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...

A number of processes contributing to important cellular functions such as cellular signal transduction, cytoskeletal organization, and membrane trafficking are known to be localized at membrane surfaces of the plasma membrane and organelles. For instance, extracellular signals are often transmitted from activated receptors to peripheral membrane proteins, such heterotrimeric G-proteins, and subsequently amplified and ⁄ or passed to successors by a number of peripheral membrane proteins located at the membrane surface. Because the interface between the aqueous phase and the hydrophobic interior of the membrane provides a unique molecular environment completely different from the homogeneous solution phase environment [1,2], one would expect induction of unique structurefunction relationships for the peripheral membrane The membrane binding affinity of the pleckstrin homology (PH) domain of phospholipase C (PLC)-d1 was investigated using a vesicle coprecipitation assay and the structure of the membrane-associated PH domain was probed using solid-state 13 C NMR spectroscopy. Twenty per cent phosphatidylserine (PS) in the membrane caused a moderate but significant reduction of the membrane binding affinity of the PH domain despite the predicted electrostatic attraction between the PH domain and the head groups of PS. Solid-state NMR spectra of the PH domain bound to the phosphatidylcholine (PC) ⁄ PS ⁄ phosphatidylinositol 4,5-bisphosphate (PIP 2 ) (75 : 20 : 5) vesicle indicated loss of the interaction between the amphipathic a2-helix of the PH domain and the interface region of the membrane which was previously reported for the PH domain bound to PC ⁄ PIP 2 (95 : 5) vesicles. Characteristic local conformations in the vicinity of Ala88 and Ala112 induced by the hydrophobic interaction between the a2-helix and the membrane interface were lost in the structure of the PH domain at the surface of the PC ⁄ PS ⁄ PIP 2 vesicle, and consequently the structure becomes identical to the solution structure of the PH domain bound to d-myo-inositol 1,4,5-trisphosphate. These local structural changes reduce the membrane binding affinity of the PH domain. The effects of PS on the PH domain were reversed by NaCl and MgCl 2 , suggesting that the effects are caused by electrostatic interaction between the protein and PS. These results generally suggest that the structure and function relationships among PLCs and other peripheral membrane proteins that have similar PH domains would be affected by the local lipid composition of membranes.Abbreviations DD-MAS, single pulse excitation dipolar decoupled-magic angle spinning; GST, glutathione-S-transferase; IP 3 , D-myo-inositol 1,4,5-trisphosphate; PC, phosphatidylcholine; PH domain, pleckstrin homology domain; PIP 2 , phosphatidylinositol

The effects of geometric properties of membranes on the structure of the phospholipase C-delta1 (PLC-delta1) pleckstrin homology (PH) domain were investigated using solid state (13)C NMR spectroscopy. Conformations of the PLC-delta1 PH domain at the surfaces of multilamellar vesicles (MLV), small unilamellar vesicles (SUV), and micelles were examined to evaluate the effects of membrane curvature on the PH domain. An increase in curvature of the water-hydrophobic layer interface hinders membrane-penetration of the amphipathic alpha2-helix of the PH domain that assists the membrane-association of the PH domain dominated by the phosphatidylinositol 4,5-bisphosphate (PIP(2)) specific lipid binding site. The solid state (13)C NMR signal of Ala88 located at the alpha2-helix indicates that the conformation of the alpha2-helix at the micelle surface is similar to the solution conformation and significantly different from those at the MLV and SUV surfaces which were characterized by membrane-penetration and re-orientation. The signal of Ala112 which flanks the C-terminus of the beta5/beta6 loop that includes the alpha2-helix, showed downfield displacement with decrease in the interface curvature of the micelles, SUV and MLV. This reveals that the conformation of the C-terminus of the beta5/beta6 loop connecting the beta-sandwich core containing the PIP(2) binding site and the amphipathic alpha2-helix is sensitive to alterations of the curvature of lipid bilayer surface. It is likely that these alterations in the conformation of the PLC-delta1 PH domain contribute to the regulatory mechanisms of the intracellular localization of PLC-delta1 in a manner dependent upon the structure of the molecular complex containing PIP(2).

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