Structure of L44H and V33G mutants.

<p>(A) Crystal structure of Aer2 1–172 proteins, with HAMP1 colored blue (WT), purple (L44H), and green (V33G). (B) Superposition of WT HAMP1 and CW locked L44H HAMP1 mutant. The L44H side chain redirects from the bundle core towards AS1, causing a 15° tilt of AS1 away from AS2 and a 5 Å shift at the top of AS1, which disrupts the upstream helical coiled coil. The positions of the AS2 output helices are identical to WT, which confirms that a HAMP1 structure generates CW output. (C) Superposition of HAMP1 in WT and in the inverted signaling V33G mutant. Removal of HR2 in V33G does not affect the helical position of AS1 or AS2, suggesting that HR2 is dispensable to generate a HAMP1 conformer. 2Fo-Fc electron density maps of V33G (contoured at 2 σ) lack density in the surrounding HR2 connector region, suggesting increased flexibility of this region. Increased flexibility of HR2 due to Gly substitution would destabilize a HAMP2 conformer and thereby favor CW output.