Structure of a key intermediate of the SMN complex reveals Gemin2's crucial function in snRNP assembly - PubMed (original) (raw)

Structure of a key intermediate of the SMN complex reveals Gemin2's crucial function in snRNP assembly

Rundong Zhang et al. Cell. 2011.

Abstract

The SMN complex mediates the assembly of heptameric Sm protein rings on small nuclear RNAs (snRNAs), which are essential for snRNP function. Specific Sm core assembly depends on Sm proteins and snRNA recognition by SMN/Gemin2- and Gemin5-containing subunits, respectively. The mechanism by which the Sm proteins are gathered while preventing illicit Sm assembly on non-snRNAs is unknown. Here, we describe the 2.5 Å crystal structure of Gemin2 bound to SmD1/D2/F/E/G pentamer and SMN's Gemin2-binding domain, a key assembly intermediate. Remarkably, through its extended conformation, Gemin2 wraps around the crescent-shaped pentamer, interacting with all five Sm proteins, and gripping its bottom and top sides and outer perimeter. Gemin2 reaches into the RNA-binding pocket, preventing RNA binding. Interestingly, SMN-Gemin2 interaction is abrogated by a spinal muscular atrophy (SMA)-causing mutation in an SMN helix that mediates Gemin2 binding. These findings provide insight into SMN complex assembly and specificity, linking snRNP biogenesis and SMA pathogenesis.

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Figures

Figure 1. Gemin2 binds the Sm pentamer, D1, D2, E, F, and G

Recombinant GST-SMN or GST-SMN/Gemin2 was used for the binding to recombinant Sm proteins (left panel). Similarly in a separate experiment, recombinant GST-Gemin2 or GST-Gemin2 with C-terminus truncated SMN (residues 1–194) was used for the binding to Sm proteins (right panel). Either individual subcore complex (SmD1/D2, SmF/E/G or SmB/D3), or the five (SmD1/D2/F/E/G) or all seven Sm proteins (SmD1/D2/F/E/G/B/D3) were used in binding. For efficient expression, SmB/D3 proteins do not contain their C-terminal RG-rich domain. The asterisk indicates degradation products of GST-Gemin2. The total panel shows 5% of the proteins used for binding.

Figure 2. Overall structure of the Gemin2-SMNGe2BD-Sm pentamer complex

Four rotational views of the ribbon diagram of the complex structure (PDB ID code 3S6N) are shown with the surface representation. The five Sm proteins, SmD1, D2, F, E, and G are colored in lime, lemon, pink, dark green and orange, respectively. Gemin2 and SMNGe2BD peptide are colored in red and blue, respectively. Gemin2’s domains and secondary structures are labeled. In panel (A), the five Sm proteins are arranged in the clockwise order of SmD1, D2, F, E and G to form a 5/7 of doughnut shape with their canonical N-terminal helices facing out. The structure contains visible structures of SmD1 (residues 1–81), SmD2 (residues 23–76 and 90–116), SmF (residues 3–76), SmE (residues 14–90), SmG (residues 14–51 and 56–72), Gemin2 (residues 22–31, 47–69, 83–123, 134–149 and 179–276) and SMN (residues 37–51). The disordered loop connecting the N-terminal tail and α1 of Gemin2 is shown in a red dashed line in panel (A). The disordered loop connecting the N- and C-terminal domains of Gemin2 are also shown in red dashed lines in panels (A), (B) and (D). The figures are prepared using PyMOL (The PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC). The rotation of the views is indicated by arrow and the degree of the rotation.

Figure 3. Multiple sequence alignment of Gemin2 orthologs from diverse organisms

The sequence alignment was performed using the Praline program (Simossis and Heringa, 2005). Human, Homo sapiens (accession number NP_003607); mouse, Mus musculus (NP_079932); chicken, Gallus gallus (NP_989530); zebrafish, Danio rerio (NP_001017608); hydra, Hydra magnipapillata (XP_002160350); S. pombe, Schizosaccharomyces pombe (CAB88094). Conservation of sequence is represented based on the BLOSUM score matrix. Partially conserved residues (conservation score 4–7) are highlighted as light gray; highly conserved residues (conservation score 8–10) are highlighted as dark gray. Gemin2’s secondary structure, as seen in the crystal, is indicated with red boxes for α-helices, blue box for the β-sheet and green lines for the loops. Positions of the mutations (Tyr52 and Arg213) are indicated by arrows.

Figure 4. Detailed views of Gemin2 structure and its interactions with the Sm pentamer

Ribbons are colored as in Figure 2. Amino acid residues involved in the interactions as described in the text are shown in sticks (A–C) or spheres (D). Side chain carbon atoms are labeled in the same color as their respective protein chains. Oxygen and nitrogen are colored in red and blue, respectively. Potential hydrogen-bonds and salt bridges are shown as dashed lines. Water molecules are shown as red spheres. (A) Interactions between Gemin2’s C-terminal domain and SmD1/D2. (B) Interactions between α1 of Gemin2 and SmF/E. (C) Interactions between β1 of Gemin2 and β2 of SmF. (D) Gemin2’s N-terminal tail inserts into the Sm pentamer’s RNA-binding pocket and interacts with the five Sm proteins. The five amino acids shown in blue spheres, Ser35, His62, Tyr39, Tyr53 and Phe32 in Sm D1, D2, F, E and G, respectively, are involved in the interactions with RNA by stacking nucleotide bases from the top. Gemin2’s Leu24, Met25, and Leu28 block the RNA nucleotide binding pockets of SmF, E, and G, respectively.

Figure 5. Gemin2 prevents the Sm pentamer from binding RNA

(A) Gemin2 dose-dependently inhibits the Sm pentamer from binding RNA and its N-terminus is critical for this inhibition. Gemin2 or Gemin2ΔN39 (1 μg) co-purified with GST-SMNGe2BD was pre-incubated with increasing amounts of the five Sm proteins (D1/D2/F/E/G; 10–50 ng of each) and then assayed for binding to [32P]-α-UTP labeled U4 or U4ΔSm snRNA. SmD1/D2 or SmF/E/G was used as controls (left panel). In a parallel experiment, SmD1/D2/F/E/G was pre-incubated with increasing amounts (1–9 μg) of Gemin2 or Gemin2ΔN39 that were co-purified with GST-SMNGe2BD and assayed for RNA-binding. The positions of the free RNA and the Sm pentamers assembled on the RNA are indicated. (B) The effects of wild type or various mutant Gemin2 proteins on the Sm pentamer’s binding to RNA. Increasing amounts (1–9 μg) of GST, GST-Gemin2 wild type (WT), or various GST tagged Gemin2 mutants (Y52D and R213D) were pre-incubated with SmD1/D2/F/E/G and assayed for binding to U4 snRNA as in (A). (C) Protein binding between wild type or various Gemin2 mutants and the Sm pentamer. Recombinant wild type and mutant Gemin2 proteins (2.5–5 μg) used in (A) and (B) were assayed for binding to SmD1/D2/F/E/G (1 μg). The total panel shows 5% of the proteins used for binding.

Figure 6. Critical residues involved in SMNGe2BD-Gemin2 interaction and an SMA-causing patient mutation in SMN abrogates its interaction with Gemin2

(A) A detailed view of the SMNGe2BD-Gemin2 polar interactions. Ribbons are colored as in Figure 2. Amino acid residues, side chain carbon atoms, oxygen and nitrogen, and potential hydrogen-bonds and salt bridges are labeled as in Figure 4. Asp44 (D44) of SMN is mutated in an SMA patient. Lys41 of SMN is not involved in interactions with Gemin2 and is therefore used as a control for the binding experiment in (C). Additional hydrophobic interactions, involving Leu39, Tyr43, Val47 and Phe50 in SMN interacting with a hydrophobic pocket in Gemin2, are shown in Figure S4. (B) Electron density map of an additional view of the interaction regions between Gemin2 and SMNGe2BD. SigmaA-weighted 2Fo-Fc electron density map (gray mesh) is contoured at 1.2σ. The color schemes are the same as in (A). Protein models are shown in cartoon representation with contacting residues in sticks. (C) The SMA patient mutation, D44V, abrogates SMN-Gemin2 interaction. In vitro translated [35S]-Met labeled wild type SMN and the indicated mutant proteins were incubated with recombinant GST-Gemin2. SMNΔN51 lacks the Gemin2 binding domain (Wang and Dreyfuss, 2001) and is used as a negative control. The total panel shows 5% of the proteins used for the binding. (D) Reciprocal binding reactions were performed as in (C). In vitro translated [35S]-Met labeled wild type Gemin2 or mutants (Y52D and R213D) were incubated with recombinant GST-SMN that was immobilized on beads.

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Structure of a key intermediate of the SMN complex reveals Gemin2's crucial function in snRNP assembly - PubMed (original) (raw)