Molecular basis of Diamond Blackfan anemia

29 Pages·2017·1.82 MB·English

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