Musashi protein is supposed to be involved in the regulation of differentiation of neural stem cells. Musashi binds to 3' untranslated region of target mRNA and represses the translation of mRNA. Musashi has two tandem RNA-binding domains (RBDs), RBD1 and RBD2. Both RBDs cooperatively bind to the target mRNA. Here, we determined the structure of RBD1-RBD2 in complex with target RNA. First, the structures of two RBDs in the complex were determined on the basis of short-range distance restrains derived from NOEs. However, the relative position of two RBDs was not determined due to the lack of long-range distance restraints across two RBDs. In order to overcome the situation, we introduced the paramagnetic center into Musashi by attaching MTSL carrying the NO radical. The long-range distance restraints (ca. 20-40 A) between two RBDs were derived from paramagnetic relaxation enhancement (PRE) caused by the paramagnetic center. The relative position of two RBDs was successfully determined on the basis of these long-range distance restraints. The change in the relative position of two RBDs on binding to the target RNA was also detected by PRE. The determined structure of RBD1-RBD2 in the complex has suggested how Musashi recognizes its target mRNA.
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