Orientation of the central domains of KSRP and its implications for the interaction with the RNA targets

Orientation of the central domains of KSRP and its implications for the interaction with the RNA targets Irene Dı ´az-Moreno 1,2 , David Hollingworth 1 , Geoff Kelly 3 , Stephen Martin 4 , Marı ´aFlor Garcı ´a-Mayoral 1 , Paola Briata 5 , Roberto Gherzi 5 and Andres Ramos 1, * 1 Molecular Structure Division, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK, 2 Instituto de Bioquı ´mica Vegetal y Fotosı ´ntesis, US-CSIC, Avda. Americo Vespucio 49, Sevilla 41092, Spain, 3 MRC Biomedical NMR Centre, 4 Physical Biochemistry Division, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK and 5 Istituto Nazionale per la Ricerca sul Cancro, Largo R. Benzi 10, Genova, Italy Received September 29, 2009; Revised February 23, 2010; Accepted March 15, 2010 ABSTRACT KSRP is a multi-domain RNA-binding protein that recruits the exosome-containing mRNA degradation complex to mRNAs coding for cellular proliferation and inflammatory response factors. The selectivity of this mRNA degradation mechanism relies on KSRP recognition of AU-rich elements in the mRNA 30UTR, that is mediated by KSRP’s KH domains. Our structural analysis shows that the inter-domain linker orients the two central KH domains of KSRP—and their RNA-binding surfaces—creating a two-domain unit. We also show that this inter-domain arrangement is important to the interaction with KSRP’s RNA targets. INTRODUCTION Gene regulation by adenosine-uridine-rich element (ARE)-mediated mRNA decay (AMD) is important for cellular proliferation, immune response and cardiovascular toning. AMD malfunction has been linked to cancer (1) and to inflammatory diseases such as Crohn-like inflammatory bowel disease and inflammatory arthritis (2). The effect of cis-acting AREs on the stability of specific mRNAs is mediated by the binding of regulatory proteins (ARE-binding proteins, ARE-BPs). Some of these proteins (e.g. TTP, BRF1 and KSRP) promote mRNA degradation while others (e.g. HuD and HuR) act as antagonists, stabilizing the mRNA (3). K-homology splicing regulator protein/fuse-binding protein 2 (KSRP/FBP2) is a multi-functional protein of the far upstream element (FUSE)-binding protein (FBP) family that has been involved in several steps of transcriptional and post-transcriptional gene control. In AMD, KSRP binds to a subset of AREs, recruiting the exosome and de-adenylation factors to the mRNA targets (4,5), defining a functional model for ARE-dependent mRNA degradation. The central part of KSRP is organized in four single-stranded nucleic acid-binding domains, the so-called K-homology domains (Figure 1A, KH1–4) which are responsible for its nucleic acid-binding activity. Using cross-linking and mRNA decay assays, we have previously shown that a minimum of two domains of KSRP are required for mRNA binding and degradation (5). Furthermore, in vitro studies (6–8) have confirmed that the isolated domains bind to AU-rich sequences with low affinity (K d >100 mM) and that simultaneous binding of at least two domains is necessary to obtain K d values in the nanomolar-to-micromolar range. The selectivity of KSRP action in promoting mRNA degradation depends on its ability to recognize its ARE targets. However, the protein interacts with a broad range of AU-rich sequences. We dissected the sequence preference of the single domains showing not only the presence of both positive and negative sequence selectivity, but also that only one of the four domains of KSRP has a preference for AU-rich sequences (7). This hints that sequence specificity is only one of several criteria involved in target discrimination. KSRP-RNA recognition is a multi-domain event and inter-domain interactions, if they take place, could play an important role. The single-stranded regions recognized by the KH domains of KSRP are embedded within different 30untranslated regions (UTRs). Inter-domain contacts could modulate RNA recognition by relating orientation/accessibility of the RNA-binding surfaces of the domains to the single-stranded regions of the RNA targets. Thus, we need to understand if the four domains of KSRP act as structurally independent binding units and we need to evaluate the role of inter-domain linkers in protein– RNA interaction. *To whom correspondence should be addressed. Tel: +44 020 88162550; Fax: +44 020 89064477; Email: [email protected] Published online 12 April 2010 Nucleic Acids Research, 2010, Vol. 38, No. 15 5193–5205 doi:10.1093/nar/gkq216 ßThe Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. We have recently shown that the KH1–KH2 and KH3– KH4 domains of KSRP are connected by long and flexible linkers and that their relative orientation is not fixed (6,8). Here we examine the relation between KH2 and KH3 and show that the two domains interact and form the structural core of the KSRP protein. The structure of the KH23 protein reveals that the two domains assume a well-defined orientation with the linker playing an important role. We discuss the implication of this novel inter-domain arrangement for RNA binding and test our conclusions in the cellular environment. MATERIALS AND METHODS Protein and RNA oligonucleotides preparation Recombinant KH domains shown in Figure 1 were constructed by PCR from a plasmid containing full-length KSRP (5). The PCR products were ligated into NcoI and HindIII sites of pETM-30 to be expressed with TEV-cleavable His-GST tags. Unlabelled and isotopically 15 N- and 13 C-labelled proteins were expressed in Escherichia coli BL21 (DE3) (Invitrogen) in LB or minimal (M9) medium supplemented with 15 NH 4 Cl or 15 NH 4 Cl and 13 C-D-glucose. All His-GST-fusion proteins were initially purified by nickel affinity chromatography. The bulky His-GST-fusion tags were then cleaved with TEV protease and removed by using a second nickel affinity step. The constructs were further purified on a Superdex-75 (Pharmacia) gel filtration column. Samples were concentrated to 0.3–1.5 mM in 10 mM Tris-HCl buffer (pH 7.4) with 50 mM NaCl and 1 mM TCEP. Protein concentration was determined by a combination of spectrophotometry with predicted extinction coefficients and ninhydrin analysis of protein hydrolysates. All RNA oligonucleotides were chemically synthesized (Curevac and Dharmacon). Circular dichroism spectroscopy All circular dichroism (CD) spectra were recorded on Jasco J-715 spectropolarimeter equipped with a PTC-348 Peltier temperature-control system. CD intensities are presented as the CD absorption coefficient calculated by using the molar concentrations of the proteins. Thermal unfolding was monitored between 10 or 20C and 90 or 95C, depending on the constructs. Temperature was increased at a rate of 1C/min and unfolding was monitored by recording the signal at 220 nm. Reversibility of the unfolding was assessed by cooling to 10 or 20C at the same rate. Protein concentrations were 1–2 mM in 10 mM Tris–HCl buffer (pH 7.4), 100 mM NaCl, 1 mM TCEP. The data were fit to a two-state native-denatured model, while two independent unfolding transitions where used for the KH23 didomain. Data fit was performed with in house software as described in (9). RNA binding was monitored by adding increasing amounts of protein to 1–2 mM AU-12mer RNA in 10 mM Tris–HCl pH 7.4, 100 mM NaCl, 1 mM TCEP. A temperature of 5C was chosen to optimize the signal change upon protein binding. The average signal between 255 and 265 nm was fitted against the protein concentration using in house software (9). Figure 1. KSRP protein. (A) Domain organization of KSRP and constructs used in this study. (B) Sequence alignment of the inter-domain KH2– KH3 linker. Top—sequence alignment of KSRP KH1–KH2, KH2–KH3 and KH3–KH4 inter-domain linkers (red box). Bottom—sequence alignment of the KH2–KH3 linker of KSRP (Homo sapiens) with the ones of the MARTA1 (Rattus norvegicus), ZBP2 (Gallus gallus) and VgRBP71 (Xenopus laevis) protein homologues and of the human FUSE-binding protein (FBP1) (red box). Residues conserved in two or more constructs are coloured in green and blue, respectively; conservative substitutions of hydrophobic and polar residues are marked in grey. Yellow boxes highlight the positions of the two F311 and Y317 residues; red boxes define the boundaries of the linker between KH2 and KH3. The KH2–KH3 linker is conserved in the KSRP/FBP family and very different from the KH1–KH2 and KH3–KH4 linkers. 5194 Nucleic Acids Research, 2010, Vol. 38, No. 15 Nuclear magnetic resonance spectroscopy The nuclear magnetic resonance (NMR) samples of the different KH constructs were prepared in 90% H 2 O/ 10% D 2 O solutions of 10 mM Tris-HCl buffer (pH 7.4), 100 mM NaCl, 1 mM TCEP, 0.02% NaN 3 at concentrations in the range 0.3–1.5 mM. NMR spectra were recorded at 300K on Varian Inova and Bruker Avance spectrometers operating at 800 and 600 MHz 1 H frequencies. The spectra were processed with NMRPipe (10) and analysed with Sparky (11). Standard 3D NMR experiments (HNCACB, HNCA and HNCO) were used to obtain sequence specific 1 HN, 15 N, 13 C a , 13 C b and 13 C’ backbone assignments (12). Side-chain aliphatic proton and carbon assignments were achieved using data from a combination of 3D 15 N and 13 C-edited TOCSY and NOESY-HSQC spectra (13) with 70 and 100 ms mixing time for TOCSY and NOESY experiments respectively, plus a HCCH–TOCSY experiment (14). Water suppression was achieved by the WATERGATE pulse sequence (15). 3 J HN-Ha scalar couplings were measured from HNHA experiment as described previously (16). 15 N relaxation parameters (T 1 ,T 2 and { 1 H}- 15 N NOE) were obtained from standard experiments (17) recorded at 600 MHz 1 H frequency and 300K and analysed using NMRPipe routines (10). The program TENSOR (18) was used to determine amplitude and rhombicity for the diffusion tensor of KH2, KH3 and KH4 in isolation and in two-domain constructs. The same program was used to estimate the reported rotational correlation times ( c ). Estimates of the rotational correlation times of wild-type (8.5 ns) and double mutant (7.3 ns) were also obtained using a fast method that relies on changes in the intensity of the downfield region of 1D proton spectrum (Kelly et al., manuscript in preparation). 15 N- 1 H amide residual dipolar coupling (RDCs) were measured using a magnetically oriented binary mixture of 5% (v/v) alkyl-poly(ethylenglycol) C 12 E 5 and 1% (v/v) hexanol and IPAP experiments as described in (6). The fit between the 15 N– 1 H amide RDCs predicted from the KH23 structure and the ones measured experimentally was calculated using the program Module (19). The thermal stability of the domains was monitored by recording 15 N-HSQC spectra on 600 MHz (single domains) and 800 MHz (KH23) Varian Inova spectrometers on 0.3 mM protein samples in 10 mM Tris–HCl buffer (pH 7.4), 50 mM NaCl, 1 mM TCEP. Spectra were recorded at 3C intervals between 27 and 69C. In all cases, except for KH2 + linker (233–324), the protein unfolding was fully reversible. The effects of RNA complex formation on KH constructs were followed by acquiring 15 N-HSQC spectra during titrations of a 50 mM sample of 15 N-labelled KH domain(s) in 10 mM Tris-HCl buffer (pH 7.4), 50 mM NaCl, 1 mM TCEP with the three (50-UAUUUAUU-30, 50-UAUUUAUUAU-30and 50-UAUUUAUUAUUU-30) RNA oligos. Titration curves were obtained by plotting chemical shift perturbations (d Bind ) against the molar ratio of RNA/protein. Non-linear least squares fits to a 1:1 binding model (20) were performed in Origin 7.5 (Microcal. Inc.). This model accounts for a dilution effect of both molecules during the titration, with the ratio RNA/protein and d Bind as the independent and dependent variables, respectively. The dissociation binding constant (K d ) and the maximum chemical shift change (d max ) were the fitted parameters. A global fit of the data was performed in which the curves from different amide resonances were fitted simultaneously to a single K d value, whereas the d max for each resonance was allowed to vary. Weighted average values of 15 N and 1 H chemical shift perturbations (d avg ) of each amide was calculated as follows: d avg = (([d H ] 2 + [d N /5] 2 )/2) 1/2 . Structure calculation and analysis Structure calculations for KH2 (221–305) and KH23 (233– 418) were performed with ARIA 1.2 (21) using distance and dihedral angles restraints. Dihedral restraints were obtained from experimentally measured scalar couplings (f; (22)) or from the chemical shift-based TALOS database (f/u; (23)). Experimental distance restraints (Table 1) were derived from the integration (XEASY; 24) of Sparky (11)-derived NOE peak lists (6). H-bond constraints were added when unambiguously identified by the structural analysis of preliminary structures. Table 1. NMR and water refinement statistics KH2 (221–305) KH23 (233–418) NMR distance and dihedral constraints Distance restraints 2149 3229 Intra-residue 886 1373 Inter-residue 1263 1856 Sequential (|i–j| = 1) 428 551 Medium range (1 <|i–j|<4) 321 401 Long-range (|i–j|>4) 514 904 Experimental fconstraints 20 66 TALOS fand uconstraints 52 107 Hydrogen bond constraints 28 51 Structure statistics (25 conformers) (20 conformers) Mean total energy (kcal/mol) 3498 ± 53 6857 ± 132 NOE violations >0.3 A ˚0.2 ± 0.4 0.9 ± 0.2 Mean NOE energy (kcal/mol) 33 ± 7 81 ± 9 RMSD from idealized covalent geometry Bond lengths (A ˚) 0.0036 ± 0.0001 0.0039 ± 0.0009 Bond angles () 0.483 ± 0.019 0.542 ± 0.015 RMSD from the mean structure (A ˚) Whole structured domain a,b Backbone atoms 0.79 ± 0.18 1.43 ± 0.33 Heavy atoms 1.45 ± 0.25 2.08 ± 0.37 Secondary structure c,d Backbone atoms 0.49 ± 0.10 0.95 ± 0.26 Heavy atoms 1.19 ± 0.22 1.52 ± 0.29 Ramachandran plot analysis Most favoured regions (%) 87.9 85.5 Additional allowed regions (%) 11.1 12.0 Generously allowed regions (%) 0.1 1.0 Disallowed regions (%) 0.9 1.5 a KH2 whole structured domain: 234–304. b KH23 whole structured domain: 234–392. c KH2 secondary structure: 234–249, 254–270, 281–304. d KH23 secondary structure: 234–249, 254–270, 281–304, 325–338, 344–359, 368–392. Nucleic Acids Research, 2010, Vol. 38, No. 15 5195 Two hundred randomized conformers underwent simulated annealing with a standard CNS protocol as described in (25) and the 25/20 lowest energy structures for KH2/KH23 were refined in water (21). Structural statistics are shown in Table 1, structure quality was evaluated by using the program PROCHECK-NMR (26). The program MOLMOL (27) was used to visualize the conformers, and to create figures. Multiple sequence alignment was executed within the CLUSTALX program (28). Cell transfections and ribonucleoprotein complexes immunoprecipitation HEK293 cells were transfected (using Lipofectamine Plus, Invitrogen) with empty pCMV-TAG2B vector (mock) or the same vector expressing either wild-type KSRP (KSRP) or KSRP F311A/Y317A double mutant (KSRP F311A/Y317A ). ribonucleoprotein complexes immunoprecipitation (RIP) assays were performed as previously described by (29) with some modifications. Briefly, cells lysates were immunoprecipitated with either Protein A- or ProteinA/ ProteinG-Sepharose-coupled antibodies at 4C overnight. Pellets were sequentially washed with the following buffers: Buffer I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.1, 150 mM NaCl); Buffer II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.1, 500 mM NaCl) and Buffer III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris–HCl, pH 8.1). Total RNA was prepared using Trizol (Invitrogen, Carlsbad, CA, USA) retro-transcribed using random primers and amplified by PCR. The primer sequences are: hsa.PP2ACA forward 50— GAT GGA GGG ATA TAA CTG GTG CC —30 hsa.PP2ACA reverse 50— ACG AGG TGC TGG GTC AAA CT —30 hsa.CTNNB1 forward 50— TTG GAA CCT TGT TTT GGA CA —30 hsa.CTNNB1 reverse 50— ACC GCA TTT TCT CTT GAA GC —30 RESULTS Characterization of the KH2–KH3 protein We have used NMR data on chemical shift perturbation (CSP), hydrodynamic behaviour and stability to define the relation between the KH2 and KH3 domains. Our data indicate that an interaction between KH2 and KH3 is taking place in which the linker plays a pivotal role. The superposition of the 15 N-HSQC spectra (Supplementary Figure S1) of the isolated KH2 (233–305) and KH3 (317–418) domains on that of the two-domain KH23 construct (233–418) shows significant chemical shift differences (d avg >0.02 ppm) for resonances of both domains which are mapped in Figure 2A. The largest chemical shift changes are observed for residues of KH2’s a 1 /a 3 and KH3’s b 1 (see the structure of KH2 in the next paragraph and ref. 6), indicating that the linker is likely to be sandwiched between the two domains. Indeed, the addition of the linker to the KH2 domain (KH2+linker construct) results in very substantial chemical shift changes in the KH2 resonances (Supplementary Figure S1), while inclusion of KH3 causes only small additional chemical shift perturbations. Furthermore, resonances of the last seven amino acids of the linker (317–324), which are not visible when this short stretch is attached to KH3 in the (317–418) construct, become clearly visible in the KH23 construct, indicating that further contacts are taking place in the full two-domain construct. Importantly, no significant chemical shift differences are observed between the isolated KH23 and the two domains within the KH1234 construct (Figure 2B), which confirms that the features of the KH23 interaction are conserved in the longer protein. Figure 2. Interaction between KH2 and KH3 in the two- and four- (KH1–KH4) domain constructs. (A) Mapping of the residues of KH2 and KH3 whose 15 N and 1 H amide chemical shift changes in the two domain construct on a space filling representation of KH23. Residues of KH2 (233–305) and KH3 (317–418) whose amide resonances d avg (ppm) is <0.2 are in light grey, while residues whose d avg 0.2 are in orange for KH2 and magenta for KH3. Residues in the linker are in green, unassigned residues in dark grey. (B) The KH2–KH3 arrangement is conserved in KH1234 Superimposition of 15 N-HSQC spectra of KH23 (233–418), green, and KSRP KH1234, purple. Grey labels correspond to KH2 signals which shift upon addition of the KH2–KH3 linker and/or KH3 binding. The absence of significant chemical shift changes in the resonances of KH2, KH3 and the linker indicate that the relative orientation of the KH2 and KH3 domains and their contacts with the linker are conserved in the longer protein. 5196 Nucleic Acids Research, 2010, Vol. 38, No. 15 15 N T1, T2, and heteronuclear NOE values recorded on the KH2, KH3 and KH23 proteins indicate that KH23 encompasses two relatively rigid regions (corresponding to the two KH domains) joined by a more mobile 18-residue long linker (Figure 3 and Supplementary Figure S2). The intra-domain trends of 15 N T1, T2 and heteronuclear NOE values for KH2 and KH3 are consistent with what is observed for other KH domains and do not change from the single to the di-domain constructs (Figure 3). The linker comprises two distinct regions corresponding to amino acids 306–310 and 311–324. The trend and values of the relaxation parameters of amino acids 306–310 are consistent with a fully flexible protein chain (30), while the ones of residues 311–324 are in between those of the structured KH domains and those of the flexible 306–310 residues (Figure 3). This second part of the linker is much more flexible in the KH2+linker construct (Supplementary Figure S2) in agreement with the linker being sandwiched between the two KH domains. The KH23 construct has a rotational correlation time (t c ) of 8.92 ns, in the range of t c /MW reported by Dayie and co-workers (31) and significantly larger than the one of the isolated KH2 and KH3 domains (5.27 and 6.36 ns in the isolated domains), consistently with the two domains interacting. The rhombicity (r) values of KH2 and KH3 in solution change very significantly between isolated domains and domains within the two-domain construct (Supplementary Table S1). This contrasts with what we observe for the KH34 construct, where the non-interacting KH3 and KH4 (6) have similar r values to the isolated KH3 and KH4 domains (Supplementary Table S1). Finally, significant changes in the stability of one of the two domains confirm that an inter-domain interaction is taking place (32). CD spectroscopy showed that the isolated KH2 and KH3 domains have T m of 69.1 (Figure 4A) and 62C (6), respectively. The unfolding curve for KH23 (Figure 4A) cannot be reproduced by summation of the curves for the individual domains, which should be the case if no interaction between domains was present. It is worth noting that addition of the KH2-KH3 linker to KH2 does not change its T m . A change in KH2 stability in the KH23 construct is confirmed by the NMR spectra recorded during the thermal unfolding of KH2 and KH23 (3C intervals over the temperature range of 27– 69C), where unfolding of KH2 and KH3 domains can be followed separately. Comparison of the KH2 resonances in the two experiments shows that the domain is 10C more stable when in isolation (Figure 4B). Fittingly, no such change is observed in the unfolding of KH1, KH2, KH3 and KH4 in isolation and in the KH12 and KH34 constructs (6,8)—where no inter-domain contact is present. Structure of KH2 and KH23 and role of the linker in inter-domain arrangement Next, we solved the structures of the KSRP KH2 (221–305) and KH23 (233–418) proteins using NMR spectroscopy. In KH23, KH2 and KH3 are positioned at a 90angle to form an L-shaped structure, in a KH–KH orientation never observed before (Figure 5A–C). The KH2–KH3 linker starts with a short four-amino acid flexible turn followed by a partially structured stretch that runs anti-parallel to KH2 a3 and lies between by KH2 a1, a2 and a3. Then the linker turns and makes contact with KH3. The conformation of the section of the linker interacting with KH2 is not extended; the polypeptide backbone forms one helical turn and a broad double turn (Figure 5A). Subsequently, the backbone is arranged in two kinks that form the joint of the KH23 L-shaped structure and define domain–domain orientation. The surface buried by the contacts between KH2 and the linker is 1500 A ˚ 2 and the one between KH3 and the linker is 800 A ˚ 2 . This difference explains the more extensive chemical shift changes for KH2 described in the previous paragraph. The relative orientation of KH2 and KH3 is defined mainly by domain(s)–linker Figure 3. Internal motions in KH2, KH3 and KH23. T 2 (A) and Heteronuclear NOE (B) values of KH2 (233–305) and KH3 (317– 418) amide resonances, in grey (as reported in 8 and 6, respectively), are compared with equivalent data on KH23 (233–418) in black. The positions of the variable loop(s) and inter-domain linker in the protein sequence are indicated at the bottom of the figure. The arrows span the KH2 and KH3 domains and two vertical lines define the boundaries of the more and less flexible parts of the linker. Residues in the invariant loops of KH2 and KH3 are characterized by T 2 and heteronuclear values shorter than the average, suggesting conformational exchanges with rates near msto ms time scale (30) while residues in the variable loops displays a behaviour consistent with motions in the nanosecond range. Nucleic Acids Research, 2010, Vol. 38, No. 15 5197 contacts but also by a few domain–domain ones that bury 285 A ˚ 2 of surface. Domain–domain and domain–linker interactions are mediated by side chain–side chain interactions and do not rely on the formation of stable H-bonds or any extension of the secondary structure of the protein. In particular, two hydrophobic amino acids from the linker (F311 and Y317) make extensive contacts with KH2 (Figure 5C). The two aromatic side chains sandwich the side chain of R314 creating two cation–p interactions and stabilizing the relative position of the three residues. The inter-domain arrangement is defined by 151 linker-domain (KH2 a 1 ,a 2 and a 3 and KH3 b 1 and b 3 ), 83 linker–linker and 5 domain–domain NOEs (Figure 5C, Supplementary Table S2). The orientation of the two domains is also confirmed by residual dipolar coupling (RDC) analysis. RDCs recorded on a sample of KH23 fit to the values predicted from the KH23 structure (Supplementary Figure S4). It is interesting to note that the relaxation measurements reported above indicate that the backbone of residues in the linker shows a significant flexibility. This flexibility could be related to the observed lack of H-bonds involving backbone moieties of the linker or/and could indicate the existence of minor conformers with a less structured linker. The comparison of the structures of the isolated KH2 (this article, Figure 5D) and KH3 (6) with the one of the two domains in the KH23 construct shows no major changes in the arrangements of the secondary structure elements, with the most significant differences observed at the C-end of KH2 a 3 and the N-end of KH3 b 1 (Figure 5E). Differences were also observed in the average conformation of the flexible (Figure 3) b 2 –b 3 loops of both domains, most likely reflecting the scarce structural information available for these regions. The structures of the isolated KH2 and KH3 domains (Figure 5D) are superimposable to the ones of the domains within the KH23 construct with an RMSD of 0.95 A ˚(KH23–KH2, backbone atoms) and 1.05 A ˚(KH23–KH3, backbone atoms). Both structures are well defined and the Ramachandran plots indicate that the very few residues in the disallowed regions (1.5% for KH23 and 0.9% for KH2) (Table 1) belong to loops that are only partially assigned. Importantly the orientation of the two domains is also well defined (Figure 5A and B). Figure 4. Thermal denaturation of KH2 and KH23. (A) 220 nm CD signal of KH2 (233–324) (left) and KH23 (233–418) (right). Smoothed data are in black, fitted curves in colour and the transition midpoints are indicated by dashed lines. (B) NMR superimposition of four 15 N- 1 H HSQC NMR spectra recorded during the thermal unfolding of the KH2 (233–305) (left) and KH23 (233–418) (right) proteins. The spectra are recorded at 27C (purple), 48C (red), 51C (orange) and 63C (yellow). KH2 resonances shift towards the random-coil region of the spectrum and disappear at temperatures above 51C in the KH23 construct, but they are still visible at 63C in the isolated domain. 5198 Nucleic Acids Research, 2010, Vol. 38, No. 15 Figure 5. Structure of KH2 and KH23. (A)90 rotated ribbon representations of the solution structure of the KSRP KH23 (233–418) di-domain. KH2 is displayed in orange, KH3 in pink. The backbone of the KH2–KH3 linker is coloured in light blue for the more flexible (306–310) residues and in dark green for the less flexible (311–324) ones. The side-chains of F311 and Y317 are shown in light green. Domain–linker and domain– domain contacts define the inter-domain orientation. (B)90 rotated superpositions of the KH23 backbone (C a trace, in dark green) for the 20 lowest energy conformers on the average structure. (C) 180rotated close ups of the domain–linker and domain–domain interactions within the KH23 structure. KH2 residues interacting with the linker and with KH3 are in orange, while linker residues are in green and KH3 residues interacting with the linker and KH2 are in pink. A selection of long range NOEs between the linker and the two domains and between domains are represented as Nucleic Acids Research, 2010, Vol. 38, No. 15 5199 The structure presented here provides the first example of an intra-molecular KH–KH interaction mediated by the inter-domain linker. The high level of conservation of the linker amino acids in the KSRP (and FBP) family (Figure 1B)—especially of the two aromatic residues F311 and Y317—strongly suggests that the orientation of the KH2 and KH3 domains is conserved in man, rat and chicken. Inter-domain arrangement and RNA binding The structure of the KH23 construct shows that the nucleic acid binding grooves of KH2 and KH3 are 90 rotated and non-contiguous, adopting an orientation very different from the ones reported so far in KH–KH di-domains (33–36) (Figure 6). To understand how the KH2–KH3 orientation relates to RNA binding we tested the interaction of KH23 with a 12mer RNA (UAUUUAU UAUUU) that recapitulates the sequence of the TNFa ARE in vitro. The same RNA had been previously used to test the RNA binding of the KH3–KH4 domain pair, while shorter oligos spanning the 12mer sequence had been used to test the four isolated domains (6–8). NMR CSP data obtained titrating 15 N-labelled KH23 with the TNFa12mer (Figure 7A) revealed not only that both KH2 and KH3 bind RNA using the ‘classical’ nucleic acid-binding grooves (Figure 7B) (37), but also that KH2- and KH3-binding surfaces do not coalesce. The lack of significant chemical shift changes in the resonances of the KH2–KH3 linker indicate that the linker is not directly involved in RNA binding and that no significant structural rearrangement is taking place in the domains interface. Smaller but clearly detectable chemical Figure 5. Continued blue lines, while yellow lines represent the intra linker NOEs connecting the side chains of the two F311 and Y317 aromatic residues with the positively charged R314. (D) Left and middle—90rotated ribbon representations of the solution structure of KH2 (221–305). Right—superposition of the KH2 backbone (C a trace, in orange) for the 25 lowest energy conformers and the average structure. The KH2 structure is well defined and shows that the domain assumes a typical KH fold. (E)90 rotated superimpositions of the backbone traces of KH23 di-domain (green), KH2 (yellow) and KH3 (magenta) (lowest energy conformers). N- and C-termini are labelled. Figure 6. Comparison between the structure of KH23 and the ones of the other two-KH domain constructs available in the PDB.The ribbon representation of the KH23 structure (grey) is compared with similar representations of the (A) NusA KH12 in magenta; (B) FMRP KH12 in red; (C) FBP1 KH34 in blue and (D) PCPB KH12 in green, structures. As the topologies of the E.coli NusA KH domains are different from the eukaryotic ones, this structure was displayed above KH23, while the amino terminal domains of FMRP KH12, FBP1 KH34 and PCBP KH12 were superimposed to KH2 within KH23. 5200 Nucleic Acids Research, 2010, Vol. 38, No. 15 Figure 7. Binding of KH23 to the 50UAUUUAUUAUUU 30RNA. (A) Superposition of a series of 15 N-HSQC spectra recorded during a titration of KH23 (233–418) with increasing amounts of UAUUUAUUAUUU RNA. The spectra are coloured according to RNA:protein ratios: grey for free protein, yellow for 0.5:1 RNA:protein ratio, magenta for 0.75:1, green for 1:1 and cyan for 1.5:1. Resonances that shift most during the titration (d avg 0.05 ppm) are labelled in black. (B) Left, top—KH23 (233–418) protein surface, coloured according to the chemical shift changes undergone by the NH resonances upon RNA binding. Residues with d avg (ppm) <0.05 are in blue, while residues with d avg (ppm) 0.05 are in orange. Red indicates residues whose resonances are broadened beyond detection. Left, bottom—90rotated surface. Right—Molmol ribbon representation of the KH23 (233–418) structure with an equivalent colour coding and orientation. The RNA binds in the classical nucleic acid-binding grooves of KH2 Nucleic Acids Research, 2010, Vol. 38, No. 15 5201