Molecular mechanisms of phospholipase C β3 autoinhibition

Abstract

Phospholipase C β (PLCβ) enzymes are dramatically activated by heterotrimeric G proteins. Central to this response is the robust autoinhibition of PLCβ by the 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 important,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.

Figure 1

The PLCβ X–Y linker and proximal C-terminal domain (CTD) are highly conserved elements that regulate basal activity. (A) Primary structure. Numbers above the diagram correspond to domain boundaries. In the sequence alignment (human PLCβ1 (AAF86613), PLCβ2 (NP_004564), and PLCβ3 (NP_000923)), residues in the acidic stretch are shown in red, and the C-terminal ends of X–Y linker internal deletions in PLCβ3 are noted below. The primary Gα_q binding site in the proximal CTD is shown in blue. (B) Crystal structure of the Gα_q–PLCβ3-Δ892_Δacid complex. AlF₄⁻-activated Gα_q is shown as a gray surface and the domains of PLCβ3 are colored as in (A). The observed N- and C-termini of PLCβ3-Δ892_Δacid and Gα_q are labeled N and C, and N′ and C′, respectively. Dashed lines correspond to disordered loops. (C) View of the PLCβ3 catalytic core (PDB entry 3OHM) and proximal CTD from the perspective of the membrane plane. PLCβ3 is colored as in (A). Circled minus signs indicate the relative position of the acidic stretch in the X–Y linker. See also Supplemental Figures 1,2.

Figure 2

Basal activity and DSF measurements of PLCβ3 variants. (A) Deletions within the X–Y linker, but not the Gα_q binding site, alter basal activity. Increased activity is observed when the acidic stretch or entire linker is removed. Data represent at least five experiments performed in duplicate. (B) Addition of 5 mM IP₃ to hyperactive PLCβ3 variants increases their thermal stability (see Supplemental Figure 3A). T_m values were determined by monitoring the increase in fluorescence of ANS as a function of temperature. See Supplemental Table 2. Data represent at least five experiments performed in triplicate. (C) Deletions including the acidic stretch show decreased thermal stability compared to variants containing intact X–Y linkers. The T_m of each PLCβ3 variant was determined with respect to PLCβ3-Δ847, used as a control on each plate (Supplemental Table 3). PLCβ3-Δ892 and its variants have higher thermal stability relative to PLCβ3-Δ847 variants due to Hα2′. Data represents at least four experiments performed in triplicate. In panels B and C, * = P≤0.5, ** = P≤0.01, *** = P≤0.001, **** =P≤0.0001.

Figure 3

Heterotrimeric G protein activation of PLCβ3 variants. The activity of PLCβ3 variants was measured at 30 °C for 5 min at increasing concentrations of AlF₄⁻-activated Gα_q or Gβ₁γ₂. (A) Gα_q activates PLCβ3-Δ892 3–60 fold. As expected, loss of the primary Gα_q binding site (PLCβ3-Δ892_ΔLINPI) abolishes Gα_q activation. PLCβ3-Δ892_Δdisorder has the lowest basal activity, and thus shows the greatest fold increase, whereas PLCβ3-Δ892_Δacid and PLCβ3-Δ892_Δall variants are weakly activated (Table 2, Supplemental Figure 3B). Data represents at least five experiments in duplicate. (B) Gβ₁γ₂-mediated activation of PLCβ3 variants. Gβ₁γ₂ robustly activates PLCβ3_Δdisorder variants due to their low basal activity, whereas PLCβ3_Δacid and PLCβ3_Δall variants are activated only 3–5 fold. Deletion of the Gα_q binding site in PLCβ3-Δ892_ΔLINPI had no effect on Gβ₁γ₂ activation (Supplemental Figure 3C, D, Table 2). Data represent at least four experiments performed in duplicate.

Figure 4

Structural characterization of PLCβ3 variants. PLCβ3 variants are colored as in Figure 1A, and the observed ends of the X–Y linker are marked with pink asterisks. The Hα2′ helix is shown in cyan. (A) Active site of the Gα_q–PLCβ3-Δ882 complex (PDB entry 3OHM). Residues Leu341, Phe381, Met383, and Val654 form the hydrophobic ridge, which anchors the active site at the membrane for catalysis (Essen et al., 1997; Lyon and Tesmer, 2013). The lid helix at the C-terminal end of the X–Y linker physically occludes the active site. (B) The active site of the Gα_q–PLCβ3-Δ892_Δacid complex is open. Residues that would form the lid helix are present in this variant, but deletion of the acidic stretch leads to their disorder. (C) Active site of the Gα_q–PLCβ3-Δ892_Δacid·IP₃ complex. Electron density is observed for most atoms of the ligand, as seen in a 3σ |Fo|-|Fc| omit map (green wire cage). Electron density for the X–Y linker is not observed until residue 589. (D) Active site of the Gα_q–PLCβ3-Δ892_Δall·IP₃ complex. Strong 3σ |Fo|-|Fc| omit map density is observed for the entire IP₃ molecule. Deletion of the entire X–Y linker in this variant has no effect on the position of the bound ligand. See also Supplemental Figure 4.

Figure 5

A model of PLCβ autoinhibition by the X–Y linker. Under basal conditions, the lid helix at the C-terminal end of the X–Y linker blocks access to the active site. The acidic stretch interacts with conserved solvent-exposed basic residues on one face of the TIM barrel domain. In this model, based on PDB entry 2FJU, the last two residues of the PLCβ2 acidic stretch, Glu512 and Glu513, interact with conserved lysines and arginines. Breaking the electrostatic interactions between the TIM barrel domain and the acidic stretch, such as by interactions with the cell membrane, leads to loss of structure in the lid helix, increasing activity. Meanwhile, the Hα2′ helix remains bound to the catalytic core, acting as a brake on PLCβ activity via an independent mechanism until displaced by the binding of Gα_q.