Phospholipase C-β (PLCβ) is directly activated by Gαq, but the molecular basis for how its distal C-terminal domain (CTD) contributes to maximal activity is poorly understood. Herein we present both the crystal structure and cryo-EM 3D reconstructions of human full-length PLCβ3 in complex with murine Gαq. The distal CTD forms an extended, monomeric helical bundle consisting of three anti-parallel segments with structural similarity to membrane-binding bin–amphiphysin–Rvs (BAR) domains. Sequence conservation of the distal CTD identifies putative membrane and protein interaction sites, the latter of which bind the N-terminal helix of Gαq in both the crystal structure and cryo-EM reconstructions. Functional analysis suggests the distal CTD plays roles in membrane targeting and in optimizing the orientation of the catalytic core at the membrane for maximal rates of lipid hydrolysis.

Phospholipase C-β (PLCβ) is a key regulator of intracellular calcium levels whose activity is controlled by heptahelical receptors that couple to Gq. We have determined atomic structures of two invertebrate homologs of PLCβ (PLC21) from cephalopod retina and identified a helix from the C-terminal regulatory region that interacts with a conserved surface of the catalytic core of the enzyme. Mutations designed to disrupt the analogous interaction in human PLCβ3 dramatically increase basal activity and diminish stimulation by Gαq. Gαq binding requires displacement of the autoinhibitory helix from the catalytic core, thus providing an allosteric mechanism for activation of PLCβ.

Eukaryotic translation initiation factor-4E (eIF4E) recognizes and binds the m 7 guanosine nucleotide at the 5# end of eukaryotic messenger RNAs; this protein-RNA interaction is an essential step in the initiation of protein synthesis. The structure of eIF4E from wheat (Triticum aestivum) was investigated using a combination of x-ray crystallography and nuclear magnetic resonance (NMR) methods. The overall fold of the crystallized protein was similar to eIF4E from other species, with eight b-strands, three a-helices, and three extended loops. Surprisingly, the wild-type protein did not crystallize with m 7 GTP in its binding site, despite the ligand being present in solution; conformational changes in the cap-binding loops created a large cavity at the usual cap-binding site. The eIF4E crystallized in a dimeric form with one of the cap-binding loops of one monomer inserted into the cavity of the other. The protein also contained an intramolecular disulfide bridge between two cysteines (Cys) that are conserved only in plants. A Cys-to-serine mutant of wheat eIF4E, which lacked the ability to form the disulfide, crystallized with m 7 GDP in its binding pocket, with a structure similar to that of the eIF4E-cap complex of other species. NMR spectroscopy was used to show that the Cys that form the disulfide in the crystal are reduced in solution but can be induced to form the disulfide under oxidizing conditions. The observation that the disulfide-forming Cys are conserved in plants raises the possibility that their oxidation state may have a role in regulating protein function. NMR provided evidence that in oxidized eIF4E, the loop that is open in the ligand-free crystal dimer is relatively flexible in solution. An NMR-based binding assay showed that the reduced wheat eIF4E, the oxidized form with the disulfide, and the Cys-to-serine mutant protein each bind m 7 GTP in a similar and labile manner, with dissociation rates in the range of 20 to 100 s 21 .

Phospholipase C (PLC) enzymes convert phosphatidylinositol-4,5-bisphosphate into the second messengers diacylglycerol and inositol-1,4,5-triphosphate. The production of these molecules promotes the release of intracellular calcium and activation of protein kinase C, which results in profound cellular changes. The PLCb subfamily is of particular interest given its prominent role in cardiovascular and neuronal signaling and its regulation by G protein-coupled receptors, as PLCb is the canonical downstream target of the heterotrimeric G protein Ga q . However, this is not the only mechanism regulating PLCb activity. Extensive structural and biochemical evidence has revealed regulatory roles for autoinhibitory elements within PLCb, Gbg, small molecular weight G proteins, and the lipid membrane itself. Such complex regulation highlights the central role that this enzyme plays in cell signaling. A better understanding of the molecular mechanisms underlying the control of its activity will greatly facilitate the search for selective small molecule modulators of PLCb.

SUMMARY Phospholipase C β (PLCβ) enzymes are dramatically activated by heterotrimeric G proteins. Central to this response is the robust autoinhibition of PLCβ by an X–Y linker region within its catalytic core and by the Hα2′ helix in the C-terminal extension of the enzyme. The molecular mechanism of each and their mutual dependence are poorly understood. Herein it is shown that distinct regions within the X–Y linker have specific roles in regulating activity. Most importantly, an acidic stretch within the linker stabilizes a lid that occludes the active site, consistent with crystal structures of variants lacking this region. Inhibition by the Hα2′ helix is independent of the X–Y linker and likely regulates activity by limiting membrane interaction of the catalytic core. Full activation of PLCβ thus requires multiple independent molecular events induced by membrane association of the catalytic core and by the binding of regulatory proteins.