Abstract
Human plasma platelet-activating factor (PAF) acetylhydrolase functions by reducing PAF levels as a general anti-inflammatory scavenger and is linked to anaphylactic shock, asthma, and allergic reactions. The enzyme has also been implicated in hydrolytic activities of other pro-inflammatory agents, such as sn-2 oxidatively fragmented phospholipids. This plasma enzyme is tightly bound to low and high density lipoprotein particles and is also referred to as lipoprotein-associated phospholipase A2. The crystal structure of this enzyme has been solved from x-ray diffraction data collected to a resolution of 1.5 angstroms. It has a classic lipase alpha/beta-hydrolase fold, and it contains a catalytic triad of Ser273, His351, and Asp296. Two clusters of hydrophobic residues define the probable interface-binding region, and a prediction is given of how the enzyme is bound to lipoproteins. Additionally, an acidic patch of 10 carboxylate residues and a neighboring basic patch of three residues are suggested to play a role in high density lipoprotein/low density lipoprotein partitioning. A crystal structure is also presented of PAF acetylhydrolase reacted with the organophosphate compound paraoxon via its active site Ser273. The resulting diethyl phosphoryl complex was used to model the tetrahedral intermediate of the substrate PAF to the active site. The model of interface binding begins to explain the known specificity of lipoprotein-bound substrates and how the active site can be both close to the hydrophobic-hydrophilic interface and at the same time be accessible to the aqueous phase.
Highlights
Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero3-phosphocholine)2 is a phospholipid messenger synthesized by a variety of cells involved in host defense, such as endothelial cells, platelets, neutrophils, monocytes, and macrophages [1]
Overall Structure of platelet-activating factor (PAF)-AH—We have solved the crystal structures of human plasma PAF-AH in a ligand-free form and as a complex with the organophosphate compound paraoxon reacted with the active site Ser273 (Fig. 1)
The structure of plasma PAF-AH was submitted to the DALI website to obtain structural homologues [28]; the S. exfoliatus lipase structure [20] was the top hit with a Z score of 23.8, C␣ RMSD of 2.4 Å, and 19% identity over a subset of 228 amino acids aligned
Summary
Protein Preparation and Crystallization—We obtained 160 mg of pure human plasma PAF-AH (PAFase, residues 47– 429, NCBI accession Q13093) overexpressed from Escherichia coli from ICOS Corporation. The data sets for native ligand-free and paraoxon inhibited crystals were collected at Beamline X29 of the National Synchrotron Light Source (Brookhaven National Laboratory, Upton, NY). Crystal Structure Solution—A single-wavelength anomalous dispersion data set to a resolution of 2.71 Å of a MeHgCl derivative of PAF-AH was used to phase the structure. The noncrystallographic symmetry axis was used to improve initial electron density maps using the CCP4 suite program Dm [24] These medium resolution electron density maps, which contained regions of well defined ␣-helices and -sheets, allowed the initial model building of roughly half the 766 amino acids/asymmetric unit with the program COOT [25]. The partial model was used as initial phases together with the native data set to a resolution of 1.5 Å for the automated program ARP/wARP [26]. A CIF parameter file for the DEP group, which is covalently attached to Ser273, was prepared using the monomer library sketcher module of the program CCP4 [24]
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