RNA plays a central role in all organisms and can fold into complex structures to orchestrate function. Visualization of such structures often requires crystallization, which can be a bottleneck in the structure-determination process. To promote crystallization, an RNA-recognition motif (RRM) of the U1A spliceosomal protein has been co-opted as a crystallization module. Specifically, the U1-snRNA hairpin II (hpII) single-stranded loop recognized by U1A can be transplanted into an RNA target to promote crystal contacts and to attain phase information via molecular replacement or anomalous diffraction methods using selenomethionine. Herein, we produced the F37M/F77M mutant of U1A to augment the phasing capability of this powerful crystallization module. Selenomethionine-substituted U1A(F37M/F77M) retains high affinity for hpII (KD of 59.7 ± 11.4 nM). The 2.20 Å resolution crystal structure reveals that the mutated sidechains make new S-π interactions in the hydrophobic core and are useful for single-wavelength anomalous diffraction. Crystals were also attained of U1A(F37M/F77M) in complex with a bacterial preQ1-II riboswitch. The F34M/F37M/F77M mutant was introduced similarly into a lab-evolved U1A variant (TBP6.9) that recognizes the internal bulged loop of HIV-1 TAR RNA. We envision that this short RNA sequence can be placed into non-essential duplex regions to promote crystallization and phasing of target RNAs. We show that selenomethionine-substituted TBP6.9(F34M/F37M/F77M) binds a TAR variant wherein the apical loop was replaced with a GNRA tetraloop (KD of 69.8 ± 2.9 nM), laying the groundwork for use of TBP6.9(F34M/F37M/F77M) as a crystallization module. These new tools are available to the research community.
The Hedgehog pathway is an essential cell-signaling paradigm implicated in cancer tumorigenesis and the developmental disorder holoprosencephaly, making it an attractive target for therapeutic design. The N-terminal domain of the Sonic Hedgehog protein (Shh-N) is the essential signaling molecule in the Hedgehog pathway. In this role Shh-N interacts with its cognate membrane receptor Patched, as well as the regulatory proteins HHIP and CDO, by utilizing interfaces harboring one or more divalent ions. Here, the crystal structure of human Shh-N is presented at 1.43 Å resolution, representing a landmark in the characterization of this protein. The structure reveals that the conserved Zn 2+binding site adopts an atypical octahedral coordination geometry, whereas an adjacent binding site, normally occupied by binuclear Ca 2+ , has been supplanted by a single octahedrally bound Mg 2+ . Both divalent sites are compared with those in previous Shh-N structures, which demonstrates a significant degree of plasticity of the Shh-N protein in terms of divalent ion binding. The presence of a high Mg 2+ concentration in the crystallization medium appears to have influenced metal loading at both metal ion-binding sites. These observations have technical and design implications for efforts focused on the development of inhibitors that target Shh-N-mediated protein-protein interactions.
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