Characterization of the membrane interactions of phospholipase Cγ reveals key features of the active enzyme

Abstract

PLCγ enzymes are autoinhibited in resting cells and form key components of intracellular signaling that are also linked to disease development. Insights into physiological and aberrant activation of PLCγ require understanding of an active, membrane-bound form, which can hydrolyze inositol-lipid substrates. Here, we demonstrate that PLCγ1 cannot bind membranes unless the autoinhibition is disrupted. Through extensive molecular dynamics simulations and experimental evidence, we characterize membrane binding by the catalytic core domains and reveal previously unknown sites of lipid interaction. The identified sites act in synergy, overlap with autoinhibitory interfaces, and are shown to be critical for the phospholipase activity in cells. This work provides direct evidence that PLCγ1 is inhibited through obstruction of its membrane-binding surfaces by the regulatory region and that activation must shift PLCγ1 to a conformation competent for membrane binding. Knowledge of the critical sites of membrane interaction extends the mechanistic framework for activation, dysregulation, and therapeutic intervention.

A frequent PLCγ1 mutation in adult T-cell leukemia/lymphoma determines functional properties of the malignant cells

“…PLCγ1 proteins (the wild type and S345F variant) were expressed and purified as described in Le Huray et al (31). The proteins were expressed in E. coli and purified by a combination of Ni 2+ -chelating, heparin affinity and gel filtration chromatography.…”

“…PLC-catalysed hydrolysis of phosphatidylinositol incorporated in liposomes was monitored by quantifying the production of inositol phosphate (IP1) using the IPone kit (CisBio) in an endpoint assay format as described in Liu et al (17), using the following assay buffer: 20 mM HEPES.KOH, 70 mM KCl, 3 mM EGTA, 2.97 mM CaCl2, 2 mM TCEP, 50 μg/mL fatty acid-free BSA, pH 7.0. Liposomes were prepared as outlined in Le Huray et al (31) and stored at a final concentration of 2 mg/mL and were composed of 20% PS, 45% PE, 15% PC, 10% cholesterol, 5% sphingomyelin and 5 % phosphatidylinositol (PI).…”

“…The only reported replacement of Ser345 is Phe; this substitution abolishes interaction with Tyr506 in the regulatory region, compromising autoinhibition and thus increasing PLC activity (Figure 1A, right panels). It is also possible that, in activated PLCγ1, the region on the catalytic TIM-barrel domain containing the Phe345 has a stronger interaction with the membrane or directly with the substrate, contributing further to an increase in hydrolysis of the membrane resident PIP2 (31).…”

Molecular dynamics simulations reveal membrane lipid interactions of the full-length lymphocyte specific kinase (Lck)

“…MD simulation studies of the PLCγ1 core domain revealed its orientation with the membrane. Mutation of the residues that were identified by the simulations to be important for the interaction of PLCγ1 core domain with the membrane reduced the binding of PLCγ1 to liposomes 22 . MD simulations have also been used to study the association of other peripheral membrane proteins to the membrane including the talin head domain (or parts of the talin head) 23 , 24 , the KRas-4B 25 and PH domains 20 .…”

Rapid Prediction of Lipid Interaction Sites on Pleckstrin Homology Domains Using Deep Graph Neural Networks and Molecular Dynamics Simulations

“…We recently presented a dataset of high-throughput MD simulations of the interactions of 100 pleckstrin homology (PH) domains with a complex plasma membrane model, and demonstrated the capability of the simulations to correctly identify experimentally verified sites of interaction with anionic phosphoinositide lipids, and to provide insights missing from the experimental datasets (49,50). PH domains are the largest family of membrane binding protein domains present in the Pfam database (56)(57)(58).…”