Full-length Gαq–phospholipase C-β3 structure reveals interfaces of the C-terminal coiled-coil domain

Abstract

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.

Membrane-induced Allosteric Control of Phospholipase C-β Isozymes

“…The C-terminal domain was previously thought to interact directly with G␣ q in the activation of PLC-␤ isozymes, but our measurements using soluble proteins indicate that PLC-␤3 lacking the C-terminal domain binds active G␣ q with an affinity only 2-fold lower than that of full-length PLC-␤3 (22). These affinity measurements are supported by a recent crystal structure of full-length PLC-␤3 and G␣ q showing minimal interaction between G␣ q and the C-terminal domain (18). Nevertheless, G␣ q -dependent activation of PLC-␤ isozymes at membranes clearly is supported by the C-terminal domain (23)(24)(25).…”

“…2E). A corollary to this proposition is that the C-terminal domain probably does not substantially stabilize the XY-linker to support autoinhibition, as previously suggested by electron microscopy studies of full-length PLC-␤3 (18).…”

“…3A). This result is perhaps superficially surprising given the fact that complex formation is guaranteed to disrupt the interface between the HTH and the TIM barrel previously shown to mediate autoinhibition (18,22). However, if we posit that the extension results in autoinhibition of the phospholipase solely by colliding with the membrane surface, then autoinhibition is only operative within the context of membrane-resident PIP 2 , and no contradiction arises from the enzymology carried out with soluble WH-15 and PIP 2 in vesicles or cellular membranes.…”

“…Whereas it is reasonable to posit that membranes facilitate interactions between the C-terminal domain and G␣ q to support phospholipase activity, the nature of these potential interactions remains poorly understood. In contrast to the C-terminal domain, a conserved region that immediately follows the C2 domain and is induced to form a helix-turn-helix (HTH) upon interaction with active G␣ q provides the major interface with G␣ q in several crystal structures (18,22,26). This interface involves the switch regions of G␣ q such that only the active form of G␣ q engages the HTH.…”

“…For instance, the PLC-␤ isozymes contain a unique C-terminal domain that is structurally similar to Bin-amphiphysin-Rvs domains (17,18). This region promotes membrane association and is required for efficient activation of PLC-␤ isozymes by G␣ q and related G␣ subunits (G␣ 11 , G␣ 14 , and G␣ 16 ) of heterotrimeric G proteins (19).…”