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

Figure 5. Structural analysis of hsPURA I–II bearing the K97E patient-derived variant.

(A) Circular dichroism (CD) spectroscopic analyses of hsPURA I–II, K97E, and R140P variants. Shown are the mean of three CD measurements for each of the proteins (n = 3). The spectra of hsPURA I–II K97E show a very different profile, indicating an altered fold of this variant compared to the wild-type form. (B, C) Crystal structures of hsPURA K97E repeats I (green) and II (blue) at 2.45 Å resolution, showing a high overall similarity to hsPURA (Figure 4A). Two independent hsPURA I–II K97E chains (A, B) in the asymmetric unit are shown in (B) and (C), respectively. The mutated amino acid K97E is indicated as red sticks. Positional shifts of amino acids in β5 (D) and β6 (E) strands of the chain B in the crystal structure of hsPURA I–II K97E. 2Fo–Fc electron density map (contour 1σ) is shown for the selected fragment of β5 and β6 strands in chain B (light pink). The superimposed chain A (gray) shows a register shift in chain B by +1 amino acid in the β5 strand and by +2 in the β6 strand. (F) Nuclear magnetic resonance (NMR) experiment with wild-type hsPURA I–II and hsPURA I–II K97E. Overlay of the 1H, 15N-HSQC spectra of hsPURA I–II (red) and K97E (black). Blue boxes indicate examples of changes between both spectra.

Figure 5.

Figure 5—figure supplement 1. Structure of hsPURA I–II R140P variant.

Figure 5—figure supplement 1.

(A, B) An overlay of chains A (red), B (green), C (blue), D (magenta) from the asymmetric unit of the crystals of hsPURA I–II R140P, and the wild-type hsPURA I–II chains A, B, C, D shown in gray. The structures are shown in two different orientations as ribbons, residue P140 is shown as red sticks. The missing fragment of the loop is shown as gray dots. The most variable part of the secondary structure elements, strands β4 and β8, have been labeled accordingly. (C) Comparison of all independent chains in the asymmetric unit of hsPURA I–II R140P structure (PDB ID: 8CHV) with the wild-type hsPURA I–II (PDB ID: 8CHT). Root mean square deviation (r.m.s.d.) values are expressed in Å; the number of superimposed Cα atoms is indicated.
Figure 5—figure supplement 2. Comparison of structural features between wild-type and K97E-variant hsPURA protein.

Figure 5—figure supplement 2.

(A) Comparison of all independent chains in the asymmetric unit of hsPURA I–II WT structure (PDB ID: 8CHT) with hsPURA I–II K97E (PDB ID: 8CHU). Root mean square deviation (r.m.s.d.) values are shown in Å; the number of superimposed Cα atoms has been indicated. (B, C) The β-ridge in the hsPURA I–II crystal structure. The superposition of all four human hsPURA I–II chains from the asymmetric unit of the wild-type structure as well as the two chains from the asymmetric unit of the K97E variant. The structure is shown as ribbons in two different orientations, K97 in the wild-type structure and E97 in the mutated form is shown as sticks. (D) The overlay of crystal structures of hsPURA I–II shown in gray (chains A, B, C, D), hsPURA I–II K97E chain A shown in cyan, and hsPURA I–II K97E chain B shown in magenta. Comparison of the interaction of helix α1 in the chain B (E) and the chain A (F) in the crystal structure of hsPURA I–II K97E. The color code is repeat I: green, repeat II: blue. In chain B, the helix α1 bends and forms a new interaction between Y121 and E161 with 2.3 Å distance. This interaction is not present in chain A.
Figure 5—figure supplement 3. Comparison of structural features between wild-type and K97E-variant hsPURA protein.

Figure 5—figure supplement 3.

(A) 15N-heteronuclear NOE experimental spectra for hsPURA I–II K97E, with blue frames indicating changes shown in Figure 5F. These peaks do not show any shifts compared to the WT control (black), which has no added NOEs, whereas in the experiment with K97E-variant protein (red) additional NOEs are observed. (B) Summary graph of molecular dynamics simulation for hsPURA I–II (blue) and hsPURA I–II K97E variant (orange). The graph shows root mean square fluctuation in Å (RMSF) for every residue (carbon alpha) of the investigated fragment. RMSF was computed using the average position of the carbon alpha during the simulation as the reference point. Asterisks indicate significance level: * for p ≤ 0.05, ** for p ≤ 0.01, *** for p ≤ 0.001, and **** for p ≤ 0.0001.