Catalytic domain of phosphoinositide-specific phospholipase C (PLC). Mutational analysis of residues within the active site and hydrophobic ridge of plcdelta1.

Structural studies of phospholipase C delta1 (PLCdelta1) in complexes with the inositol-lipid headgroup and calcium identified residues within the catalytic domain that could be involved in substrate recognition, calcium binding, and catalysis. In addition, the structure of the PLCdelta1 catalytic domain revealed a cluster of hydrophobic residues at the rim of the active site opening (hydrophobic ridge). To assess a role of each of these residues, we have expressed, purified, and characterized enzymes with the point mutations of putative active site residues (His311, Asn312, Glu341, Asp343, His356, Glu390, Lys438, Lys440, Ser522, Arg549, and Tyr551) and residues from the hydrophobic ridge (Leu320, Phe360, and Trp555). The replacements of most active site residues by alanine resulted in a great reduction (1,000-200,000-fold) of PLC activity analyzed in an inositol lipid/sodium cholate mixed micelle assay. Measurements of the enzyme activity toward phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4, 5-bis-phosphate (PIP2) identified Ser522, Lys438, and Arg549 as important for preferential hydrolysis of polyphosphoinositides, whereas replacement of Lys440 selectively affected only hydrolysis of PIP2. When PLC activity was analyzed at different calcium concentrations, substitutions of Asn312, Glu390, Glu341, and Asp343 resulted in a shift toward higher calcium concentrations required for PIP2 hydrolysis, suggesting that all these residues contribute toward Ca2+ binding. Mutational analysis also confirmed the importance of His311 ( approximately 20,000-fold reduction) and His356 ( approximately 6,000-fold reduction) for the catalysis. Mutations within the hydrophobic ridge, which had little effect on PIP2 hydrolysis in the mixed-micelles, resulted in an enzyme that was less dependent on the surface pressure when analyzed in a monolayer. This systematic mutational analysis provides further insights into the structural basis for the substrate specificity, requirement for Ca2+ ion, catalysis, and surface pressure/activity dependence, with general implications for eukaryotic phosphoinositide-specific PLCs.

Solution structure of the human pp60c-src SH2 domain complexed with a phosphorylated tyrosine pentapeptide.

Human pp60c-src is a cellular nonreceptor tyrosine kinase that participates in cytosolic signal transduction and has been implicated in the development of malignant tumors in the human breast and colon. Signal transduction is mediated by highly specific interactions between the SH2 domain and receptor phosphorylated tyrosine binding motifs. To elucidate the molecular conformation and interactions in solution, a family of highly resolved nuclear magnetic resonance (NMR) structures was determined for the src SH2 domain complexed with a high-affinity phosphorylated pentapeptide, acetyl-p YEEIE-OH. The 23 structures, generated with a distance geometry (DG) and a dynamical simulated annealing (SA) procedure, satisfied 2072 experimental restraints derived from a variety of multifrequency/multidimensional and isotope-filtered NMR data. Superimposition of residues 143-245 upon the mean coordinate set yielded an atomic rmsd of 0.58 +/- 0.09 A for the N, C alpha, C' atoms and 1.04 +/- 0.08 for all the non-hydrogen atoms. Residues in the ordered secondary structure regions superimpose to 0.29 +/- 0.04 A for the N, C alpha, C' and 0.73 +/- 0.08 A for all the non-hydrogen atoms. The angular order parameter calculated for the phi, psi angles was > 0.9 for 81 of the 106 protein residues. The main protein conformational features are three antiparallel beta-strands that traverse a compact core with an alpha-helix on each side of the core near the N- and C-termini. The observed intermolecular nuclear Overhauser effects (NOE) from the pY, +1E, and +3I residues positioned the ligand in an extended conformation across the SH2 domain surface with the pY and +3I side chains inserted into the protein binding pockets. In general, the protein conformation is consistent with previously reported structures of different SH2 domain complexes determined by X-ray crystallography. However, inter- or intramolecular interactions involving the guanidinium side chains of the solvated R alpha A2 or the buried R beta B5 were not observed at pH = 5.5 or 7.0. If such interactions exist in solution, the absence of any confirming data probably arises from rapid exchange with solvent and/or undetermined dynamic components. Thus, the unrestrained R alpha A2 side chain did not show an amino-aromatic interaction or a hydrogen bond to the -1 carbonyl oxygen as observed in the crystal structures. This result is consistent with the solution structure of a different SH2 domain complex. A more detailed comparison between the crystal structure and the NMR-derived solution structures of the same src SH2 domain complex is presented.(ABSTRACT TRUNCATED AT 400 WORDS)

Crystal structure of phospholipase C from Bacillus cereus complexed with a substrate analog.

We report the first crystal structure of a complex between PLC from Bacillus cereus (PLCBc) and a competitive inhibitor that is an analog of the natural phospholipid substrate. The structure has been determined at 1.9 A resolution and refined to a final R-factor of 15.7%. The inhibitor binds with its phosphonyl group to the three Zn ions in the active site of the enzyme and is also involved in a hydrogen bonded network including several water molecules and amino acid side-chains which appear to help orient the substrate for productive binding. The interactions within this complex provide some important information regarding the mechanism of PLC-catalyzed hydrolysis of membrane phospholipids. A water molecule, located approximately apical to the diacylglycerol leaving group, seems to be the most likely candidate for the attacking nucleophile which initiates the reaction.

Solution structure of the SH3 domain of phospholipase C-gamma.

SH3 (Src homology 3) domains are found in many signaling proteins and appear to function as binding modules for cytoplasmic target proteins. The solution structure of the SH3 domain of human phospholipase C-gamma (PLC-gamma) was determined by two-dimensional 1H NMR analysis. This SH3 domain is composed of eight antiparallel beta strands consisting of two successive "Greek key" motifs, which form a barrel-like structure. The conserved aliphatic and aromatic residues form a hydrophobic pocket on the molecular surface, and the conserved carboxylic residues are localized to the periphery. The hydrophobic pocket may serve as a binding site for target proteins. Analysis of the slowly exchanging amide protons by NMR measurements indicates that despite containing a high content of beta structure, the SH3 domain of PLC-gamma is flexible.