An autoinhibitory helix in the C-terminal region of phospholipase C-β mediates Gαq activation

Abstract

The enzyme phospholipase C-β (PLCβ) is a crucial regulator of intracellular calcium levels whose activity is controlled by heptahelical receptors that couple to members of the Gq family of heterotrimeric G proteins. 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 considerably 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.

Figure 1

Primary and tertiary structures of PLCβ family members, and comparison of cephalopod PLC21 with the Gα_q–PLCβ3 complex. (a) Primary structure of human PLCβ3. PLCβ3 truncations used in this paper are indicated below the diagram. Numbers above the diagram correspond to amino acid positions at domain boundaries. (b) Crystal structure of LPLC21. LPLC21 crystallized as a dimer with pseudo two-fold symmetry. Domains are colored as in a. The Hα2′-Hα3 hairpin from the proximal CTR is shown in cyan, and the catalytic Ca²⁺ is shown as a black sphere. Disordered loops are drawn as dashed lines, with the exception of the connection between the C2 domain and the beginning of Hα2′, which is ambiguous in the dimer interface. The C-terminus of the C2 domain and start of Hα2′ are marked with pink and blue asterisks, respectively. N- and C-terminal ends of the protein fragment resolved in the crystal structure are labeled N and C, respectively. (c) Crystal structure of SPLC21. Domains are colored as in b. (d) Crystal structure of the Gα_q–PLCβ3 complex (PDB entry 3OHM)²³. Hα1 and Hα2, which form the primary Gα_q binding site, are shown in dark blue. Residues corresponding to Hα2′ in the PLC21 structures are shown in cyan. Activated Gα_q is shown in light gray, with GDP and AlF₄ colored red, and Mg²⁺ black.

Figure 2

Interactions of Hα2′ with the catalytic core. (a) The SPLC21 Hα2′ helix docks in a conserved cleft formed between the TIM barrel and C2 domains, in close proximity to the active site and the X–Y linker. The shorter Hα3 helix forms a hairpin interaction with Hα2′ stabilized by hydrophobic interactions. Domains are colored as in 1a. (b) Specific interactions of SPLC21 Hα2′ with the catalytic core. Side chains that make large contributions to the binding interface are shown as sticks with carbons colored according to their respective domains and nitrogens blue. (c) The Hα2′-catalytic core interaction is recapitulated in a crystal contact of the Gα_q–PLCβ3 structure (PDB entry 3OHM)²³. Domains are colored as in 1d. The subunit of Gα_q shown is in complex with a different catalytic core in the crystal lattice. (d) Specific interactions between Hα2′ and the catalytic core in human PLCβ3 (Fig. 1c). Residues analogous to those of SPLC21 shown in b are drawn as sticks, and site-directed mutations created in this study to perturb the interface are indicated. The SPLC21 Hα2′ helix is continuous, whereas Hα2′ in human PLCβ3 is kinked at Ala877, as if to optimize the interactions of the Leu879 side chain, which is smaller than that of the corresponding Phe804 residue in SPLC21.

Figure 3

Functional studies of PLCβ3 variants. (a) The proximal CTR stabilizes the catalytic core. ThermoFluor assays were used to measure the melting point of three PLCβ3 variants by monitoring the change in fluorescence of ANS. Representative curves are shown for PLCβ3 (circles), PLCβ3-Δ892 (squares), and PLCβ3-Δ847 (triangles). PLCβ3-Δ847 is 5–7 °C less stable (left-shifted) than PLCβ3 or PLCβ3-Δ892. See Supplementary Table 1. AU, arbitrary units. (b) Comparison of the basal activity of PLCβ3 variants. Deletion of the proximal CTR in PLCβ3-Δ847 increases basal activity relative to PLCβ3-Δ892. The higher basal activity of PLCβ3 reflects the contribution of more distal regions of the CTR to maximal activity. Activity was measured by counting free [³H]-IP₃ released from liposomes containing [³H]-PIP₂ at 30 °C in the presence of ~200 nM free Ca²⁺ at 4–5 time points. The data shown represent at least four individual experiments performed in duplicate ± SEM. (c) Mutation of PLCβ3 at positions that contribute to the Hα2′-catalytic core interface dramatically increase basal activity, indicating that this interaction is involved in autoinhibition. (d) Distal regions of the PLCβ3 CTR enhance binding to Gα_q. FCPIA was used to quantify the ability of PLCβ3 truncations to displace AlexaFluor488-labeled PLCβ3-Δ892 (R872A L876A L879A triple mutant) from biotinylated, AlF₄-activated Gα_i/q bound to avidin beads. Representative curves for PLCβ3 (circles), PLCβ3-Δ892 (squares) and PLCβ3-Δ847 (triangles) are shown. See Table 3 for measured inhibition constants.