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. 2024 Apr 24;13:RP93561. doi: 10.7554/eLife.93561

Figure 3. Crystal structure of C-terminal PUR domain from hsPURA and effects of mutations on its function.

(A) Crystal structure of hsPURA III at 1.7 Å resolution (PDB ID: 8CHW). Two hsPURA III molecules (magenta and gray, respectively) form a homodimer. Like other PUR domains (e.g. hsPURA I–II), hsPURA repeat III consists of four β-sheets and one α-helix. (B) In electrophoretic mobility shift assays (EMSAs) the interaction of hsPURA FL and hsPURA III with a 24-mer (CGG)8 RNA fluorescently labeled with Cy5 fluorophore at 5′-end was observed. The amount of the RNA was kept constant at 8 nM while the protein concentration increased from 0 to 16 μM. The unbound RNA used for quantification (see E) is indicated with red arrows. (C) Apparent affinities derived from EMSAs (C) indicate that the C-terminal PUR domain is also able to interact with the nucleic acids. Pairwise t-tests of the hsPURA fragments showed significantly lower RNA binding compared to the full-length protein (hsPURA III: p = 9.6E−4). Three replicates were measured for each experiment and protein variant, and the standard deviations have been calculated and shown as bars. (D, E) Strand-separation activity of hsPURA. The graphs show the averaged values of three independent experiments as dots with standard deviations as error bars. (D) The hsPURA III fragment shows sigmoidal increase of the ssDNA concentration measured as a fluorescent signal. For the quantification of the sigmoidal curves, we utilized Boltzmann function implemented in the Origin software. Calculated x0, which corresponds to the Km in this assay yields 4.26 ± 0.74 µM. (E) Strand-separation activity of full-length hsPURA was calculated with Michaelis–Menten kinetic, yielding an average Km value of 0.83 ± 0.05 µM, respectively. For both hsPURA samples at least three independent measurements have been performed. (F) NanoBRET experiments in HEK293 cells with different hsPURA fragment-expressing constructs. Milli-BRET units (mBU) were measured for dimerization of hsPURA I–III with hsPURA I–III, hsPURA I–III F233del, and hsPURA I–III R245P. Pairwise t-tests of the mutant hsPURA I–III versions F233del and R245P yielded significantly lower signals compared to the wild-type protein (hsPURA I–III F233del: p = 3.3E−4; hsPURA I–III R245P: p = 1.5E−7), indicating impaired interactions between the proteins. Black horizontal line shows reference of mBU obtained for hsPURA I–II as negative control. Of note, since the BRET signal is the ratio of donor and acceptor signal, it does not require normalization for expression levels. Asterisks in (C) and (F) indicate significance level: *** for p ≤ 0.001.

Figure 3—source data 1. Electrophoretic mobility shift assays (EMSAs) for the interaction of hsPURA variants with a 24-mer (CGG)8 RNA fluorescently labeled with Cy5 fluorophore at 5′-end.
Uncropped, raw EMSA gel image for hsPURA III.
Figure 3—source data 2. Electrophoretic mobility shift assays (EMSAs) for the interaction of hsPURA variants with a 24-mer (CGG)8 RNA fluorescently labeled with Cy5 fluorophore at 5′-end.
Uncropped, labeled EMSA gel image for hsPURA III.
Figure 3—source data 3. Electrophoretic mobility shift assays (EMSAs) for the interaction of hsPURA variants with a 24-mer (CGG)8 RNA fluorescently labeled with Cy5 fluorophore at 5′-end.
Uncropped, raw EMSA gel image for hsPURA FL.
Figure 3—source data 4. Electrophoretic mobility shift assays (EMSAs) for the interaction of hsPURA variants with a 24-mer (CGG)8 RNA fluorescently labeled with Cy5 fluorophore at 5′-end.
Uncropped, labeled EMSA gel image for hsPURA FL.

Figure 3.

Figure 3—figure supplement 1. Analysis and comparison of the crystal structure of hsPURA repeat I-II and III.

Figure 3—figure supplement 1.

(A) Superposition of the crystal structure of the hsPURA repeat III homodimer at 1.7 Å resolution (PDB ID: 8CHW; chains shown as magenta and gray ribbons) with its D. melanogaster homologue dmPURA III (PDB ID: 5FGO; both chains shown as yellow ribbons). The hsPURA III dimer shows high structural similarity to dmPURA III with root mean square deviation (r.m.s.d.) of 1.26 Å for 125 superimposed Cα atoms and 50% of sequence identity. (B) The surface electrostatic potentials for hsPURA III (left), hsPURA I–II (middle), and hsPURA I–II K97E (right). All three structures are oriented with the nucleic binding sites facing toward the reader. The position of K97 in wild-type hsPURA I–II and E97 in hsPURA II–II K97E is encircled.
Figure 3—figure supplement 2. Structural features likely contributing to the PURA-dependent unwinding of dsRNA.

Figure 3—figure supplement 2.

Superposition of the hsPURA I–II crystal structure (PDB ID: 8CHT; repeat I in green and II in blue) with the previously published structure of the PURA I–II fragment from D. melanogaster (gray ribbon model) in complex with DNA (shown in red) (PDB ID: 5FGP). The structure is presented in two different orientations. The β-ridge is highlighted by the magenta square and label. The position of the residue 97 is shown in yellow and labeled accordingly.
Figure 3—figure supplement 3. The strand-separation activity assay DNA probe.

Figure 3—figure supplement 3.

The primary and secondary structures of the DNA oligo with 5′-labeled FAM fluorophore and 3′-labeled Dabcyl quencher.
Figure 3—figure supplement 4. The crystal structure of the hsPURA III homodimer.

Figure 3—figure supplement 4.

(A) The crystal structure of the hsPURA III homodimer at 1.7 Å resolution (PDB ID: 8CHW) with the residues F233 shown as green sticks and labeled. (B) The crystal structure of the hsPURA III homodimer at 1.7 Å resolution (PDB ID: 8CHW) with the residues R245 shown as green sticks and labeled.