Molecular basis of Diamond Blackfan anemia: structure and function analysis of RPS19. Lynn A. Gregory, Almass-Houd Aguissa-Touré, Noël Pinaud, Pierre Legrand, Pierre-Emmanuel Gleizes, Sébastien Fribourg To cite this version: Lynn A. Gregory, Almass-Houd Aguissa-Touré, Noël Pinaud, Pierre Legrand, Pierre-Emmanuel Gleizes, et al.. Molecular basis of Diamond Blackfan anemia: structure and function analysis of RPS19.. Nucleic Acids Research, 2007, 35 (17), pp.5913-5921. ￿10.1093/nar/gkm626￿. ￿inserm- 00175611￿ HAL Id: inserm-00175611 https://www.hal.inserm.fr/inserm-00175611 Submitted on 28 Sep 2007 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Nucleic Acids Research Molecular basis of Diamond-Blackfan anemia: Structure and function analysis of RPS19 F Journal: Nucleic Acids Research o Manuscript ID: draft r Manuscript T ype: 2Standard Manuscript - UK Editorial Office Key WorPds: pre-rRNA maturation, ribosome, disease, DBA e e r R e v i e w This is a pre-copy-editing, author-produced PDF of an article accepted for publication in Nucleic Acids research following peer review. The definitive publisher-authenticated version Nucleic Acids Res. 2007 Sep 1;35(17):5913-5921 is available online at: http://nar.oxfordjournals.org/cgi/content/full/35/17/5913 Page 1 of 27 Nucleic Acids Research 1 2 3 4 5 Molecular basis of Diamond-Blackfan anemia: 6 7 8 Structure and function analysis of RPS19 9 10 11 12 Running title : Crystal structure of RPS19 13 14 15 16 17 Lynn A. Gregory* 1,2,Almass-Houd Aguissa-Touré* 3,Noël Pinaud 1,2, 18 19 20 Pierre Legrand4,Pierre-Emmanuel Gleizes § 3,Sébastien Fribourg § 1,2 F 21 22 o 23 24 r 1 INSERM U869, Institut Européen de Chimie et Biologie, 2 rue Robert Escarpit 25 26 Pessac, F-33607, France. 27 P 28 2 Université Victor Segalen, Bordeaux 2, F-33076, France 29 e 30 3 Laboratoire de Biologie Moléculaire des eucaryotes (UMR5099) and Institut 31 e d’Exploration Fonctionnelle des Génomes (IFR109), CNRS and Université Paul 32 r 33 Sabatier, 118 route de Narbonne F-31062 Toulouse, France. 34 35 4 Synchrotron SOLEIL L’Orme des Merisiers, Saint Aubin- BP48, 91192 Gif sur R 36 37 Yvette Cedex, France. e 38 39 v 40 i *Equal contribution 41 42 e §Correspondence should be addressed to S.F or P.E.G. 43 44 e-mail: [email protected] w 45 46 tel/fax: 00 33 5 40 00 30 63/68 47 48 49 e-mail: [email protected] 50 51 tel/fax: 00 33 5 61 33 59 26 / 58 86 52 53 Keywords: pre-rRNA maturation, ribosome, disease, DBA. 54 55 56 57 58 59 60 1 Nucleic Acids Research Page 2 of 27 1 2 3 ABSTRACT (146 words) 4 5 6 7 8 Diamond-Blackfan anemia (DBA) is a rare congenital disease linked to 9 10 mutations in the ribosomal protein genes rps19 and rps24. It belongs to the 11 12 emerging class of ribosomal disorders. To understand the impact of DBA 13 14 15 mutations on RPS19 function, we have solved the crystal structure of RPS19 16 17 from Pyrococcus abyssi. The protein forms a five (cid:1)-helix bundle organized 18 19 20 around a central amphipathic (cid:1)-helix, which corresponds to the DBA mutation F 21 22 hot spot. From thoe structure, we classify DBA mutations relative to their 23 24 r 25 respective impact on pro tein folding (class I) or on surface properties (class II). 26 27 Class II mutations clusterP into two conserved basic patches. In vivo analysis in 28 29 e yeast demonstrates an essential role for class II residues in the incorporation into 30 31 e 32 pre-40S ribosomal particles. This drata indicate that missense mutations in DBA 33 34 primarily affect the capacity of the protein to be incorporated into pre- 35 R 36 ribosomes, thus blocking maturation of the pre-40S particles. 37 e 38 39 v 40 i 41 42 e 43 44 w 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2 Page 3 of 27 Nucleic Acids Research 1 2 3 INTRODUCTION 4 5 Diamond Blackfan anemia (DBA) is a rare congenital disorder characterized by the 6 7 8 defective differentiation of pro-erythroblasts, the precursors of red blood cells. 9 10 Patients suffer severe anemia and display heterogeneous clinical features including 11 12 malformations, growth failure and predisposition to cancer (1,2). Linkage analysis has 13 14 15 revealed that a quarter of all DBA reported cases are connected to the heterozygous 16 17 mutation of the gene encoding the ribosomal protein RPS19 (3,4). The RPS19 protein 18 19 20 is a component of the 40S ribosomal subunit and belongs to a family of ribosomal F 21 22 proteins restricted to eukaryotes and archeon. It is essential for yeast viability and for o 23 24 r early stages of development in mice (5) (6). Disruption, as well as point mutations of 25 26 27 the RPS19 gene in yeast andP human cells, affect maturation of the pre-ribosomal RNA 28 29 (pre-rRNA) and block producteion of the 40S ribosomal subunits (5,7-9). Why the 30 31 e mutation of a ribosomal protein primarily affects pro-erythroblast differentiation 32 r 33 34 remains a central question. However, recent linkage of a second ribosomal protein 35 R 36 gene, rps24, to DBA (10) strongly supports the hypothesis that DBA is the 37 e 38 consequence of a ribosomal disorder (8,11). 39 v 40 i 41 Over 60 different mutations affecting the rps19 gene have been reported, including 42 e 43 deletions, insertions, frameshifts, premature stop codons, and missense mutations (12- 44 w 45 46 14). Some mutations, like very early stop codons or modification of the promoter 47 48 clearly result in RPS19 haplodeficiency by hampering synthesis of RPS19 from the 49 50 mutated allele. However, for more subtle mutations like misense mutations, the 51 52 53 question arises as to whether they affect the folding of the protein or whether they are 54 55 milder mutations affecting the function while preserving the overall fold. Since there 56 57 is no homolog of RPS19 in bacteria, for which high-resolution structures of the small 58 59 60 ribosomal subunit are available, the structure of RPS19 and its precise location within 3 Nucleic Acids Research Page 4 of 27 1 2 3 the 40S subunit remain unknown. The crystal structure of RPS19 from Pyroccocus 4 5 abyssi presented herein fills this gap and provides a rationale for the impact of RPS19 6 7 8 mutations in Diamond-Blackfan anemia. 9 10 11 12 MATERIAL AND METHODS 13 14 15 16 17 Protein expression, purification and crystallization 18 19 20 F 21 22 Pyrococcus abyssi RPS19 was cloned into a pET-15b (Novagen) modified plasmid. o 23 24 r The expression was carried out in BL21 (DE3) Rosetta cells (Novagen) at 15°C. 25 26 27 Bacterial cells were sonicatePd and centrifuged for 30 min at 50 000 g. The supernatant 28 29 was heated up for 20 min at 5e0°C and centrifuged for another 30 min at 50 000 g. 30 31 e After incubation and elution from the cobalt-affinity resin, the tag was cleaved from 32 r 33 34 the protein by an overnight digest with 1/200 w/w ratio with TEV. A step of 35 R 36 purification on Hi-S (Pharmacia) was carried out and the protein was eluted at about 37 e 38 600 mM NaCl. The protein was concentrated up to 10 mg/ml in 50 mM Tris-HCl pH 39 v 40 i 41 7.5, 600 mM NaCl. Crystals were obtained 20°C by the hanging drop vapor diffusion 42 e 43 method by mixing equal amounts of the protein solution and of a reservoir composed 44 w 45 46 of 30 % - 36 % PEG 2000 MME, pH 6.8 – 7.5 over a couple days to a size of 50 x 47 48 200 x 200 microns. They diffracted to 1.15Å on synchrotron beamline and belonged 49 50 to the space group P2 2 2 with cell dimensions a= 32 Å, b=57 Å, c=82 Å and 51 1 1 1 52 53 contained one molecule per asymmetric unit and 48 % solvent. 54 55 56 57 Structure solution and refinement 58 59 60 4 Page 5 of 27 Nucleic Acids Research 1 2 3 Native and derivative data were collected at the ESRF synchrotron and processed with 4 5 XDS (15). Data collection statistics are shown in Table 1. A HgBr2 derivative 6 7 8 resulting from a 12 hours soaks in 2.5 % (v/v) HgBr2 (prepared from a saturated 9 10 solution) was collected at the LIII-edge og Hg. Derivative dataset was combined with 11 12 the most isomorphous native dataset, SHELXD and SHARP were used to locate and 13 14 15 refine the heavy atom site position (16,17). The resulting phases had an overall figure 16 17 of merit (F.O.M) of 0.33 at a resolution of 2.0 Å. After solvent flattening with 18 19 20 Solomon (SHARP), automatic building was carried out with ARP/wARP (18). 125 F 21 22 residues out of a total of 150 were initially placed. Further building and refinement o 23 24 r cycles were carried out with Coot and REFMAC (19,20). Last cycles of refinement 25 26 27 were carried out including hPydrogens and using individual anisotropic B factors. 28 29 The final model has a good steereochemistry with an R-free value of 15.5 % and a R- 30 31 e factor value of 13.7 % (Table 1). 32 r 33 34 35 R 36 Analysis of RPS19 mutations in yeast 37 e 38 39 v 40 i 41 Site-directed mutagenesis on yeast RPS19 was performed by PCR. For 42 e 43 complementation experiments, the mutated alleles were subcloned into vector pFL38- 44 w 45 46 Ps15 (URA3), downstream of the constitutive RPS15 promoter. The resulting 47 48 plasmids were introduced in strain GAL-RPS19 (ura3M; (5)). Cells were cultured in 49 50 liquid synthetic medium containing galactose, but no uracile, and spotted at different 51 52 53 densities on agar plates using the same medium, with either galactose or glucose as 54 55 the carbon source to modulate RPS19 expression. 56 57 To evaluate incorporation of wild-type or mutated RPS19A into ribosomes, the 58 59 60 corresponding open-reading frames were also cloned in frame downstream the TAP 5 Nucleic Acids Research Page 6 of 27 1 2 3 tag coding sequence in the pFL38-Ps15 vector and expressed in Euroscarf strain 4 5 Y06271 (rps19A::KanMX, RPS19B). Whole cell extracts were fractionated by 6 7 8 ultracentrifugation on sucrose gradient for ribosome analysis. Two hundred milliliters 9 10 of yeast culture were grown in YPD medium to an OD of 0.5 and cycloheximide 600 11 12 13 was added at a final concentration of 100 µg/ml. After 10 min incubation, yeast cells 14 15 were harvested by centrifugation at 5 000 rpm and washed in 20 ml ice-cold buffer A 16 17 (20 mM HEPES [pH 7.5], 10 mM KCl, 5 mM MgCl2, 1 mM EGTA, 1 mM DTT, and 18 19 20 100 µg/ml cycloheximide). Cells were broken with glass beads and resuspended in F 21 22 150 microliters buffer A. The suspension was clarified by centrifugation for 5 min at o 23 24 r 25 10 000 rpm. An amount of the extract corresponding to 1 mg of protein was loaded on 26 27 a 10.5 ml 10%–50% sucProse gradient in buffer A without cycloheximide and 28 29 e centrifuged for 12 hours at 26 000 rpm in a SW41 rotor. A gradient collector 30 31 e 32 (ÄKTAprime – Amersham Bioscienrces), in combination with a Pharmacia UV- 33 34 detector LKB.UV-M II, was used to record the UV profile. Twenty 0.5 ml fractions 35 R 36 were collected and 150 microliters of each fraction were slot-blotted on nitrocellulose 37 e 38 39 membrane to detect TAP-tagged RPS19A. The vmembrane was then incubated with 40 i 41 peroxidase anti-peroxidase complexes (Sigma), which was revealed by 42 e 43 44 chemoluminescence (ECL, GE Healthcare). After scanning of the film to obtain a w 45 46 digital image, the labeling intensity for each fraction was quantified by densitometry 47 48 using MetaMorph (Universal Imaging). The UV profiles were superimposed with the 49 50 51 blot quantifications to obtain figure 5. 52 53 Ribosomal RNAs were co-immunoprecipitated with RPS19-TAP and analyzed as 54 55 published previously (21,22). Pre-rRNAs were detected on northern-blots with probe 56 57 58 D-A2 (5'-GAAATCTCTCACCGTTTGGAATAGC-3') and A2-A3 (5(cid:8)- 59 60 ATGAAAACTCCACAGTG-3(cid:8)). 6 Page 7 of 27 Nucleic Acids Research 1 2 3 4 5 6 7 8 RESULTS AND DISCUSSION 9 10 11 12 Overall structure of RPS19 13 14 15 16 17 The structure of RPS19 from P. abyssi (RPS19Pa) was determined by SIRAS on a 18 19 20 mercury derivative and refined against the best native. The final model has an R-free F 21 22 value of 15.5 % (Table 1) and comprises residues 2 to 150 with the exception of two o 23 24 r disordered loops between residues 37 to 44 and 79 to 84 (Figure 1a). The structure of 25 26 27 RPS19 is almost entirely (cid:1)P-helical and folds around a five (cid:1)-helix bundle. Knowing 28 29 that human RPS19 (RPS19Hs) sehares 36% identity and 57% homology with P. abyssi 30 31 e 32 RPS19 (RPS19Pa) sequence (Figure 1rc), we may assume that they display a similar 33 34 fold. In the rest of the text, RPS19 residues in P. abyssi, S. cerevisiae and H. sapiens 35 R 36 are labeled “Pa”, “Sc”and “Hs” respectively (see also Table 2). 37 e 38 39 v 40 i 41 Two distinct classes of mutations in DBA patients cluster around an amphipathic 42 e 43 helix 44 w 45 46 47 48 Fourteen amino acids of RPS19 are the targets of missense mutations in DBA 49 50 patients. Although these mutations are spread along the entire primary sequence 51 52 53 (Figure 1c), the structure of RPS19Pa shows that most missense mutations cluster 54 55 within or around the (cid:1)-helix 3 (Figure 1). This (cid:1)-helix, made of residues 50 to 65 of 56 57 58 RPS19Pa (52 to 67 in human numbering), corresponds to the mutation hot spot. It is 59 60 located at a central position in the structure where it bridges (cid:1)-helices 1 and 6 on one 7 Nucleic Acids Research Page 8 of 27 1 2 3 side with (cid:1)-helices 4 and 5 on the other side (Figure 1a). The apolar side of (cid:1)-helix 3 4 5 6 is engaged into hydrophobic interactions with residues of the neighboring (cid:1)-helices, 7 8 thus forming the hydrophobic core of the protein (Figure 1b). Strikingly, three 9 10 mutations on (cid:1)-helix 3 (A57PHs, A61S/EHs, L64PHs), two on (cid:1)-helix 1 (V15FHs and 11 12 13 L18PHs) and two on (cid:1)-helix 6 (G127EHs and L131RHs) affect residues involved in this 14 15 hydrophobic core (green residues on Figure 1). Thus, although distant on the primary 16 17 18 sequence, these seven amino acids are functionally related and are involved in the 19 20 folding and the stability of the protein. F 21 22 23 In contrast, mutationos P47LHs, W52RHs, R56QHs, S59FHs, R62W/QHs, R101HHs, and 24 r 25 G120SHs affect residues lo cated on the surface of RPS19 (red residues on Figure 1). 26 27 P Remarkably, these residues show a much higher degree of inter-species conservation 28 29 e 30 than the amino acids within the hydrophobic core (Figure 1c). These residues are 31 e 32 located within two highly conserved brasic patches at the surface of the protein (Figure 33 34 2). On the polar face of (cid:1)-helix 3, four exposed mutated residues (W52Hs, R56Hs, 35 R 36 37 S59Hs and R62Hs) form the floor of a central ebasic groove (patch A on Figure 2a, 2b 38 39 v and 2c). In addition, residues from (cid:1)-helices 4 and 5 and from the (cid:3)-sheet define the 40 i 41 42 conserved patch B (Figure 2d, 2e and 2f). It comperises residues R99Pa (R101Hs), 43 44 K100Pa (R102Hs), Q103Pa (Q105Hs), K113Pa (K115Hs), Gw118Pa (G120Hs) and R119Pa 45 46 (R121Hs). DBA mutations R101H Hs and G120S Hs affect this conserved surface. 47 48 49 Interestingly, both conserved surfaces have a positive electrostatic charge: patch A 50 51 harbors a localized positive charge (Figure 2c), whereas patch B is embedded in a 52 53 54 larger positive area (Figure 2f). 55 56 Based on these observations, we propose to subdivide DBA mutations into two 57 58 classes: class I encompasses structural residues affecting the folding of the protein; 59 60 class II mutations affect surface residues and would impair the function of RPS19 8