Ligand-induced Conformational Changes via Flexible Linkers in the Amino-terminal region of the Inositol 1,4,5-Trisphosphate Receptor

Cytoplasmic Ca²⁺ signals are highly regulated by various ion transporters, including the inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R), which functions as a Ca²⁺ release channel on the endoplasmic reticulum membrane. Crystal structures of the two N-terminal regulatory regions from type 1 IP₃R have been reported; those of the IP₃-binding core (IP₃R_CORE) with bound IP₃, and the suppressor domain. This study examines the structural effects of ligand binding on an IP₃R construct, designated IP₃R_N, that contains both the IP₃-binding core and the suppressor domain. Our circular dichroism results reveal that the IP₃-bound and IP₃-free states have similar secondary structure content, consistent with preservation of the overall fold within the individual domains. Thermal denaturation data show that, while IP₃ has a large effect on the stability of IP₃R_CORE, it has little effect on IP₃R_N, indicating that the suppressor domain is critical to the stability of IP₃R_N. The NMR data for IP₃R_N provide evidence for chemical exchange, which may be due to protein conformational dynamics in both apo and IP₃-bound states: a conclusion supported by the small-angle X-ray scattering data. Further, the scattering data show that IP₃R_N undergoes a change in average conformation in response to IP₃ binding and the presence of Ca²⁺ in the solution. Taken together, these data lead us to propose that there are two flexible linkers in the N-terminal region of IP₃R that join stably folded domains and give rise to an equilibrium mixture of conformational sub-states containing compact and more extended structures. IP₃ binding drives the conformational equilibrium toward more compact structures, while the presence of Ca²⁺ drives it to a more extended set.