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The structure of iPLA2? reveals dimeric active sites and suggests mechanisms of regulation and localization.

ABSTRACT: Calcium-independent phospholipase A2? (iPLA2?) regulates important physiological processes including inflammation, calcium homeostasis and apoptosis. It is genetically linked to neurodegenerative disorders including Parkinson's disease. Despite its known enzymatic activity, the mechanisms underlying iPLA2?-induced pathologic phenotypes remain poorly understood. Here, we present a crystal structure of iPLA2? that significantly revises existing mechanistic models. The catalytic domains form a tight dimer. They are surrounded by ankyrin repeat domains that adopt an outwardly flared orientation, poised to interact with membrane proteins. The closely integrated active sites are positioned for cooperative activation and internal transacylation. The structure and additional solution studies suggest that both catalytic domains can be bound and allosterically inhibited by a single calmodulin. These features suggest mechanisms of iPLA2? cellular localization and activity regulation, providing a basis for inhibitor development. Furthermore, the structure provides a framework to investigate the role of neurodegenerative mutations and the function of iPLA2? in the brain.

SUBMITTER: Malley KR

PROVIDER: S-EPMC5823874 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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The structure of iPLA2β reveals dimeric active sites and suggests mechanisms of regulation and localization.

Malley Konstantin R KR Koroleva Olga O Miller Ian I Sanishvili Ruslan R Jenkins Christopher M CM Gross Richard W RW Korolev Sergey S

Nature communications 20180222 1

Calcium-independent phospholipase A2β (iPLA2β) regulates important physiological processes including inflammation, calcium homeostasis and apoptosis. It is genetically linked to neurodegenerative disorders including Parkinson's disease. Despite its known enzymatic activity, the mechanisms underlying iPLA2β-induced pathologic phenotypes remain poorly understood. Here, we present a crystal structure of iPLA2β that significantly revises existing mechanist ...[more]

PMID: 29472584

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Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is a ?-herpesvirus closely associated with Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman disease. Open reading frame 57 (ORF57), a viral early protein of KSHV promotes splicing, stability and translation of viral mRNA and is essential for viral lytic replication. Previous studies demonstrated that dimerization of ORF57 stabilizes the protein, which is critical for its function. However, the detailed structural basis of dimerization was not elucidated. In this study, we report the crystal structures of the C-terminal domain (CTD) of ORF57 (ORF57-CTD) in both dimer at 3.5 Å and monomer at 3.0 Å. Both structures reveal that ORF57-CTD binds a single zinc ion through the consensus zinc-binding motif at the bottom of each monomer. In addition, the N-terminal residues 167-222 of ORF57-CTD protrudes a long "arm" and holds the globular domains of the neighboring monomer, while the C-terminal residues 445-454 are locked into the globular domain in cis and the globular domains interact in trans. In vitro crosslinking and nuclear translocation assays showed that either deletion of the "arm" region or substitution of key residues at the globular interface led to severe dimer dissociation. Introduction of point mutation into the zinc-binding motif also led to sharp degradation of KSHV ORF57 and other herpesvirus homologues. These data indicate that the "arm" region, the residues at the globular interface and the zinc-binding motif are all equally important in ORF57 protein dimerization and stability. Consistently, KSHV recombinant virus with the disrupted zinc-binding motif by point mutation exhibited a significant reduction in the RNA level of ORF57 downstream genes ORF59 and K8.1 and infectious virus production. Taken together, this study illustrates the first structure of KSHV ORF57-CTD and provides new insights into the understanding of ORF57 protein dimerization and stability, which would shed light on the potential design of novel therapeutics against KSHV infection and related diseases.

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The structure of iPLA2? reveals dimeric active sites and suggests mechanisms of regulation and localization.

Publications

The structure of iPLA<sub>2</sub>β reveals dimeric active sites and suggests mechanisms of regulation and localization.

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