Structure, backbone dynamics and interactions with RNA of the C-terminal RNA-binding domain of a mouse neural RNA-binding protein, Musashi1

Musashil is an RNA-binding protein abundantly expressed in the developing m ouse central nervous system. Its restricted expression in neural precursor cells suggests that it is involved in the regulation of asymmetric cell div ision. Musashil contains two ribonucleoprotein (RNP)-type RNA-binding domai ns (RBDs), RBD1 and RBD2. Our previous studies showed that RBD1 alone binds to RNA, while the binding of RBD2 is not detected under the same condition s. Joining of RBD2 to RBD1, however, increases the affinity to greater than that of RBD1 alone, indicating that RBD2 contributes to RNA-binding. We ha ve determined the three-dimensional solution structure of the C-terminal RB D (RBD2) of Musashil by NMR. It folds into a compact alpha beta structure c omprising a four-stranded antiparallel beta-sheet packed against two alpha- helices, which is characteristic of RNP-type RBDs. Special structural featu res of RBD2 include a beta-bulge in beta 2 and a shallow twist of the beta- sheet. The smaller H-1-N-15 nuclear Overhauser enhancement values for the r esidues of loop 3 between beta 2 and beta 3 suggest that this loop is flexi ble in the time-scale of nano- to picosecond order. The smaller N-15 T-2 va lues for the residues around the border between alpha 2 and the following l oop (loop 5) suggest this region undergoes conformational exchange in the m illi- to microsecond time-scale. Chemical shift perturbation analysis indic ated that RBD2 binds to an RNA oligomer obtained by in vitro selection unde r the conditions for NMR measurements, and thus the nature of the weak RNA- binding of RBD2 was successfully characterized by NMR, which is otherwise d ifficult to assess. Mainly the residues of the surface composed of the four -stranded P-sheet, loops and C-terminal region are involved in the interact ion. The appearance of sidechain NH proton resonances of arginine residues of loop 3 and imino proton resonances of RNA bases upon complex formation s uggests the formation of intermolecular hydrogen bonds. The structural arra ngement of the rings of the conserved aromatic residues of beta 2 and beta 3 is suitable for stacking interaction with RNA bases, known to be one of t he major protein-RNA interactions, but a survey of the perturbation data su ggested that the stacking interaction is not ideally achieved in the comple x, which may be related to the weaker RNA-binding of RBD2. (C) 1999 Academi c Press.