Solution structure of the two N-terminal RNA-binding domains of nucleolin and NMR study of the interaction with its RNA target.
Nucleolin is an abundant 70 kDa nucleolar protein involved in many aspects of ribosomal RNA biogenesis. The central region of nucleolin contains four tandem consensus RNA-binding domains (RBD). The two most N-terminal domains (RBD12) bind with nanomolar affinity to an RNA stem-loop containing the consensus sequence UCCCGA in the loop. We have determined the solution structure of nucleolin RBD12 in its free form and have studied its interaction with a 22 nt RNA stem-loop using multidimensional NMR spectroscopy. The two RBDs adopt the expected beta alpha beta beta alpha beta fold, but the position of the beta 2 strand in both domains differs from what was predicted from sequence alignments. RBD1 and RBD2 are significantly different from each others and this is likely important in their sequence specific recognition of the RNA. RBD1 has a longer alpha-helix 1 and a shorter beta 2-beta 3 loop than RBD2, and differs from most other RBDs in these respects. The two RBDs are separated by a 12 amino acid flexible linker and do not interact with one another in the free protein. This linker becomes ordered when RBD12 binds to the RNA. Analysis of the observed NOEs between the protein and the RNA indicates that both RBDs interact with the RNA loop via their beta-sheet. Each domain binds residues on one side of the loop; specifically, RBD2 contacts the 5' side and RBD1 contacts the 3'.
Allain
F H
FH
Department of Chemistry and Biochemistry, University of California, 405 Hilgard Avenue, Los Angeles, 90095-1569, USA.
Gilbert
D E
DE
Bouvet
P
P
Feigon
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D018583:Pliability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D016415:Sequence Alignment; D012996:Solutions; D013379:Substrate Specificity; D000097796:Nucleolin
D010750:Phosphoproteins; D012313:RNA; D016601:RNA-Binding Proteins; D012996:Solutions; D000097796:Nucleolin