Resonance assignments and solution structure of the second RNA-binding domain of sex-lethal determined by multidimensional heteronuclear magnetic resonance.
The RNA-binding protein Sex-lethal (Sxl) is a critical regulator of sexual differentiation and dosage compensation in Drosophila. This regulatory activity is a consequence of the ability of Sxl to bind uridine-rich RNA tracts involved in pre-mRNA splicing. Sxl contains two RNP consensus-type RNA-binding domains (RBDs). A structural study of a portion of Sxl (amino acids 199-294) containing the second RNA-binding domain (RBD-2) using multidimensional heteronuclear NMR is presented here. Nearly complete 1H, 13C, and 15N resonance assignments have been obtained from 15N- and 13C/15N-uniformly labeled protein. These assignments were used to analyze 3D 15N-separated NOESY and 13C/13C-separated 4D NOESY spectra which produced 494 total and 169 long-range NOE-derived distance restraints. Along with 41 backbone dihedral restraints, these distance restraints were employed to generate an intermediate-resolution family of calculated structures, which exhibits the beta alpha beta-beta alpha beta tertiary fold found in other RBD-containing proteins. The RMSD to the average structure for the backbone atoms of residues 11-93 is 1.55 +/- 0.30 A, while the RMSD for backbone atoms involved in secondary structure is 0.76 +/- 0.14 A. A capping box [Harper, E.T., & Rose, G.D. (1993) Biochemistry 32, 7605-7609] was identified at the N-terminus of the first helix and has been characterized by short- and medium-range NOEs. Finally, significant structural similarities and differences between Sxl RBD-2 and other RBD-containing proteins are discussed.
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Department of Chemistry, University of California, Berkeley 94720-1460.
Kanaar
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Wemmer
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004330:Drosophila; D029721:Drosophila Proteins; D004926:Escherichia coli; D005812:Genes, Switch; D007301:Insect Hormones / Q000737:chemistry* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D012996:Solutions
D029721:Drosophila Proteins; D007301:Insect Hormones; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012996:Solutions; C067777:Sxl protein, Drosophila; D012313:RNA