Biochemical characterization and crystal structure of a Dim1 family associated protein: Dim2.
The U4/U6*U5 tri-snRNP complex is the catalytic core of the pre-mRNA splicing machinery. The thioredoxin-like protein hDim1 (U5-15 kDa) constitutes an essential component of the U5 particle, and its functions have been reported to be highly conserved throughout evolution. Recently, the Dim1-like protein (DLP) family has been extended to other proteins harboring similar sequence motifs. Here we report the biochemical characterization and crystallographic structure of a 149 amino acid protein, hDim2, which shares 38% sequence identity with hDim1. The crystallographic structure of hDim2 solved at 2.5 A reveals a classical thioredoxin-fold structure. However, despite the similarity in the thioredoxin fold, hDim2 differs from hDim1 in many significant features. The structure of hDim2 contains an extra alpha helix (alpha3) and a beta strand (beta5), which stabilize the protein, suggesting that they may be involved in interactions with hDim2-specific partners. The stability and thermodynamic parameters of hDim2 were evaluated by combining circular dichroism and fluorescence spectroscopy together with chromatographic and cross-linking approaches. We have demonstrated that, in contrast to hDim1, hDim2 forms stable homodimers. The dimer interface is essentially stabilized by electrostatic interactions and involves tyrosine residues located in the alpha3 helix. Structural analysis reveals that hDim2 lacks some of the essential structural motifs and residues that are required for the biological activity and interactive properties of hDim1. Therefore, on the basis of structural investigations we suggest that, in higher eukaryotes, although both hDim1 and hDim2 are involved in pre-mRNA splicing, the two proteins are likely to participate in different multisubunit complexes and biological processes.
Simeoni
Federica
F
Department of Molecular Biophysics and Therapeutics, Centre de Recherches de Biochimie Macromoléculaire, FRE-2593 CNRS, 1919 Route de Mende, 34293 Montpellier, France.
Arvai
Andy
A
Bello
Paul
P
Gondeau
Claire
C
Hopfner
Karl-Peter
KP
Neyroz
Paolo
P
Heitz
Frederic
F
Tainer
John
J
Divita
Gilles
G
D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D019281:Dimerization; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D017415:Ribonucleoprotein, U5 Small Nuclear; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
D018797:Cell Cycle Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; D017415:Ribonucleoprotein, U5 Small Nuclear; D012996:Solutions; C506012:TXNL4A protein, human; C506013:TXNL4B protein, human