Fig. 5: The core network is critical for NTD structural integrity and specific RNA-recognition.
a Comparison of MST-derived KD values of viral Ext and SL4 RNAs (3’-Cy5 labeled), respectively, when titrated with WT, the two des_mutants Q58I and S105I, or the negative control R107A. The transition point (50% bound) at the KD of NTD_R107A to Ext RNA is shown by dotted lines. Data are presented as mean values +/− SD from three biological replicates (N = 3), each measured in technical duplicates. See Supplementary Fig. 11b and c for details on fitting S105I binding to SL4. b Reduced affinities indicated by fold-change of des_mutant KD values from panel (a) normalized to the WT. Error bars represent standard errors propagated during normalization. Individual replicates are grouped by colored circles representing each biological replicate (yellow – N1, green – N2, and white – N3). c Apparent specificity was determined as the relative affinity increase for Ext RNA (target) versus SL4 RNA (non-target) by quantification of the fold-change of respective KD values. A high value correlates with a strong preference for Ext over SL4. For error bars, see the explanation for panel (b). d Differential 1H/15N CSP plots (ΔΔδ) for Q58I and S105I in the presence of 1.2 equivalents Ext and SL4, respectively, obtained by subtraction from the corresponding WT CSP plot (see Supplementary Fig. 11a). e, f Superimpositions of Q58I and S105I with our WT crystal structure, respectively. For S105I, no consecutive electron density was obtained for hairpin residues from 94 to 102, and the loop was modeled with residual gaps (indicated by the dotted lines, see Supplementary Fig. 3b and d). g Zoom-ins on WT, Q58I, and S105I crystal structures highlighting the core network region. Residues and contacts (orange – H-bond; gray – vdW; black – vdW/π-π) are labeled in the WT structure only. h Superimposition of core network residue F171 in both mutants and the WT.
