Extended Data Fig. 6: Ammonia binding induces SCAP dissociation from Insig.

a–d, Comparison of the coupling, tilting and kink angles of S3, S5 and S6 helices during the 1 μs simulations of SCAP bound with NH₄⁺ vs. SCAP without bound NH₄⁺. In panel (a), S3 and S6 helices from the NH₄⁺ unbound simulation (in light gray) is aligned with the NH₄⁺ bound simulation (in dark gray). NH₄⁺, D428, S326 and S330 are shown in the stick representation. The coupling of the S3 and S6 helices was altered by the binding of NH₄⁺ (a). In the NH₄⁺ bound simulation, the S3 helix had a smaller tilting angle (b) and S5 and S6 helix had a larger tilting angle (c and d). Inset in panel (b) illustrates a helix titling angle. Insets in panel (c) and (d) illustrate a helix kink conformation with the lower part of the helix aligned (white), and the top part of the helix showing a difference between NH₄⁺ bound and NH₄⁺ unbound SCAP. Only converged data from the last 500 ns of each simulation were used for the histogram analysis. e, Comparison of the interface contact area between SCAP and Insig during the simulations of the NH₄⁺ bound SCAP vs. the NH₄⁺ unbound SCAP. f, A schematic model for NH₄⁺ regulated SCAP activation. Left: Insig-SCAP binding in the absence of 25-HC and NH₄⁺. Top: Binding of 25-HC blocks NH₄⁺ binding to prevent SCAP activation (orange). Middle: Absence of 25-HC opens the channel, which permits the entry of NH₄⁺ to bind to D428 first, then to S326/S330 to form a stable binding site, leading to significant conformational changes of SCAP (red) and its dissociation from Insig for subsequent translocation and SREBP activation. Bottom: D428A mutant is unable to bind NH₄⁺, preventing NH₄⁺ from inducing conformational changes required for SCAP dissociation from Insig in the absence of 25-HC; thus, it cannot be activated by NH₄⁺.