Crystal structures of the RNA-bound and unbound states of all three VP35 IIDs are nearly identical.

A) Crystal structures of Zaire IID alone (3FKE, brown) and bound to 8-bp dsRNA (3L25, gray) show that both the blunt end and backbone binding poses of Zaire are nearly identical to the unbound (apo) structure. B) Overlay of Zaire IID bound to 8-bp dsRNA (3L25, gray) with unbound structures of Reston IID (3L2A, red) and Marburg IID (4GH9, blue)

Simulations predict the probability of pocket opening is lowest in Reston and highest in Marburg.

Each curve is the probability distribution of the distance between two residues that serves as a proxy for pocket opening. Reston VP35 IID (red) shows the least probability of opening the pocket. Zaire VP35 IID shows a greater probability of opening the pocket as well as an increased maximum distance of pocket opening. Marburg VP35 IID shows the highest probability of pocket opening and the highest maximum distance of pocket opening. The structure with the largest pocket (i.e. largest distance between the two residues) for each variant is shown in ribbon with a transparent surface, using the same color scheme as for the probability distributions. Residues 225 (236 in Zaire) and 295 (306 in Zaire) are shown in yellow sticks.

Thiol labeling of C296 confirms that the cryptic pocket in Marburg has the highest probability of being open while Reston has the lowest probability of opening.

(A-C) Plots of the distance between residues 225 and 295 vs. the Solvent Accessible Surface Area (SASA) of (A) C296 of Reston IID, (B) C307 of Zaire IID,(C) C296 of Marburg IID. Each point on the plot represents an MSM center and is colored according to its equilibrium probability.(D) Observed thiol labeling rates for C296/C307 of Zaire IID (black circles), Marburg IID (blue squares), and Reston IID (red triangles) at a range of DTNB concentrations.

(E-G) Plots of the distance between residues 225 and 295 vs. the SASA of (E) C315 of Reston IID, (F) C326 of Zaire IID, and (G) C315 of Marburg IID calculated from our MSMs.The SASA of C296 is more correlated with the opening of the cryptic pocket than the SASA of C315, so we focus on thiol labeling of C296 to experimentally characterize pocket opening.(H) Observed thiol labeling rates for C315/C326 of Zaire IID (black circles), Marburg IID (blue squares), and Reston IID (red triangles) at a range of DTNB concentrations. Fits to the Linderstrøm–Lang model are shown in colored lines and the expected labeling rate from the unfolded state is shown as blue dotted lines for Marburg IID and red dotted lines for Reston IID. This rate is estimated from the stability and unfolding rate measured for these homologs shown in Fig S6. The mean and standard deviation from three measurements are shown for D and H. Error bars are smaller than the marker used.

Binding to different length dsRNAs suggests closed conformations preferentially bind dsRNA blunt ends while open conformations prefer binding the backbone.

(A-C) Binding to fluorescently labeled 8bp (dashed lines) and 25bp RNA (solid lines) with and without a 3’ 2 nucleotide overhang of (A) Wild Type (WT) Reston IID (B) WT Zaire IID (C) WT Marburg IID.The anisotropy was measured via a fluorescence polarization assay, converted to anisotropy, and fit to a one-dimensional lattice binding model. The mean and standard deviation from three replicates is shown but error bars are generally smaller than the symbols.(D) Comparison of binding affinities obtained from the global fits. The mean and standard deviation from fits to each of the three replicates are shown.(E-F) Fraction of the backbone of 25 bp RNA covered by the open states (empty markers) and closed states (colored markers) calculated from the binding parameters obtained from the fits for (E) Reston WT IID and (F) Zaire WT IID.

Single amino acid substitutions at residue 280 (291 in Zaire) modulate the probability of pocket opening.

A) Probability distribution of the distance between residues 225 and 295 obtained from MSMs built from FAST adaptive sampling simulations of Reston IID WT (solid red), Reston IID P280A (dashed red), Zaire IID WT (solid black), and Zaire IID A291P (dashed black). B) Observed labeling rates of C296 for Reston IID WT (transparent solid red) and Reston IID P280A (dark dashed red) Zaire IID WT (transparent solid black), and Zaire IID A291P (dashed black).

Single amino acid substitutions that alter the probability of pocket opening also induce differential binding to blunt ends and the backbone.

(A-D) Binding of Zaire A291P IID (empty black circles), WT IID (solid red triangles), WT Zaire IID (solid black circles), Reston P280A IID (empty red triangles), WT Marburg IID (blue squares) to fluorescently labeled (A) 8bp blunt-ended RNA (B) 8bp RNA with two nucleotide overhangs on 3’ ends (C) 25bp blunt-ended RNA (D) 25bp RNA with two nucleotide overhangs on 3’ ends.

The anisotropy was measured via a fluorescence polarization assay, converted to anisotropy, fit to a one-dimensional lattice binding model. The mean and standard deviation from three replicates is shown but error bars are generally smaller than the symbols. Lines indicate the global fits.

(E) Comparison of Koc obtained from the global fits to Keq for C296 exposure obtained from DTNB labeling experiments shown in Fig 3D.

(F) Comparison of dissociation constants obtained from the global fits.

(G) Comparison of cooperativity between backbone binding modes obtained from the global fits.(H) Comparison of cooperativity between end and backbone binding modes obtained from the global fits. The mean values of fits to three individual replicates are shown for E, F, G and H. Standard deviations are shown as error bars.