Characterization of the Dynamics of an Essential Helix in the U1A Protein by Time-Resolved Fluorescence Measurements

The RNA recognition motif (RRM) is the one of the most common RNA binding domains and is found in nearly all proteins involved in post-transcriptional gene expression.1-6 The βαββαβ secondary structure of the RRM assembles into an RNA binding platform made up of a four-stranded antiparallel β-sheet, supported through an extensive hydrophobic core by two α helices.7 The RRM is modulated in different proteins to recognize single-stranded RNAs with diverse sequences and in a variety of structural contexts. While specific amino acid side-chains are observed to contact RNA in structures of RRM–RNA complexes, more subtle structural factors, including conformational changes and dynamic processes, may also be important contributors to binding affinity and specificity.4,8,9 A C-terminal helix that contributes to complex formation is often present in RRMs. This C-terminal helix plays diverse roles in different RRMs. For example, in the U1A protein, helix C changes position upon binding RNA.10,11 In contrast, in CstF-64, helix C unfolds upon binding,12,13 while in the Sex lethal, HuD, nucleolin, and Poly(A)-binding protein RRMs, unstructured C-terminal regions form helices upon binding RNA.14-18 Thus, the C-terminal amino acids of RRMs comprise a variable region that may contribute to specific RNA recognition through participation in dynamical processes. In this paper, we report the characterization of the dynamics of the C-terminal helix of the U1A protein using time-resolved fluorescence anisotropy. The U1A protein is a component of U1 snRNP, a subunit of the spliceosome.19,20 U1A binds with high specificity to two related target sites, stem loop 2 of U1 snRNA (SL2 RNA) and an internal loop region in the pre-mRNA of U1A.21-23 Although U1A contains two RRMs, only the N-terminal RRM has been observed to bind RNA.24,25 The N-terminal RRM of U1A has been structurally characterized free and bound to SL2 RNA by X-ray crystallography and NMR spectroscopy.10,11,26 A comparison of the free and bound structures suggests that recognition requires extensive conformational changes in both the protein and RNA. A significant component of the protein conformational change occurs in the orientation of helix C (D90–K98) (Figure 1). In the NMR structure of U1A in the absence of RNA, helix C interacts with conserved residues in the RNA binding region on the surface of the β sheet that is the primary site of RNA recognition,10 while in the complex helix C is positioned away from the β-sheet surface.11 Figure 1 Diagram of the superposition of the free and bound conformations of the U1A protein.10,11 The darker diagram is the structure of the free form of U1A, while the lighter diagram is the structure of U1A in complex with SL2 RNA. The role of the conformational change of helix C in complex formation is not clear. For example, the protein may be captured by the RNA while helix C is transiently in the open conformation or the RNA may induce the observed conformational change upon binding. Energy calculations based on molecular dynamics (MD) simulations have suggested that the stabilities of the open and closed structures are comparable.27 The X–ray structure of a construct of the free protein with a relatively short helix C sequence showed that helix C is in the open conformation, supporting the result from MD studies that the open and closed structures are of similar stabilities.26 Fluorescence and NMR experiments and MD simulations have suggested that helix C retains significant flexibility in the free U1A, although the time scale and range of dynamical motion has not been well-characterized.28-30 Mutational studies have shown that helix C contributes to binding affinity and specificity of the U1A protein for SL2 RNA and participates in cooperative networks of interactions with other residues involved in RNA binding.31,32 Studies using NMR, MD, and reorientational eigenmode dynamics techniques have suggested that these cooperative networks may originate in correlated dynamics involving helix C.30,33-35 Thus, the dynamical properties of helix C in the free protein are likely to make important cooperative contributions to RNA recognition by the U1A protein. In this paper, we report time-resolved fluorescence anisotropy experiments of a U1A construct containing Trp in helix C to directly investigate the dynamics of helix C on the picosecond to nanosecond time scale and to probe the influence of protein mutations on the dynamics of helix C. The identification of the segmental dynamics of helix C in the U1A protein is supported by comparison to a U1A protein labeled with Trp in the stable β-sheet, rather than the C-terminal helix. The data suggest the cone angle of motion of helix C to be 20°, which is similar to the cone angle predicted from molecular dynamics simulations performed on the U1A protein.27,36 Mutation of an amino acid on the surface of the β-sheet that contacts helix C or an amino acid in the hinge region between helix C and the remainder of the protein destabilizes the complex, but does not dramatically alter the dynamics of helix C. Together these results suggest that helix C is not equilibrating between the closed and open form on the nanosecond time scale, but is undergoing a more limited degree of dynamical motion within the closed conformation that is relatively insensitive to mutation of residues that contact the helix or link the helix to the remainder of the protein.