Journal of Molecular Biology
CommunicationAlternative Conformations at the RNA-binding Surface of the N-terminal U2AF65 RNA Recognition Motif
Section snippets
High-Resolution Structure of U2AF65-RRM1
The structure of human U2AF65-RRM1 (residues 148–229) was determined by multiwavelength anomalous dispersion (MAD) phasing (Supplementary Data Table 1). The final U2AF65-RRM1 coordinates consist of 87 residues, including five N-terminal residues from the protease cleavage site (Gly-Pro-Leu-Gly-Ser), 193 water molecules, two zinc ions and a partial molecule of PEG MME 550 (Figure 1(a) and (b)). The secondary structural arrangement of U2AF65-RRM1 (βαββαβ) corresponds to the canonical RRM fold,1
Comparison of the Overall apo and RNA-Bound U2AF65-RRM1 Structures
The major differences between the apo and RNA-bound U2AF65 RRM1 structures are summarized in Table 1. Overall, the positions of the U2AF65-RRM1 backbone atoms are largely unchanged by association with RNA (0.6 Å r.m.s.d. for 82 matching Cα atoms) (Figure 2(a)). The β2/β3 loop is the most variable region between the apo and RNA-bound U2AF65-RRM1 X-ray structures (Figure 2(b)). Despite its minimal length, the β2/β3 loop is highly flexible, as reflected by poorly defined electron density and the
Comparison of Bound Water Molecules in the apo and RNA-Bound U2AF65-RRM1 Structures
The lower resolution of the RNA-bound U2AF65-RRM1 hinders a comprehensive analysis of the positions of water molecules bound to the protein or RNA surface. However, five water molecules interacting with four of the seven bound nucleotides (U3, U4, U5, and U7) were observed in the electron density for the U2AF65/poly(U) complex.11 One of these water molecules is pre-positioned in the apo-U2AF65-RRM1 structure for water-mediated interactions with the RNA strand (Figure 2(b); Supplementary Data
Overview of Alternative Side-Chain Conformations
The high resolution of the apo-U2AF65-RRM1 X-ray structure revealed that six side-chains (Arg150, Glu162, Met165, Leu175, Lys225, and Arg227), or 7% of the total residues in the domain, have two alternative conformations with nearly equivalent occupancies (0.55, 0.66, 0.50, 0.57, 0.68, 0.62, for the major conformations) (Figure 3). Only one of these (Met165) is found in the protein interior. The alternative conformations of Met165 seek to fill a hydrophobic pocket in the protein core (Figure 3
Alternative Side-Chain Conformations at the RNA Binding Surface
The most striking of the RNA-dependent conformations involves Arg150 at the 3′ terminus of the RNA (Figures 2(d) and 3(a)). In the apo-structure, one alternative conformation of Arg150 is located with the positively charged guanidinium group stacking against the negatively charged Glu201 side-chain, and the NH1 atom donating hydrogen bonds to both a bound water molecule and the backbone carbonyl group of Gly146. The second, slightly lower occupancy conformation is turned towards the body of the
Implications for pre-mRNA Splice site Recognition
To fulfil its role as an essential splicing factor, U2AF65 must accurately specify the Py-tract and recruit the splicing machinery to the appropriate 3′ splice site. The majority of U2AF65-RRM1 residues adopt similar conformations in the presence and in the absence of the poly(U) ligand, providing a prearranged molecular shape that may contribute to the preference of U2AF65 for binding Py-tracts.21., 22. However, U2AF65 also must adapt to deviations from the uridine-rich consensus sequence in
Acknowledgements
We thank S. K. Burley, in whose laboratory this research was initiated, for support and advice. We thank the BioCARS staff for use of Station 14-ID-B at the Advanced Photon Source, which receives support (through grant RR07707) from the National Center for Research Resources of the National Institutes of Health. We thank C. Wolberger, L. M. Amzel, and D. Leahy for advice and generous access to X-ray equipment. M. M. Benning (Bruker, AXS) and J. D. Ferrara (MSC) assisted with collection of
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- 1
Present address: K. R. Thickman, Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburg, PA 15261, USA; E. A. Sickmier, Department of Molecular Structure, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA.