Direct observation of conformational dynamics of the PH domain in phospholipases Cɛ and β may contribute to subfamily-specific roles in regulation

Abstract

Phospholipase C (PLC) enzymes hydrolyze phosphatidylinositol lipids to produce second messengers, including inositol‐1,4,5‐triphosphate (IP3) and diacylgycerol (DAG), which increase intracellular calcium and activate protein kinase C (PKC), respectively. PLCɛ contributes to cardiac hypertrophy and contractility, as well as to oncogenic and inflammatory signaling pathways following activation of G protein‐coupled receptors and receptor tyrosine kinases. PLCɛ shares a conserved core with the PLC superfamily, but the roles of individual domains in regulation of activity and membrane binding have not been established. We used functional assays to show that the PLCɛ PH domain significantly increases basal lipase activity, but is dispensable for stability. We provide the first structural insights into domain organization of PLCɛ using small‐angle X‐ray scattering (SAXS) and electron microscopy (EM) to reveal that the PH domain is conformationally heterogeneous in solution. Comparisons of the PLCɛ solution structure to that of the closely‐related PLCβ enzyme demonstrate that the PLCβ PH domain is also mobile in solution, in contrast to previously reported crystal structures. We propose that the dynamic nature of the PLC PH domain and resulting conformational heterogeneity contributes to subfamily‐specific differences in activity and regulation by G proteins. We are now using cryo‐EM to expand on these findings and obtain higher resolution structures. Support or Funding Information This work is supported by the Purdue Center for Cancer Research, AHA grant 16SDG29930017, NIH NLHBI 1R01HL141076‐01 to A.M.L., and Purdue College of Science Staff and Administrative & Professional Staff Advisory Council Professional Development awards to E.E.G. SAXS experiments used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE‐AC02‐06CH11357. This project was supported by grant 9 P41 GM103622 from the National Institute of General Medical Sciences of the National Institutes of Health. Use of the Pilatus 3 1M detector was provided by grant 1S10OD018090‐01 from NIGMS. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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

“…Rap1A-dependent activation of PLCe has been demonstrated in cellbased assays, but not using purified components (20,21,23). As full-length PLCe has not been purified in sufficient quantities for biochemical analysis, we relied on the PLCe PH-COOH variant for these studies ( Figure 1A) (24), which retains both RA domains and is thus expected to be responsive to Rap1A. In a liposome-based activity assay, the addition of constitutively active and prenylated Rap1A G12V increased the specific activity of PH-COOH ~3-fold over basal, with a maximum specific activity of 1,900 ± 300 nmol IP3/min/nmol PLCe variant ( Figure 1B, Table 1, Table S1), which is similar to the fold activation reported in cell-based assays using full-length PLCe (17,23,25).…”

“…The PLCe EF3-COOH fragment lacks these elements, and has similar stability and basal activity to PH-COOH (Table 1, Figure S1). As a negative control, we also investigated the PH-C2 variant, which has reduced stability and activity relative to PH-COOH (Table 1, Figure S1) (24) and lacks both RA domains. Indeed, PLCe PH-C2 was not activated by Rap1A G12V at any concentration tested, consistent with the absence of the RA2 domain ( Figure 1B, Table 1).…”

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

“…Rap1A-dependent activation of PLCe has been demonstrated in cellbased assays, but not using purified components (20,21,23). As full-length PLCe has not been purified in sufficient quantities for biochemical analysis, we relied on the PLCe PH-COOH variant for these studies ( Figure 1A) (24), which retains both RA domains and is thus expected to be responsive to Rap1A. In a liposome-based activity assay, the addition of constitutively active and prenylated Rap1A G12V increased the specific activity of PH-COOH ~3-fold over basal, with a maximum specific activity of 1,900 ± 300 nmol IP3/min/nmol PLCe variant ( Figure 1B, Table 1, Table S1), which is consistent with the fold activation reported in cell-based assays using fulllength PLCe (17,23,25).…”

“…The dialysate was applied to Roche cOmplete NiNTA resin or a GE HisTrap, and the flowthrough containing the TEV-cleaved EF3-COOH was collected and passed over the column two more times. The protein in the collected flowthrough was then purified as previously described (24).…”

“…Melting temperatures (Tm) of PLCe variants were determined as previously described (24,30). A final concentration of 0.5 mg/mL was used for each PLCe variant.…”