Interdomain communication in the phosphatidylcholine regulatory enzyme, CCTα, relies on a modular αE helix

Remodeling of the interdomain allosteric linker upon membrane binding of CCTα pulls its active site close to the membrane surface

“…The Trps engineered into ␣E/J did not impair the interaction of the M domain with membrane vesicles. All active ␣E/J Trp variants had approximately the same affinity as the WT CCT for the highly curved vesicles used in the fluorescence experiments (13).…”

“…engineered tryptophans by vesicles containing Br-PC. The preparation and analysis of the impact of many of the ␣E/Jengineered Trps on CCT activity and thermal stability were presented in the companion paper (13). In addition, we analyzed single Trps engineered into the catalytic domain at F121W and F124W that were characterized previously (14).…”

“…When the helices were constrained from bending, activation of the membranebound enzyme (CCT mem ) was impaired. The activity-promoting impact of mutations in the ␣E hinge that destabilize interactions between the ␣E helix pair underscored the importance of helix ␣E malleability (13). The solvent accessibility of residues in the ␣E hinge in CCT mem was consistent with a hinge that exists within a folded ensemble (13).…”

“…The activity-promoting impact of mutations in the ␣E hinge that destabilize interactions between the ␣E helix pair underscored the importance of helix ␣E malleability (13). The solvent accessibility of residues in the ␣E hinge in CCT mem was consistent with a hinge that exists within a folded ensemble (13). The working hypothesis is that the membrane-triggered displacement of the AI helices frees the ␣E C to sample other conformations, including ones more productive for catalysis.…”

“…Bridging the C and M domains is an allosteric linker we refer to as the ␣E C /J (residues 216 -234, mammalian numbering; Fig. 1) that is highly conserved across phyla (13), contains both charged and nonpolar residues, and is lacking in cytidylyltransferases that are not lipid-regulated. In the companion paper (13), we showed via analysis of the impacts of mutations in this region on CCT activity that the ␣E C /J is vital for lipid-dependent activity.…”