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. 2020 Dec 4;295(49):16562-16571.
doi: 10.1074/jbc.RA120.015685. Epub 2020 Sep 18.

Functional and structural characterization of allosteric activation of phospholipase Cε by Rap1A

Monita Sieng  1 , Arielle F Selvia  1 , Elisabeth E Garland-Kuntz  1 , Jesse B Hopkins  2 , Isaac J Fisher  1 , Andrea T Marti  1 , Angeline M Lyon  3
Affiliations

Functional and structural characterization of allosteric activation of phospholipase Cε by Rap1A

Monita Sieng et al. J Biol Chem. .

Abstract

Phospholipase Cε (PLCε) is activated downstream of G protein-coupled receptors and receptor tyrosine kinases through direct interactions with small GTPases, including Rap1A and Ras. Although Ras has been reported to allosterically activate the lipase, it is not known whether Rap1A has the same ability or what its molecular mechanism might be. Rap1A activates PLCε in response to the stimulation of β-adrenergic receptors, translocating the complex to the perinuclear membrane. Because the C-terminal Ras association (RA2) domain of PLCε was proposed to the primary binding site for Rap1A, we first confirmed using purified proteins that the RA2 domain is indeed essential for activation by Rap1A. However, we also showed that the PLCε pleckstrin homology (PH) domain and first two EF hands (EF1/2) are required for Rap1A activation and identified hydrophobic residues on the surface of the RA2 domain that are also necessary. Small-angle X-ray scattering showed that Rap1A binding induces and stabilizes discrete conformational states in PLCε variants that can be activated by the GTPase. These data, together with the recent structure of a catalytically active fragment of PLCε, provide the first evidence that Rap1A, and by extension Ras, allosterically activate the lipase by promoting and stabilizing interactions between the RA2 domain and the PLCε core.

Keywords: G protein; Ras-related protein 1 (Rap1); calcium intracellular release; cardiovascular disease; cell signaling; conformational change; diacylglycerol; membrane enzyme; phosphatidylinositol signaling; phospholipase C; protein kinase C (PKC); small-angle X-ray scattering (SAXS); structural biology.

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Conflict of interest statement

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
Multiple domains in PLCε are required for Rap1AG12V-dependent activation.A, domain diagram of rat PLCε. The Y-box insertion and C2-RA1 linker are shown in orange and purple, respectively. Numbers above the diagram correspond to the domain boundaries most relevant to this work, with the variants under study shown below. B, PLCε PH-COOH (black circles) is activated by Rap1AG12V in a concentration-dependent manner. In contrast, PH-C2 (red squares) and EF3-COOH (blue triangles) are not activated at any concentration of Rap1AG12V tested. The data represents at least two independent experiments performed in duplicate. Error bars represent S.D.
Figure 2
Figure 2
Hydrophobic residues on the surface of the RA2 domain are critical for activation.A, the structure of H-Ras (gray) bound to the RA2 domain (blue, PDB entry 2C5L (14)) reveals conserved, hydrophobic residues (teal spheres) involved in crystal lattice contacts. Lys2171 and Lys2173 (hot pink spheres) were previously reported to be required for Rap1A-dependent activation. R. norvegicus residues are in parentheses. GTP is shown as orange sticks, and Mg2+ is shown as a black sphere. B and C, mutation of the Lys2150 or Lys2152 to alanine eliminates activation by Rap1AG12Vin vitro (B), as does mutation of the conserved hydrophobic residues distant from the Rap1A binding surface (C).
Figure 3
Figure 3
Rap1AG12V binding to PLCε PH-COOH or EF3-COOH stabilizes different conformational states.A and B, scattering profile for PLCε PH-COOH (A) and Guinier plot (B) demonstrate the variant is monomeric and monodisperse in solution. (C) Its pair–distance distribution function is consistent with a largely globular protein with some extended features. D and E, the scattering profile (D) and Guinier plot (E) for the Rap1AG12V–PH-COOH complex are also consistent with a monodisperse complex. (F) Its pair–distance distribution function shows a more compact structure upon the binding of Rap1AG12V. G–I, PLCε EF3-COOH is similar to PH-COOH in solution, as evidenced by its scattering profile (G), Guinier plot (H), and pair–distance distribution function (I). J and K, the Rap1AG12V–EF3-COOH complex does not have elevated lipase activity but is still monodisperse in solution as shown in (J) the scattering profile and (K) Guinier plot. (L) The shape of the pair–distance distribution function reveals the complex is more globular than EF3-COOH alone, and more compact, as evidenced by the smaller Dmax. The data for the PLCε PH-COOH variant are included for comparison (24).
Figure 4
Figure 4
Normalized pair–distance and dimensionless Kratky plots for PLCε variants alone and in complex with Rap1AG12V.A, the normalized P(r) functions for PH-COOH and Rap1AG12V–PH-COOH are similar, with Dmax values of ∼162 and ∼165 Å, respectively. B, comparison of PH-COOH (black circles) and Rap1AG12V–PH-COOH (gray squares) shows that the complex is more compact and globular than PH-COOH alone, as evidenced by the more bell-shaped curve and convergence at lower qRg. C, the normalized P(r) functions for EF3-COOH and Rap1AG12V–EF3-COOH reveal that binding of Rap1AG12V induces substantial conformational changes that lead to a more compact structure. This is further supported by the ∼30 Å decrease in Dmax for the Rap1AG12V–EF3-COOH complex. D, comparison of EF3-COOH (blue circles) and Rap1AG12V–EF3-COOH (light blue squares). Rap1AG12V binding induces conformational changes that result in a more compact and globular solution structure.
Figure 5
Figure 5
A model for activation of PLCε by Rap1A.Top panels, PLCε exists in multiple conformational states in solution. The PH domain, EF1/2, and RA2 domains are flexibly connected to the rest of the enzyme, as indicated by the dashed black lines, and interact transiently with one another under basal conditions (red arrows) (24). Bottom left panel, activated Rap1A binds to its high-affinity binding site on the PLCε RA2 domain. However, this interaction is insufficient on its own to activate lipase activity. Bottom right panel, the Rap1A–RA2 complex also interacts with a site on the PLCε core, potentially formed by the PH domain and EF hands, resulting in Rap1A-dependent activation.
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