Structure and Dynamics of the Phospholipase C-δ1 Pleckstrin Homology Domain Located at the Lipid Bilayer Surface

Abstract

Despite the importance of signal transduction pathways at membrane surfaces, there have been few means of investigating their molecular mechanisms based on the structural information of membrane-bound proteins. We applied solid state NMR as a novel method to obtain structural information about the phospholipase C-␦1 (PLC-␦1) pleckstrin homology (PH) domain at the lipid bilayer surface. NMR spectra of the alanine residues in the vicinity of the ␤5/␤6 loop in the PH domain revealed changes in local conformations due to the membrane localization of the protein. We propose that these conformational changes originate from a hydrophobic interaction between the amphipathic ␣-helix located in the ␤5/␤6 loop and the hydrophobic layer of the membrane and contribute to the membrane binding affinity, interdomain interactions and intermolecular interactions of PLC-␦1.Pleckstrin homology (PH) 1 domains are well defined structural modules of about 120 amino acid residues (1, 2) mainly found in proteins involved in cellular signaling and cytoskeletal functions (3-6). It has been proposed that these domains function as mediators of intermolecular interactions analogous to many other structural modules involved in cellular signaling (e.g. SH2 and SH3 domains). Many kinds of inositol lipids and inositol phosphates have been identified as important ligands of PH domains (3-6), and, in some cases, the PH domains also interact with other proteins and mediate protein-protein interactions (5).The PH domain of phospholipase C-␦1 (PLC-␦1) is one of the most extensively studied PH domains. It has been proposed that it regulates the membrane localization of PLC-␦1 (7, 8) through its high affinity specific interaction with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ), a PLC-␦1 ligand (9), and D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) (10), a product of PtdIns(4,5)P 2 hydrolysis by PLC-␦1. Despite the rather low sequence similarity among the PH domain families, the secondary and tertiary structural motifs of the PH domain are highly conserved (3-6). A high resolution structural model of the rat PLC-␦1 PH domain forming a complex with Ins(1,4,5)P 3 has been determined by x-ray diffraction study at 1.9-Å resolution (11). The model consists of a seven-stranded ␤ sandwich formed by two orthogonal anti-parallel ␤-sheets and a C-terminal amphipathic ␣-helix. These are conserved structural motifs among the PH domains whose structures have been determined by x-ray diffraction and NMR studies. The loops between the ␤ strands, particularly the ␤1/␤2, ␤3/␤4, and ␤6/␤7 loops, differ greatly among the PH domains, and, in the case of the PLC-␦1 PH domain, the ␤1/␤2 and ␤3/␤4 loops mainly interact with Ins(1,4,5)P 3 . The ␤5/␤6 loop of the PLC-␦1 PH domain includes a characteristic short amphipathic ␣-helix (␣2-helix) that is not found in other PH domain model structures studied so far.Because functionally important intermolecular interactions of PLC-␦1 with its ligand, PtdIns(4,5)P 2 , or other proteins included in the signal transduc...

The Pleckstrin Homology Domain of Phospholipase Cβ Transmits Enzymatic Activation through Modulation of the Membrane−Domain Orientation

“…So far, the mechanism through which PHβ2 binds to membranes is unknown in contrast to PHδ 1 whose membrane interactions have been clearly identified [4,50]. A structural model of PHβ 2 and subsequent simulation of its insertion into an electrically neutral membrane allows us to propose a dominant membrane orientation where the 71-88 region resides on the membrane surface (Figure 3).…”

Stimulation of phospholipase Cβ by membrane interactions, interdomain movement, and G protein binding — How many ways can you activate an enzyme?

“…This activation had been previously missed since the activator, PI(4,5)P 2 , was the same as the substrate. It should be noted that the PH-δ1 is not a rigid lipid-binding domain since a slight conformational change, involving a small amphipathic helix of β5/β6 loop occurs upon binding to PI(4,5)P 2 and this change is regulated by the phosphatidylserine content in membranes [80,81]. Roberts and collaborators also suggest that the PH domain may have an allosteric role in PLC-δ1 activity [20].…”

“…Comparing a model structure of PH-β2 with the known structure of PH-δ1 suggests that the β5/β6 loop, which is longer than in PH-δ1, (Figure 3) could act as a regulatory domain for activation by Gβγ [41]. A peptide corresponding to this loop (PHβ [71][72][73][74][75][76][77][78][79][80][81][82][83][84][85][86][87][88] ) activates the enzyme, and this activation is competitive with Gβγ activation. The structure of PLC-β2 reveals that the β5/β6 loop does not belong to the surface that is tightly packed with the EF-hands and the catalytic regions, or to the hydrophobic ridge interacting with Rac2.…”

“…The ability of the peptide corresponding to PHβ [71][72][73][74][75][76][77][78][79][80][81][82][83][84][85][86][87][88] to activate the enzyme was unexpected.…”

Mechanistic Basis of Differential Cellular Responses of Phosphatidylinositol 3,4-Bisphosphate- and Phosphatidylinositol 3,4,5-Trisphosphate-binding Pleckstrin Homology Domains

“…This is largely because of the presence of a large number of cationic residues and the apparent lack of hydrophobic residues on their membrane-binding surfaces (32,51). However, recent studies have indicated that the PH domain of phospholipase C (PLC) ␦1 significantly penetrates the membrane (52,53).…”