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. 2021 Jan 25;12:581. doi: 10.1038/s41467-020-20754-4

Fig. 2. Structural bases for FA-induced HxlR activation.

Fig. 2

a The “N-terminal helix-flipping” mechanism of HxlR protein upon FA treatment. Superposition of HxlR-WT (magenta) and HxlR-WT-FA-DNA (green) indicates that FA induces a dramatic rearrangement on helix α1. The FA-triggered Cys11-Lys13 intrahelical crosslinking and methylene bridge formation forced the residues before Cys11 to flip ~120 degree away from the original orientation, and become unstructured. This “N-terminal helix-flipping” caused a large movement of the wHTH domain, with helix α4 rotated 21.9 degree and the tips of β-wings translocated 24.6 Å, producing an optimized conformation for DNA binding. Translocations of the structure domains and residues are indicated by the red arrow. b Detailed view of the crosslinking-induced “N-terminal helix-flipping”. In order to form the crosslink, C-Cα bond in Cys11 rotated to allow the sidechain thiol group to get close to Lys13 (red arrow), leading the N atom of Cys11 and the connected N-terminal residues to flip to the opposite side (black arrow). c The “N-terminal helix-flipping” triggered the reorganization of helices α1, α2’, and α5’, with the residues before Cys11 becoming unstructured, helix α5’ moving towards helix α1 and the concurrent rotation of helix α2’. Translocations of these helices are indicated by red arrows. d The FA-induced Cys11-Lys13 intrahelical crosslinking brings helix α2’ towards helices α1. The resulting methylene bridge forced helix α2’ to move closer to helix α1, with Met26’ filling the space left by Cys11-Lys13 crosslinking (shown in sphere). Red arrows indicate the movement of Lys13 and helix α2’. e Superposition of HxlR-K13A (cyan) and HxlR-WT-FA-DNA (green) indicates the absence of “N-terminal helix-flipping” in HxlR-K13A. Similar to that of HxlR-WT, helix α1 in HxlR-K13A extends beyond Cys11 to the N-terminal residues, with Cys11 and Ala13 located on the opposite sides. Distances between the DNA-binding helices α4 and α4′ are indicated by black arrow. Residues from HxlR-K13A and HxlR-WT are marked in black and green, respectively. f “N-terminal helix-flipping” is crucial for HxlR to undertake the DNA-binding conformation. As the “N-terminal helix-flipping” does not occur in the HxlR-K13A mutant, the sidechain of Phe9 is located between that of Trp29’ and Tyr99’, which causes the distance between Trp29’ and Tyr99’ larger than that of HxlR-WT-FA-DNA and prevents further approaching of HxlR subunits. The sidechain of Phe9 is shown in sphere, and the distances between Trp29’ and Tyr99’ in different conformations are indicated by black arrows.