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 co nsensus sequence UCCCGA in the loop. We have determined the solution struct ure of nucleolin RBD12 in its free form and have studied its interaction wi th a 22 nt RNA stem-loop using multidimensional NMR spectroscopy. The two R BDs adopt the expected beta alpha beta beta alpha beta fold, but the positi on of the beta2 strand in both domains differs from what was predicted from sequence alignments. RBD1 and RBD2 are significantly different from each o thers and this is Likely important in their sequence specific recognition o f the RNA. RBD1 has a longer alpha -helix 1 and a shorter beta2-beta3 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 bin ds to the RNA. Analysis of the observed NOEs between the protein and the RN A 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 co ntacts the 5' side and RBD1 contacts the 3'. (C) 2000 Academic Press.