Journal: Nature Communications
Article Title: HEXIM1 inter-monomer autoinhibition governs 7SK RNA binding specificity and P-TEFb inactivation
doi: 10.1038/s41467-026-68285-8
Figure Lengend Snippet: A 15 N- 1 H HSQC spectral overlay of BR-L-AR(S226C) (black) and BR-L-AR(S226C MTSL ) (red). Residues that are significantly broadened by MTSL are labeled. B Paramagnetic Relaxation Enhancement (PRE) intensity ratios (I PRE /I noPRE ) for BR-L-AR(S226C) with versus without MTSL tag of resolved resonances plotted against residue number. Schematic of BR-L-AR with S226C MTSL tag location is shown above plot. C–E Plots of intermolecular PRE intensity ratios of ( C ) 14 N BR-L-AR(S237C MTSL ) (blue bars), ( D ) S233C MTSL (purple bars), and ( E ) S226C MTSL (dark gray bars) added to 15 N BR-L-AR. Schematics of BR-L-AR and individual MTSL locations are shown above each plot. For ( B – E ) solid horizontal line indicates the value of [mean-0.5 × σ] and dashed line indicates the value of [mean-σ]. Stretches of > 3 either contiguous or non-contiguous residues with intensity ratios below the dashed line [mean-σ] or solid line [mean-0.5 × σ] are highlighted with red and orange shaded boxes, respectively. Dark arrows denote proline residues, lacking amide proton, light gray boxes denote residues with overlapping resonances that are excluded for accurate intensity determination, and red box denote residues broadened below the lower Y-axis boundary. The dashed lines connecting (C-D-E) panels highlight the opposite direction of shifting of the primary broadening region relative to the directional change of the MTSL position in the protein sequence, indicating a head-to-tail conformation for the inter-molecular interactions. F Simplified cartoons illustrating the intra- and inter-molecular components of the PRE detected interactions, and a self-hugging topology of autoinhibited Hexim1 dimer based on the inter-molecular interactions.
Article Snippet: The PD-10 elution was collected into a falcon tube, containing 10-fold molar excess of S-(1-oxyl-2,2,5,5,-tetramethyl-2,5,-dihydro-1H-pyrrol-3-yl) methylmethane-sulfonothiolate (MTSL; Toronto Research Chemicals TRC-O875000) dissolved in 0.5 mL of PRE-buffer, covered with aluminum foil to avoid light.
Techniques: Labeling, Residue, Sequencing
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![A 15 N- 1 H HSQC spectral overlay of BR-L-AR(S226C) (black) and BR-L-AR(S226C <t>MTSL</t> ) (red). Residues that are significantly broadened by MTSL are labeled. B Paramagnetic Relaxation Enhancement (PRE) intensity ratios (I PRE /I noPRE ) for BR-L-AR(S226C) with versus without MTSL tag of resolved resonances plotted against residue number. Schematic of BR-L-AR with S226C MTSL tag location is shown above plot. C–E Plots of intermolecular PRE intensity ratios of ( C ) 14 N BR-L-AR(S237C MTSL ) (blue bars), ( D ) S233C MTSL (purple bars), and ( E ) S226C MTSL (dark gray bars) added to 15 N BR-L-AR. Schematics of BR-L-AR and individual MTSL locations are shown above each plot. For ( B – E ) solid horizontal line indicates the value of [mean-0.5 × σ] and dashed line indicates the value of [mean-σ]. Stretches of > 3 either contiguous or non-contiguous residues with intensity ratios below the dashed line [mean-σ] or solid line [mean-0.5 × σ] are highlighted with red and orange shaded boxes, respectively. Dark arrows denote proline residues, lacking amide proton, light gray boxes denote residues with overlapping resonances that are excluded for accurate intensity determination, and red box denote residues broadened below the lower Y-axis boundary. The dashed lines connecting (C-D-E) panels highlight the opposite direction of shifting of the primary broadening region relative to the directional change of the MTSL position in the protein sequence, indicating a head-to-tail conformation for the inter-molecular interactions. F Simplified cartoons illustrating the intra- and inter-molecular components of the PRE detected interactions, and a self-hugging topology of autoinhibited Hexim1 dimer based on the inter-molecular interactions.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1311/pmc12901311/pmc12901311__41467_2026_68285_Fig3_HTML.jpg)
![A Schematic and PRE intensity ratios (I PRE /I noPRE ) for 15 N-labeled BR-L-AR(A201C) with versus without the MTSL tag of resolved resonances plotted against residue number. B Schematic and Paramagnetic Relaxation Enhancement (PRE) intensity ratios (I PRE /I noPRE ) for 15 N-labeled BR-L-AR(S183C) with versus without MTSL tag of resolved resonances plotted against residue number in the absence (black/gray bars) and presence (cyan bars) of SL1-dI RNA (1:1.2 protein:RNA ratio). For ( A , B ), the solid horizontal line indicates the value of [mean-0.5 × σ] and the dashed line indicates the value of [mean-σ]. Stretches of > 3 either contiguous or non-contiguous residues with intensity ratios below the dashed line [mean-σ] or solid line [mean-0.5 × σ] are highlighted with red and orange shaded boxes, respectively. Shaded boxes in panel B are from the PRE profile in the absence of RNA. Dark arrows denote proline residues, lacking amide proton, and light gray boxes denote residues with overlapping resonances that are excluded for accurate intensity determination. Residues with negative values reflect that the resonances were broadened to the noise level. Simplified cartoons on the right illustrate the BR–PYNT–AR inter-molecular interactions and how RNA binding results in the release of these interactions.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1311/pmc12901311/pmc12901311__41467_2026_68285_Fig4_HTML.jpg)