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Jan Kadlec Jan Kadlec European Molecular Biology Laboratory, Grenoble Outstation, Grenoble Cedex 9, France Unit of Virus Host-Cell Interactions, UJF-EMBL-CNRS, UMI3265, Grenoble Cedex 9, France Search for more papers by this author Michael Sattler Michael Sattler Munich Center for Integrated Protein Science, Department Chemie, Technische Universität München, Garching, Germany Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Stephen Cusack Corresponding Author Stephen Cusack European Molecular Biology Laboratory, Grenoble Outstation, Grenoble Cedex 9, France Unit of Virus Host-Cell Interactions, UJF-EMBL-CNRS, UMI3265, Grenoble Cedex 9, France Search for more papers by this author Marcello Clerici Marcello Clerici European Molecular Biology Laboratory, Grenoble Outstation, Grenoble Cedex 9, France Unit of Virus Host-Cell Interactions, UJF-EMBL-CNRS, UMI3265, Grenoble Cedex 9, France Search for more papers by this author André Mourão André Mourão Munich Center for Integrated Protein Science, Department Chemie, Technische Universität München, Garching, Germany Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Irina Gutsche Irina Gutsche Unit of Virus Host-Cell Interactions, UJF-EMBL-CNRS, UMI3265, Grenoble Cedex 9, France Search for more papers by this author Niels H Gehring Niels H Gehring Molecular Medicine Partnership Unit, European Molecular Biology Laboratory and University of Heidelberg, Heidelberg, Germany Department of Pediatric Oncology, Hematology and Immunology, Children's Hospital, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Matthias W Hentze Matthias W Hentze Molecular Medicine Partnership Unit, European Molecular Biology Laboratory and University of Heidelberg, Heidelberg, Germany Search for more papers by this author Andreas Kulozik Andreas Kulozik Molecular Medicine Partnership Unit, European Molecular Biology Laboratory and University of Heidelberg, Heidelberg, Germany Department of Pediatric Oncology, Hematology and Immunology, Children's Hospital, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Jan Kadlec Jan Kadlec European Molecular Biology Laboratory, Grenoble Outstation, Grenoble Cedex 9, France Unit of Virus Host-Cell Interactions, UJF-EMBL-CNRS, UMI3265, Grenoble Cedex 9, France Search for more papers by this author Michael Sattler Michael Sattler Munich Center for Integrated Protein Science, Department Chemie, Technische Universität München, Garching, Germany Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Stephen Cusack Corresponding Author Stephen Cusack European Molecular Biology Laboratory, Grenoble Outstation, Grenoble Cedex 9, France Unit of Virus Host-Cell Interactions, UJF-EMBL-CNRS, UMI3265, Grenoble Cedex 9, France Search for more papers by this author Author Information Marcello Clerici1,2, André Mourão3,4,5, Irina Gutsche2, Niels H Gehring6,7, Matthias W Hentze6, Andreas Kulozik6,7, Jan Kadlec1,2, Michael Sattler3,5 and Stephen Cusack 1,2 1European Molecular Biology Laboratory, Grenoble Outstation, Grenoble Cedex 9, France 2Unit of Virus Host-Cell Interactions, UJF-EMBL-CNRS, UMI3265, Grenoble Cedex 9, France 3Munich Center for Integrated Protein Science, Department Chemie, Technische Universität München, Garching, Germany 4Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany 5Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg, Germany 6Molecular Medicine Partnership Unit, European Molecular Biology Laboratory and University of Heidelberg, Heidelberg, Germany 7Department of Pediatric Oncology, Hematology and Immunology, Children's Hospital, University of Heidelberg, Heidelberg, Germany *Corresponding author. Corresponding author. European Molecular Biology Laboratory, Grenoble Outstation, 6 rue Jules Horowitz, BP 181, 38042 Grenoble Cedex 9, France. Tel.: +33 476 207238; Fax: +33 476 207786; E-mail: [email protected] The EMBO Journal (2009)28:2293-2306https://doi.org/10.1038/emboj.2009.175 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Nonsense-mediated decay (NMD) is a eukaryotic quality control mechanism that degrades mRNAs carrying premature stop codons. In mammalian cells, NMD is triggered when UPF2 bound to UPF3 on a downstream exon junction complex interacts with UPF1 bound to a stalled ribosome. We report structural studies on the interaction between the C-terminal region of UPF2 and intact UPF1. Crystal structures, confirmed by EM and SAXS, show that the UPF1 CH-domain is docked onto its helicase domain in a fixed configuration. The C-terminal region of UPF2 is natively unfolded but binds through separated α-helical and β-hairpin elements to the UPF1 CH-domain. The α-helical region binds sixfold more weakly than the β-hairpin, whereas the combined elements bind 80-fold more tightly. Cellular assays show that NMD is severely affected by mutations disrupting the beta-hairpin binding, but not by those only affecting alpha-helix binding. We propose that the bipartite mode of UPF2 binding to UPF1 brings the ribosome and the EJC in close proximity by forming a tight complex after an initial weak encounter with either element. Introduction Among the different mechanisms evolved by eukaryotes to control the quality of mRNA, nonsense-mediated decay (NMD) is one of the most extensively studied (Behm-Ansmant et al, 2007; Doma and Parker, 2007; Isken and Maquat, 2007; Shyu et al, 2008). The NMD pathway involves recognition and targeting for degradation of transcripts containing premature termination codons (PTCs), which may result from DNA mutations, transcription errors or pre-mRNA-processing errors, notably splicing. Originally, it was thought that the major biological function of NMD was to protect cells from the potentially deleterious effects of truncated proteins (Behm-Ansmant et al, 2007). However, it is now clear that, in different organisms, 3–10% of transcriptome is naturally targeted by NMD (He et al, 2003; Rehwinkel et al, 2005; Behm-Ansmant and Izaurralde, 2006), and it may be a much more general mechanism for regulating transcriptome diversity arising from alternative or mis-splicing (Green et al, 2003; Isken and Maquat, 2007; Jaillon et al, 2008). Nine NMD protein factors, SMG1–9, have been identified in higher eukaryotes, two of them very recently (Yamashita et al, 2009). The three UPF (UP-frameshift) proteins, UPF1 (SMG2), UPF2 (SMG3) and UPF3 (SMG4), constitute the conserved core of the NMD machinery and are found in almost all eukaryotes with a few possible exceptions among protists (Kadlec et al, 2006; Chen et al, 2008), suggesting that NMD has an ancient evolutionary origin. Despite the universal conservation of the three UPF proteins, significantly different mechanistic models have been proposed for NMD in different organisms (Conti and Izaurralde, 2005; Lejeune and Maquat, 2005). However, an evolutionarily consistent model for PTC recognition has recently begun to emerge (Amrani et al, 2004; Kertesz et al, 2006; Schwartz et al, 2006; Muhlemann et al, 2008; Brogna and Wen, 2009) on the basis of studies in the mammalian system (Buhler et al, 2006; Eberle et al, 2008; Ivanov et al, 2008; Silva et al, 2008; Singh et al, 2008). This model proposes that NMD is triggered by an inefficient termination event caused by the failure of PABP (and/or other 3′ UTR factors) to interact with the terminating ribosome (Brogna and Wen, 2009). In mammals, the exon junction complex (EJC) works within this model as an enhancer to increase NMD efficiency, but it is not absolutely required, as previously thought (Lejeune and Maquat, 2005). EJC is a multi-protein complex that is deposited by the splicing machinery on mRNA ∼24 nt upstream of the exon boundaries and marks the sites of intron excision (Kim et al, 2001; Le Hir et al, 2001). EJC is retained during subsequent mRNA maturation events, including nuclear export, but can recruit additional factors. Notably, in the nucleus, the EJC core factors, MAGOH–Y14, recruit UPF3b (Gehring et al, 2003; Chamieh et al, 2008) and subsequently UPF3b recruits UPF2 on export into the cytoplasm (Lykke-Andersen et al, 2000). The UPF3b–UPF2 interaction has been described at the atomic level and is mediated by the N-terminal RNP domain of UPF3b interacting with the third of the three MIF4G (middle domain of eIF4G) domains of UPF2 (Kadlec et al, 2004). In mammalian cells, NMD is thought to occur during the first, 'pioneer' round of translation (Ishigaki et al, 2001). The functional link between the ribosome stalled at a PTC and the EJC involves the recruitment of different factors in a complex and dynamic molecular architecture, beginning with the translation termination factors, eRF1 and eRF3 (eRF1-3) (Czaplinski et al, 1998; Ivanov et al, 2008). Subsequently UPF1 and SMG1 join eRF1-3 to form a transient complex called SURF (named after the component proteins) (Kashima et al, 2006). The downstream EJC makes contact with the SURF complex through the interaction of UPF2 (which is bound to UPF3b on the EJC), with UPF1 and SMG1 forming the so-called DECID complex (Kashima et al, 2006). At this stage, the conserved ternary core UPF complex is formed and SMG1 is stimulated to phosphorylate UPF1 on its C-terminal SQ-motifs (Kashima et al, 2006). Hyperphosphorylated UPF1 is recognized by SMG7 by a 14-3-3-like domain, also conserved in SMG5 and SMG6 (Fukuhara et al, 2005). SMG6 carries the endonuclease activity that initiates the degradation of nonsense mRNAs in metazoans, showing that NMD machinery contributes directly to their decay (Glavan et al, 2006; Huntzinger et al, 2008; Eberle et al, 2009). SMG7 promotes further destabilization of these transcripts in a DCP2- and XRN1-dependent manner (Unterholzner and Izaurralde, 2004). It has recently been shown that the decapping enzyme, DCP1, is recruited to the phospho-UPF1 through the proline-rich nuclear receptor co-regulatory protein 2 (PNRC2) (Cho et al, 2009). The interaction between the ribosome-associated SURF complex and the downstream EJC to form the DECID complex is primarily mediated through UPF2, which bridges between UPF1 on SURF and UPF3b on EJC. However, it has been reported that NMD can also occur either in a UPF2-independent process (Gehring et al, 2005) or in a UPF3b-independent manner (Chan et al, 2007; Tarpey et al, 2007). UPF1 is a highly conserved ∼120 kDa protein that shows RNA-dependent ATPase and 5′-3′ RNA helicase activities in vitro (Cheng et al, 2007), both of which are required for NMD (Czaplinski et al, 1995; Weng et al, 1996; Bhattacharya et al, 2000). UPF1 has several additional cellular functions, including a function in maintaining genome stability (Azzalin and Lingner, 2006; Isken and Maquat, 2008), and a mouse knockout for UPF1 is embryonically lethal (Medghalchi et al, 2001). The crystal structure of the UPF1 superfamily 1 helicase domain (residues 295–914) has been determined (Cheng et al, 2007). UPF1 also has a unique highly conserved N-terminal cysteine–histidine-rich domain (CH-domain, residues 115–275) that binds three structural zinc atoms (Kadlec et al, 2006). The CH-domain contains the UPF2-binding site (Weng et al, 1996; Serin et al, 2001; Kadlec et al, 2006). UPF2 is an ∼140 kDa perinuclear protein (hUPF2 comprises 1272 residues) characterized by three MIF4G domains (Mendell et al, 2000; Serin et al, 2001). UPF3b binds to the third MIF4G domain of UPF2 (Kadlec et al, 2004), whereas the function of the preceding N-terminal part of the protein is unknown. The UPF1-binding region of UPF2 is at the C-terminus of the protein and is separated from the third MIF4G domain by a conserved Glu/Asp-rich acidic region (He et al, 1997; Serin et al, 2001). The targeted knockout of UPF2, the only known function of which is in NMD, has severe effects on mouse haematopoietic stem cells, but milder effects on differentiated ones, suggesting an important role of NMD in proliferating cells (Weischenfeldt et al, 2008). Here, we characterize the interaction between human UPF1 and UPF2 by a variety of techniques, including X-ray crystallography, electron microscopy (EM), NMR, small-angle X-ray scattering (SAXS), isothermal calorimetry and in vitro and in vivo mutagenesis. We present crystal structures of the combined CH- and helicase domains (residues 115–914) of UPF1 in complex with the C-terminal region of human UPF2 (residues 1105–1198), providing the first information on both the relative arrangement of the two UPF1 domains and the structural basis for the interaction between UPF1 and UPF2. We show that the free C-terminal region of UPF2 is unstructured but co-folds on binding to UPF1, with an α-helical element binding on one side of the CH-domain and a β-hairpin element on the other. This mode of interaction of UPF2 with UPF1 is a good example of 'clamp-type fuzzy complex' (Tompa and Fuxreiter, 2008) in which an intrinsically disordered protein region partially folds on binding to a partner protein (Dyson and Wright, 2002). Possible rationales for this mode of UPF1–UPF2 interaction will be discussed in the light of the current understanding of the mechanism of NMD. Results Overview of the X-ray and NMR structural work The UPF1–UPF2 complex was obtained by the co-expression of the two proteins in Escherichia coli or by a reconstitution using UPF2 purified under denaturing conditions. We determined several different structures of the UPF2–UPF1 complex, including two structures with the CH-domain alone (data not shown because of a relatively low resolution, see methods) and two with the combined CH- and helicase domains of UPF1 (residues 115–914) (Table I). The most complete picture of the complex emerges from a monoclinic (P21) crystal form of the complex containing both domains of UPF1 with UPF2(1105–1198) at 2.9 Å resolution. In this structure, both the helical and β-hairpin segments of UPF2 have good and unambiguous electron density (Figure 1A, Supplementary Figure S1), although the linker between the two is only poorly defined. A second orthorhombic (I222) crystal form of the same complex diffracting to the higher resolution of 2.5 Å shows a relatively poor definition of the CH-domain (probably because of mobility through a lack of strong crystal contacts) and a very weak density for only the UPF2 β-hairpin region; indeed crystal contacts preclude binding of the helical segment. We have also determined the structure of a slightly extended construct of the CH-domain of UPF1 alone (residues 115–287) at the considerable higher resolution of 1.5 Å resolution compared with the original structure (Kadlec et al, 2006) (data not shown). This CH-domain construct is better expressed and has a properly configured C-terminal region, as in the full-length UPF1 structures. It was thus used in subsequent solution work, notably in NMR studies. Figure 1.Structure of the UPF1(115–914)–UPF2(1105–1198) complex. (A) Ribbon diagram of the complete structure, with UPF1 in green and UPF2 in blue. The missing links between the UPF1 CH- and helicase domains and between the N and C-terminal parts of UPF2 are represented as dotted lines. (B) Superposition of the closed form of the helicase domain (orthorhombic crystal, green) with the previously described helicase domain in the phosphate-bound form (PDB ID 2gk7, blue). The RMSD between the two structures is 1.03 Å for 591 aligned Cα atoms. The RMSD values between the orthorhombic form and the AMPPNP (PDB ID 2gjk) and ADP (PDB ID 2gk6) forms are, respectively, 1.81 and 2.00 Å. (C) Superposition of the open form of the helicase domain (monoclinic crystal, red) with the previously described helicase domain in the ADP-bound form (PDB ID 2gk6, gold). The RMSD between the two structures is 1.35 Å for 584 aligned Cα atoms. The RMSD values between the monoclinic form and the phosphate and AMPPNP forms are, respectively, 2.48 and 3.07 Å. (D) Superposition of UPF1 from the monoclinic (red) and orthorhombic (green) crystal forms showing that the relative orientations of the CH and helicase domains are the same in each case, although the helicase conformation is different. (E) The principal interacting residues from the CH- (green) and helicase (yellow) domains of UPF1 are represented as sticks. The same interactions are found in both monoclinic and orthorhombic crystal forms. These residues are well conserved (Supplementary Figure S2). Download figure Download PowerPoint Table 1. Data collection and refinement statistics of UPF1(115–914)–UPF2(1105–1198) complex UPF1(115–914)–UPF2(1105–1198) orthorhombic form UPF1(115–914)–UPF2(1105–1198) monoclinic form Beamline (ESRF) ID29 ID14-4 Wavelength (Å) 0.976 0.940 Space group I222 P21 Cell dimensions a, b, c (Å) 64.9, 129.1, 311.3 92.4, 97.3, 124.6 α, β, γ (deg) 90.0, 90.0, 90.0 90.0, 102.4, 90.0 Resolution (Å) 30–2.5 (2.5–2.6)a 30–2.85 (2.85–2.90)a Rmerge 11.1 (76.7) 8.6 (75.8) I/σI 11.2 (1.9) 11.1 (2.1) Completeness (%) 98.4 (98.0) 97.3 (97.1) Redundancy 3.71 (3.78) 3.80 (3.88) Refinement Resolution (Å) 46.5–2.50 (2.50–2.57)a 30–2.85 (2.85–2.92)a Total no. of reflections/free 42869/2266 46534/2463 Rwork 0.196 (0.289) 0.200 (0.332) Rfree 0.240 (0.309) 0.248 (0.372) Number of atoms Protein atoms 6168 (+3 Zn) 13085 (+6 Zn) Water molecules 254 — Sulphate ions 8 23 Average B-factors (Å2) 45.9 65.6 RMSD values Bond lengths (Å) 0.012 0.011 Bond angles (deg) 1.39 1.23 Ramachandran plotb Favoured (%) 96.0 96.2 Allowed (%) 99.9 99.8 a Values in parentheses are for highest-resolution shell. b Molprobity http://molprobity.biochem.duke.edu/. In parallel, we measured two-dimensional NMR spectra on various complexes using 15N and 15N/13C-labelled proteins to derive structural and dynamic information under solution conditions. Backbone signals could be assigned for the CH-domain of UPF1 alone, allowing the mapping of the UPF2-binding site. This was carried out both in the context of the complex with the full C-terminal region of UPF2 and also by titrating synthetic peptides of the separate alpha-helical and beta-hairpin motifs to the 15N-labelled UPF1 CH-domain. These NMR chemical shift perturbation data give strong additional evidence for two separate binding sites of UPF2 on the CH-domain of UPF1, as well as the presence of residual disordered regions in bound UPF2. Structure of the combined CH- and helicase domains of UPF1 Previous structures of the UPF1 helicase core (residues 295–914) showed that it comprises two RecA-like sub-domains (denoted 1A and 2A) with two unique insertions into domain 1A, denoted 1B (a β barrel domain) and 1C (a helical domain) (Cheng et al, 2007). Three states of the enzyme were described, closed forms with either AMPPNP (PDB code 2gjk) or phosphate bound (PDB code 2gk7) and a more open form with ADP bound (PDB code 2gk6). The differences arise mainly because of rigid body motions of the four sub-domains. The orthorhombic and monoclinic crystal forms that we have determined of the UPF1(115–914)–UPF2(1105–1198) complex also show different relative arrangements of the sub-domains (Figure 1B–D). In the high-resolution orthorhombic form, the helicase is in a very well-ordered closed configuration, with only a narrow cleft between the two RecA-like domains (Figure 1B). This conformation shows the highest similarity with the previously described phosphate-bound form (Cheng et al, 2007), with a RMSD value of 1.03 Å for 591 aligned Cα atoms (Figure 1B). Indeed, we observe a tightly bound sulphate at the position of the phosphate/gamma phosphate of AMPPNP. In the monoclinic crystal form, parts of UPF1 are less well ordered, especially the β-barrel domain 1B. Domains 1A and 2A are in a more open conformation that resembles most closely the ADP-bound form with an RMSD value of 1.35 Å for 584 aligned Cα atoms (Figure 1C). The individual domains 1A and 2A do not differ significantly in structure in all crystal forms to date. However, in our orthorhombic form, some of the flexible loops in domains 1B and 1C are better defined than in the previously determined structure (Figure 1B). The different conformations observed for the helicase domain in our two crystal forms may be because of the difference in crystallization conditions and crystal packing, but highlight the intrinsic flexibility of the helicase quaternary structure. Despite the difference in the orientations of the helicase sub-domains, the two crystal forms show the same orientation and interface of the CH-domain with respect to the helicase domain (Figure 1D). One end of the elongated CH-domain (which contains both N- and C-terminal elements of the domain) packs against the external surface of the helicase sub-domain 1A (notably N-terminal helices α1, α2 and α3), with the rest of the CH-domain extending away from the helicase. Residues 280–287, forming the linker region between the two domains, are poorly ordered. The domain interface involves specific hydrogen bonds, as well as van der Waals contacts (Figure 1E). Arg253 and His129 side chains (CH domain) form, respectively, a hydrogen bond with the main chain carboxyl of Val437 and a salt bridge with Glu434 (helicase). In addition, Asp298 (helicase) forms a hydrogen bond with the main chain amino group of Gln256 (CH domain). Finally Asp117 (CH-domain) forms a salt bridge with Lys428 (helicase). Helicase Tyr300, which stacks on Arg255, and Tyr442, which stacks on Arg253, are also crucial elements in the interface. The total buried area of this interface is 1163.1 Å2 (565.7 Å2 for the helicase domain and 597.4 Å2 for the CH-domain), as determined by the PISA server (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html). Although relatively modest, the fact that the same interface is observed in two distinct crystal forms and involves largely conserved residues (Supplementary Figure S2) suggests that the observed rigid orientation of the CH- and helicase domains is biologically significant and not a crystal-packing artefact. Furthermore, as the helicase itself is in a different state (closed or open) in the two different crystal forms, this shows that the rigid attachment of the CH-domain to domain 1A is compatible with different relative configurations of domain 2A. However, it cannot be ruled out that the ability to undergo functional conformational changes between these configurations may be affected by the presence of the CH-domain. To determine whether the observed domain configuration of UPF1 depended on the presence of bound UPF2, we attempted to crystallize UPF1 in the absence of UPF2, but this was unsuccessful. Instead we carried out EM and SAXS studies on UPF1(115–914), with and without bound UPF2(1105–1198). Negatively stained EM images were of sufficient quality to allow a three-dimensional reconstruction of each sample. The reconstructions obtained for unbound and UPF2-bound UPF1 were very similar to each other and to the crystal structure of the UPF1–UPF2 complex, with, in each case, the CH-domain bound to the side of the helicase domain (Supplementary Figure S3). To confirm that this is also valid for the proteins in solution, we analysed free and UPF2-bound UPF1(115–914) by SAXS and compared the measured scattering data with the theoretical scattering curve calculated from the crystal structure models. In both cases, the calculated curves show a satisfactory fit to the measured ones (Supplementary Figure S4A,B). In the case of UPF1(115–914) alone, the best fit was obtained with the crystal structure of the helicase in the closed conformation (χ=0.87), whereas for the UPF1–UPF2 complex, the best fit was with the helicase in the open conformation (χ=0.95). For both UPF1 and the UPF1–UPF2 complex, the measured radius of gyration is very close to that calculated from the respective crystal structures (see below) (Supplementary Figure S4D). The increased radius of gyration of the complex compared with the free UPF1 and the change in form of the distance distribution (Supplementary Figure S4C), which shows that the complex clearly has more mass at large distances from the centre of the mass, are fully consistent with the crystallographically observed binding of UPF2 on the CH-domain at the periphery of UPF1. Furthermore, the model-independent ab initio envelopes calculated from the data show an elongated shape that accommodates the crystal structures well, with the CH-domain protruding away from the helicase domain, and in the case of the UPF2 complex, with more volume associated with the CH-domain (Supplementary Figure S5). Minor discrepancies in these comparisons are likely to occur for two reasons. First, the crystal structures lack some loops in UPF1 and in the flexible linker of UPF2 connecting the two UPF1-binding elements; second, in solution, helicase probably fluctuates between open and closed conformations with perhaps a broader amplitude than that sampled by the crystal structures. As a final control, we calculated the scattering curve from an atomic model in which the CH-domain is displaced towards the cleft formed by the 1A and 2A domains of UPF1 (both for free and UPF2-bound UPF1). This severely deteriorated the fits to the experimental data (respectively, χ=2.82 and χ=5.42 for free and UPF2-bound UPF1, data not shown). Taken together, the crystallographic, EM and SAXS results indicate that the UPF1 CH-domain is bound to the helicase domain in a fixed, distal orientation, pointing away from the ATPase active site, irrespective of the presence or not of bound UPF2(1105–1207). This conclusion is further reinforced by an ATPase assay conducted on free and UPF2-bound UPF1, which shows that UPF2(1105–1207) does not alter UPF1 RNA-dependent ATPase activity (Supplementary Figure S6). Structure of UPF2 in the UPF1(115–914)–UPF2(1105–1198) complex The crystal structure of the UPF1(115–914)–UPF2(1105–1198) complex in the monoclinic form allows the identification of two UPF1-interacting regions of UPF2, separated by a flexible linker, in agreement with what was originally proposed for yeast UPF2 (He et al, 1996). The N-terminal part of the UPF2 (residues 1108–1128) fragment forms a long, slightly curved, amphipathic α-helix (Figure 2A). The C-terminal part folds into a β-hairpin (residues 1167–1189) comprising strand βA, strand βB and an intervening loop, followed by a short α-helix (residues 1193–1198) (Figure 2B). Structures of the CH-domain alone with UPF2(1167–1207) show that this α-helix extends to at least residue 1203 (data not shown). Between the two, residues 1129–1166 form an extended linker, the first part of which (residues 1129–1139) is observed with weak electron density wrapping around the CH-domain, whereas the following glycine-rich peptide is not visible at all. Figure 2.UPF2 binds on two opposite surfaces of the UPF1 CH domain. (A, B) UPF2 (blue) is represented as ribbons and UPF1 (grey) as ribbons and a transparent surface. UPF1 zinc atoms are shown in green. The UPF2 missing linker is represented as a dotted line. The two views differ by a rotation of 180 degrees around the horizontal axis. (C) The principal residues of the UPF2 N-terminal helix (cyan) and the UPF1 CH-domain (yellow), which form the hydrophobic interface between the two molecules, are represented as sticks. (D) The main interacting residues of UPF2 C-terminal β-hairpin (cyan) and UPF1 CH domain (yellow) are represented as sticks. Met 1169 and Met 1190 do not interact directly with UPF1 binding but form part of a small hy
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