Intramolecular electrostatic interactions contribute to phospholipase C?3 autoinhibition.

ABSTRACT: Phospholipase C? (PLC?) enzymes regulate second messenger production following the activation of G protein-coupled receptors (GPCRs). Under basal conditions, these enzymes are maintained in an autoinhibited state by multiple elements, including an insertion within the catalytic domain known as the X-Y linker. Although the PLC? X-Y linker is variable in sequence and length, its C-terminus is conserved and features an acidic stretch, followed by a short helix. This helix interacts with residues near the active site, acting as a lid to sterically prevent substrate binding. However, deletions that remove the acidic stretch of the X-Y linker increase basal activity to the same extent as deletion of the entire X-Y linker. Thus, the acidic stretch may be the linchpin in autoinhibition mediated by the X-Y linker. We used site-directed mutagenesis and biochemical assays to investigate the importance of this acidic charge in mediating PLC?3 autoinhibition. Loss of the acidic charge in the X-Y linker increases basal activity and decreases stability, consistent with loss of autoinhibition. However, introduction of compensatory electrostatic mutations on the surface of the PLC?3 catalytic domain restore activity to basal levels. Thus, intramolecular electrostatics modulate autoinhibition by the X-Y linker.

SUBMITTER: Esquina CM 

PROVIDER: S-EPMC6686194 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature