Conformational flexibility and molecular interactions of an archaeal homologue of the Shwachman-Bodian-Diamond syndrome protein

Abstract

Background: Defects in the human Shwachman-Bodian-Diamond syndrome (SBDS) protein-coding gene lead to the autosomal recessive disorder characterised by bone marrow dysfunction, exocrine pancreatic insufficiency and skeletal abnormalities. This protein is highly conserved in eukaryotes and archaea but is not found in bacteria. Although genomic and biophysical studies have suggested involvement of this protein in RNA metabolism and in ribosome biogenesis, its interacting partners remain largely unknown.

Results: We determined the crystal structure of the SBDS orthologue from Methanothermobacter thermautotrophicus (mthSBDS). This structure shows that SBDS proteins are highly flexible, with the N-terminal FYSH domain and the C-terminal ferredoxin-like domain capable of undergoing substantial rotational adjustments with respect to the central domain. Affinity chromatography identified several proteins from the large ribosomal subunit as possible interacting partners of mthSBDS. Moreover, SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiments, combined with electrophoretic mobility shift assays (EMSA) suggest that mthSBDS does not interact with RNA molecules in a sequence specific manner.

Conclusion: It is suggested that functional interactions of SBDS proteins with their partners could be facilitated by rotational adjustments of the N-terminal and the C-terminal domains with respect to the central domain. Examination of the SBDS protein structure and domain movements together with its possible interaction with large ribosomal subunit proteins suggest that these proteins could participate in ribosome function.

Figure 1

Ribbon diagram of mthSBDS. Stereo figure is drawn with colors blending from blue (N-terminus) through to red (C-terminus). Labels indicate domains I, II and III.

Figure 2

SBDS protein flexibility. SBDS molecules are superposed by domain II. The view is as in Figure 1 but rotated by ~90° around the vertical axis. Labels indicate domains I, II and III. a) Ribbon diagrams of two mthSBDS molecules present in the asymmetric unit (A, blue and B, yellow). b) Stereo view of Ca models of the two mthSBDS molecules (A, blue and B, yellow) and afSBDS ([PDB:1P9Q], red).

Figure 3

Sequence alignment of SBDS proteins from different species. Sequences of SBDS proteins from M. thermautotrophicus (MthSBDS), Archaeoglobus fulgidus (Aful), Halobacterium marismortui (Hmar), Saccharomyces cerevisiae (Scer), Human, Rat, Chicken, Anopheles gambiae (Agam), Drosophila melanogaster (Dmel), Caenorhabditis elegans (Cele) and Arabidopsis thaliana (Atha) were aligned. Green and orange triangles designate boundaries between domains 1 and 2 and domains 2 and 3, respectively. The secondary structure of mthSBDS (molecule A) is shown on top of the aligned sequences. Positions for which the percentage of 'equivalent' residues, considering their physico-chemical properties, is higher than 70% are colored in red on a white background and framed in blue boxes. If residues are strictly conserved, they are colored in white characters on a red background.

Figure 4

Location of L1, L2, L14 and L37AE proteins within the Haloarcula marismortui 50S ribosomal subunit [PDB:1JJ2, PDB:1MZP]. rRNA is shown as a ribbon (green) and proteins are represented by their molecular surfaces. The three proteins found to interact with mthSBDS and the ribosomal L37AE protein encoded within the exosome superoperon are labeled and colored.

Figure 5

Surface electrostatic potential of mthSBDS. Three different views, each 90° apart along the vertical axis of the figure, are shown with colors ranging from red (-0.5 V) to blue (0.5 V). Labels indicate domains I, II and III. The electrostatic potential was calculated using the PARSE forcefield at an implicit ionic strength of 150 mM NaCl and mapped on the solvent accessible surface of mthSBDS. The FYSH domain is at the bottom of the figure. On the right panel, the positions of selected positively charged residues are indicated with green arrows (see text for details).

Figure 6

Analysis of mthSBDS TLS groups. The asymmetric unit of the mthSBDS crystal is represented in ribbons with molecule A in orange and molecule B colored by domains: domain I in blue, domain II in green and domain III in magenta. The principal axes of libration of each TLS group are represented as arrows; their lengths are proportional to the mean libration along them.