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. 2020 May 28;3(7):e202000725. doi: 10.26508/lsa.202000725

Figure 3. Structure of STAG1 hotspot regions in complex with RAD21 peptides.

Figure 3.

(A) Cartoon diagram of the overall structure of STAG1 N-terminal region (pink), and central region (cyan) superposed on to the STAG2 structure (gray with semi transparency), with RAD21 peptide regions in green and heat repeats numbered. (B) Close-up view of the interaction between RAD21 and STAG1 N-terminal region (sites I and II), with hydrogen bonds shown as dashed lines and key interacting residues shown in stick format. (B, C) Schematic view of the interaction between RAD21 residues 322-221 and the N-terminal region of STAG1; the orientation and coloring correspond roughly to what is shown in panel (B). Hydrogen bonds are shown as dashed lines and van der Waals contacts are shown as red-spoked arcs. (D) Close-up view of the interaction between RAD21 and STAG1 central region (site IV), with hydrogen bonds shown as dashed lines and key interacting residues shown in stick format. The first 12 residues of the RAD21 peptide, which form a further short section of α-helix in the STAG2 structure are disordered in the electron density and have not been included in the model. (D, E) Schematic view of the key interactions between RAD21 and STAG1 central region, highlighting the interacting region spanning RAD21 residues 375–382, the orientation and coloring correspond roughly to what is shown in panel (D). Hydrogen bonds are shown as dashed lines, and van der Waals contacts are shown as red-spoked arcs. (F) Comparison of STAG1 central region WT versus D797A mutant binding to a RAD21356–395 peptide by surface plasmon resonance. Biotinylated RAD21356–395 was immobilized on the chip, and increasing concentrations of STAG1 were injected in a single cycle kinetic experiment, a clear dose-dependent binding response is observed for the wild-type protein (red curve, with fit to a 1:1 binding model shown as black line), whereas only small nonspecific responses are seen for the D797A variant (blue curve). (G) Analysis of the STAG1 central region–RAD21356–395 interaction on surface plasmon resonance using equilibrium analysis. Increasing concentrations of STAG1 were injected over the sensor surface, and the response at equilibrium is fit to a dose–response curve (inset) with an apparent Kd of 148 ± 22 nM.