Crystal structure of human plasma platelet-activating factor acetylhydrolase: structural implication to lipoprotein binding and catalysis

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.

FIGURE 1.

A, stereo ribbon model of the PAF-AH structure (helix, purple, β-strands, green; loops, light gray). The catalytic triad of Ser²⁷³, His³⁵¹, and Asp²⁹⁶ are rendered in ball and stick. The predicted LDL-binding surface is oriented to the bottom of the view. B, interface-binding region and active site of PAF-AH are shown from the crystal structure reacted with the organophosphate compound paraoxon. The view shown was rendered after a 60° rotation on the x axis from the view shown in A. The catalytic triad and bound DEP moiety are shown in green. Residues predicted to penetrate into the hydrophobic portion of the LDL interface (Thr¹¹³, His¹¹⁴, Trp¹¹⁵, Leu¹¹⁶, Met¹¹⁷, Ile¹²⁰, Leu¹²³, Leu¹²⁴, Ile³⁶⁴, Ile³⁶⁵, Met³⁶⁸, and Leu³⁶⁹) are shown in yellow. C, electrostatic surface of the interfacial-binding region and active site pocket using the same view as B. The DEP ethoxy group pointing downward is sitting in a pocket where the sn-2 chain of PAF-AH substrates is predicted to bind. The DEP ethoxy group pointing to the top and outward is predicted to be where the larger lyso-PC would be oriented. D, stereo view of the active site S273 of PAF-AH covalently linked to DEP with an electron density map (coefficients 2F_o - F_c, 1.2 σ) around highlighted active site residues. The orientation shown is nearly identical to the views of B and C. The catalytic triad (Ser²⁷³, His³⁵¹, and Asp²⁹⁶) and other active site residues (Trp²⁹⁸, Phe³²², Phe²⁷⁴, Leu¹⁵³, His²⁷², Tyr¹⁶⁰, Phe¹¹⁰, and Gln³⁵²) are shown in green ball and stick, with other neighboring protein atoms in gray. The figure was prepared using the program PyMOL (51).

FIGURE 2.

A, reaction of Ser²⁷³ of PAF-AH with the organophosphate compound paraoxon. The bound complex mimics the tetrahedral intermediate of the reaction. B, first step of the esterolysis reaction of Ser²⁷³ of PAF-AH with a substrate, such as PAF, where R₁ represents lyso-PAF.

FIGURE 3.

Polymorphic sites of PAF-AH shown in gray ball and stick relative to the active site Ser²⁷³ and in a view looking directly at the interfacial binding surface of the enzyme. Three of the polymorphic sites (I198T, A379V, and R92H) are solvent-accessible. Two loss of function polymorphisms (V279F and Q281R) that lead to a loss of function in 4% of Japanese individuals are core residues. This figure was rendered using the program PyMOL (51).

FIGURE 4.

A, model of the tetrahedral intermediate of C₁₈-PAF (cyan) bound to PAF-AH with catalytic triad residues (Ser²⁷³, His³⁵¹, and Asp²⁹⁶) in green and interfacial-binding residues (Thr¹¹³, His¹¹⁴, Trp¹¹⁵, Leu¹¹⁶, Met¹¹⁷, Ile¹²⁰, Leu¹²³, Leu¹²⁴, Ile³⁶⁴, Ile³⁶⁵, Met³⁶⁸, and Leu³⁶⁹) in yellow. The coordinates of the tetrahedral intermediate were modeled based on the crystal structure of the DEP moiety complexed to PAF-AH (tetrahedral mimic). The C₁₈-alkyl chain was oriented to penetrate into the hydrophobic portion of the LDL particle. The predicted plane of the hydrophilic-hydrophobic interface, which was predicted by the OPM method (47, 48), is displayed with small gray spheres. B, the view from A was rotated by 90° on the y axis to show a side view of the interface and substrate-bound model. A prominent cluster of 10 carboxylate residues (Asp³⁷⁴, Asp³⁷⁶, Asp³⁸², Asp⁴⁰¹, Asp⁴⁰³, Asp⁴⁰⁶, Glu⁴¹⁰, Asp⁴¹², Asp⁴¹³, and Glu⁴¹⁴) are shown in red, and three basic residues (Lys⁵⁵, Arg⁵⁸, and Lys³⁶³) are shown in blue ball and stick. C, electrostatic surface view of A. D, electrostatic surface view of B. The figure was prepared using the program PyMOL (51).