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. 2020 Jul 1;48(14):7728–7747. doi: 10.1093/nar/gkaa520

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© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — Structural analysis of mTTD-PHD V1 and V2 modules. (A) Distribution of R_g determined by molecular modelling in combination with SAXS profiles of mTTD-PHD V1 (left), mTTD-PHD V2 (middle) and hTTD-PHD (right). Dashed line indicates the initial pool of structures derived from molecular modelling; solid line indicates the SAXS-fitted ensembles of TTD-PHD conformers. (B) Overlay of (¹H–¹⁵N) HSQC spectra (centre-zoomed) of mTTD-Linker 2 V1 (red) and V2 (green). Resonances were assigned for the V1 protein (85% complete and deposited in the BMRB (ID 30704)). A full spectral overlay is presented in Supplementary Figure S8B. (C) Comparison of amide chemical shifts in (¹H–¹⁵N) HSQC spectra of mTTD-Linker 2 V1 and mTTD-Linker 2 V2. Overlapping resonances (coloured blue) are mapped onto the mTTD-Linker 2 V1 structure. These are localized outside of the linker-groove interface, and TTD_N–TTD_C junction. (D) NMR structural ensembles of mTTD-Linker 2 V1 (left, PDBID: 6VEE) and hTTD-Linker 2 (right, PDBID: 6VED) showing the relative position of the Linker 2 regions with respect to the TTD. (E) NMR-derived structure of mTTD-Linker 2 V1 with surface representation of mTTD in grey and mLinker 2 V1 in green. R298 (magenta – stick representation) of mLinker 2 V1 is anchored to the R-pocket (cyan). The P-patch (i.e. P293, P294 and P295) is colored in red. mLinker 2 V2 (pink) is in flux with respect to the mTTD surface; a snapshot representation derived from molecular modeling is shown, with R293 (orange – stick representation) above the R-pocket. (F) NMR structure of the interaction between mLinker 2 V1 (green, residues labeled in black) and mTTD (grey, residues labeled in blue). Surface representation with hydrophobic (cyan) and acidic (pink) residues forming the R-pocket occupied by R298 highlighted is shown on the left. Hydrogen bonds (dashed black lines) that stabilize the mLinker 2 V1-mTTD interaction (upstream of the P-patch: N_A288–O_N134, N_Y136–O_L286, N_Q292–O_S289 and downstream of the P-patch: NH2_R298–OD1_D138, NH2_R298–OD2_D138, N_R298–OE2_E149, NH1_R298–O_W147, N_T300–OE2_E186) are shown in stick representations in the middle and right. (G) (¹H–¹⁵N) HSQC titrations of mTTD-Linker 2 V1 (left) and V2 (right) with unlabelled 4-benzylpiperidine-1-carboximidamide (BPC) at various molar ratios.