Structure of the C-terminal RNA-binding domain of hnRNP D0 (AUF1), its interactions with RNA and DNA, and change in backbone dynamics upon complex formation with DNA

Abstract

Heterogeneous nuclear ribonucleoprotein (hnRNP) D0 has two ribonucleoprotein (RNP) -type RNA-binding domains (RBDs), each of which can specifically bind to the UUAG-sequence. hnRNP D0 also binds specifically to single-stranded d(TTAGGG)n, the human telomeric DNA repeat. We have already reported the structure and interactions with RNA of the N-terminal RBD (RBD1). Here, the structure of the C-terminal RBD (RBD2) determined by NMR is presented. It folds into a compact αβ structure comprising an antiparallel β-sheet packed against two α-helices, which is characteristic of RNP-type RBDs. In addition to the four β-strands commonly found in RNP-type RBDs, an extra β-strand, termed β4−, was found just before the fourth β-strand, yielding a five-stranded β-sheet. Candidate residues of RBD2 involved in the interactions with RNA were identified by chemical shift perturbation analysis. Perturbation was detected on the β-sheet side, not on the opposite α-helix side, as observed for RBD1. It is notable that the β4− to β4 region of RBD2 is involved in the interactions in contrast to the case of RBD1. The chemical shift perturbation analysis also showed that RBD2 interacts with DNA in essentially the same way as with RNA. Changes in the backbone dynamics upon complex formation with DNA were examined by means of model free analysis of relaxation data. In free RBD2, the β4− to β4 region exhibits slow conformational exchange on the milli- to microsecond time scale. The exchange is quenched upon complex formation. The flexibility of free RBD2 may be utilized in the recognition process by allowing different conformational states to be accessed and facilitating induced fit. Additionally, faster flexibility on the nano- to picosecond time scale was observed for loop 3 located between β2 and β3 in free RBD2, which is retained by the complex as well.