Crystal structure of cPLA2 and methods of identifying...

Details Crystal structure of cPLA2 and methods of identifying... Crystal structure of cPLA2 and methods of identifying...

1999-02-15

2004-10-05

Moran, Marjorie (Department: 1631)

Data processing: measuring, calibrating, or testing

Measurement system in a specific environment

Chemical analysis

C702S019000, C435S019000

Reexamination Certificate

active

06801860

ABSTRACT:

BACKGROUND OF THE INVENTION
Leukotrienes and prostaglandins are inflammatory mediators important in asthma, arthritis, and other inflammatory diseases. Leukotrienes cause airway obstruction in asthmatics through bronchoconstriction, increased mucus secretion, and chemoattraction of inflammatory cells (O'Byrne, 1997); prostaglandins cause pain and edema associated with arthritis. Pharmacological intervention blocking either the synthesis or action of these lipid mediators is effective in treating human disease, thus confirming their importance (Simon et al., 1998; O'Byrne, 1997).
Cytosolic phospholipase A
2
(cPLA
2
) initiates the production of leukotrienes and prostaglandins by releasing arachidonic acid from cellular membranes. Arachidonic acid in turn is metabolized to prostaglandins by the cyclooxygenase pathway and to leukotrienes by the 5-lipoxygenase pathway. Concomitant with the release of arachidonic acid, lyso-platelet-activating factor (lyso-PAF) is formed, which can then be acetylated to generate PAF, a molecule also implicated in the pathophysiology of asthma and arthritis (Venable et al., 1993). Hence, the reaction catalyzed by cPLA
2
initiates the production of three classes of inflammatory mediators: leukotrienes, prostaglandins, and PAF.
cPLA
2
is a member of a diverse superfamily of phospholipase A
2
enzymes with the common ability to cleave the sn-2 ester of glycerophospholipids. The first members of the family to be characterized were the low molecular weight enzymes that are secreted either extracellularly or into granules (and are here collectively referred to as sPLA
2
s; groups I, II, III, V, VII, and IX) (Dennis, 1997). The PLA
2
family has expanded with the cloning and characterization of calcium-dependent arachidonyl-selective cPLA
2
(Clark et al., 1991; Kramer et al., 1991), the calcium-independent PLA
2
(Tang et al., 1997; Balboa et al., 1997) and the plasma and intracellular PAF-acetylhydrolases (Hattori et al., 1994, 1995). Each of these new enzymes shares no sequence homology with the low molecular weight enzymes or with each other. In addition, unlike sPLA
2
s, which use activated water to cleave the phospholipid, these enzymes appear to use a nucleophilic serine. In this respect, they have more in common with other lipases of the &agr;/&bgr; hydrolase family than with the sPLA
2
s. Two additional enzymes with 30% identity to the catalytic domain of cPLA
2
have recently been cloned; they have been termed cPLA
2
&bgr; (C. Song et al., manuscript in preparation) and cPLA
2
&ggr; (Underwood et al., 1998).
The cloning of cPLA2 is also described in U.S. Pat. Nos. 5,322,776, 5,354,677, 5,527698 and 5,593,878. The cloning of calcium-independent cPLA2 is also described in U.S. Pat. Nos. 5,466,595, 5,554,511, 5,589,170 and 5,840,511.
Numerous pieces of evidence have supported the central role of cPLA
2
in lipid mediator biosynthesis. cPLA
2
is the only enzyme which is highly selective for phospholipids containing arachidonic acid in the sn-2 position (Clark et al., 1995; Hanel & Gelb, 1993). Activation of cPLA
2
or its increased expression have been linked with increased leukotriene and prostaglandin synthesis (Lin et al., 1992b). Following activation, cPLA
2
translocates to the nuclear membrane, where it is co-localized with the cyclooxygenase and lipoxygenase that metabolize arachidonate to prostaglandins and leukotrienes (Schievella et al., Glover et al., 1995). Although these data are compelling, the most definitive evidence for the central role of cPLA
2
in eicosanoid and PAF production came from mice made deficient in cPLA
2
through homologous recombination (Uozumi et al., 1997; Bonventre et al., 1997). Peritoneal macrophages derived from these animals failed to make leukotrienes, prostaglandins, or PAF. The cPLA
2
deficient mice have also been informative of the role of cPLA
2
in disease, since these mice are resistant to bronchial hyperreactivity in an anaphylaxis model used to mimic asthma (Uozumi et al., 1997).
cPLA
2
consists of at least two functionally distinct domains: a N-terminal Ca
2+
-dependent lipid-binding (CaLB) domain and a Ca
2+
-independent catalytic domain (Nalefski et al., 1994). The N-terminal CaLB domain is a member of the C2 family and its structure has been solved (Perisic et al., 1998; Xu et al., 1998); it mediates calcium regulation by co-localizing the catalytic domain with its membrane substrate (Nalefski et al., 1994). cPLA
2
activity, in addition, is also regulated by phosphorylation of the catalytic domain (Lin et al., 1991; Leslie, 1997). Ser505 (of SEQ ID NO:2) and Ser727 (of SEQ ID NO:2) are conserved across all species and are phosphorylated in multiple cell types (de Carvalho et al., 1998). Phosphorylation of Ser505 (of SEQ ID NO:2) by members of the MAP-kinase family is a common response to extracellular stimuli that release arachidonic acid. Mutation of Ser505 (of SEQ ID NO:2) to Ala decreases activation (Lin et al., 1993) whereas the analogous mutation on Ser727 (of SEQ ID NO:2) has no effect (Leslie, 1998).
Several lines of evidence suggest that the catalytic mechanism of cPLA
2
proceeds through a serine-acyl intermediate (Trimble et al., 1993; Hanel & Gelb, 1995). Mutation of Ser228 (of SEQ ID NO:2) abolishes cPLA
2
activity against all substrates including phospholipids, lysophospholipids, and fatty acylated coumarin (Pickard et al., 1996; Huang et al., 1996). Ser228 (of SEQ ID NO:2) is present in a pentapeptide sequence, G-L-S-G-S (SEQ ID NO:3), which is similar to the classic “lipase motif” G-X-S-X-G (Schrag & Cygler, 1997) found in most lipases within the broader family of enzymes called the &agr;/&bgr; hydrolases. These enzymes possess a common core which consists of a well-conserved mixed &bgr; sheet whose strands are interspersed by &agr; helices. In all &agr;/&bgr; hydrolases, the catalytic serine is present in a tight turn between a &bgr;-strand and an &agr;-helix, termed the “nucleophilic elbow” (see review by Schrag & Cygler, 1997). This turn directs the short serine side chain away from the protein backbone, reducing the steric hindrance about the residue and requiring that the +2 and −2 sidechains be small to avoid steric clash; thus the prevalence of the G-X-S-X-G motif (Derewenda & Derewenda, 1991).
In addition to serine, &agr;/&bgr; hydrolases use a histidine and an acid (aspartate/glutamate) as the other members of a catalytic triad simliar to that present in serine proteases (Schrag & Cygler, 1997). However, although in cPLA
2
Asp549 (of SEQ ID NO:2) was shown to be essential for activity, none of the 19 histidine residues were (Pickard et al., 1996). A different residue, Arg200 (of SEQ ID NO:2), was implicated as playing a role in the enzymatic process, although the mechanism for its involvement remained unknown. These observations suggested that cPLA
2
acts through a novel catalytic mechanism for acyl hydrolases.
Like both the sPLA
2
s and the lipases of the &agr;/&bgr; hydrolase family, cPLA
2
preferentially cleaves substrates presented in an interface (Nalefski et al., 1994). This phenomenon, known as interfacial activation, has been attributed to either conformational changes in the enzyme or more favorable presentation of the substrate (Scott et al., 1990). The origin of the 1500-fold difference in cPLA
2
activity toward monomeric and micellar substrate remains unknown.
Despite the key role of cPLA
2
in inflammatory disease, its three-dimensional structure remained unsolved, leaving numerous questions unanswered. Here we report the x-ray crystal structure of human cPLA
2
at 2.5 Å resolution. The structure provides insight into the origin of arachidonate selectivity and interfacial activation, clarifies the roles of Ser228, Asp549, and Arg200 (of SEQ ID NO:2), and reveals the interplay between CaLB and the catalytic domains. Importantly, the structure is of a unique topology, distinct from that of the &agr;/&bgr; hydrolase family.
SUMMARY OF THE INVENTION
All references to amino acids in cPLA2 herein are made using residue

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