Journal of Molecular Biology
Volume 332, Issue 5, 3 October 2003, Pages 1155-1163
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The Death-domain Fold of the ASC PYRIN Domain, Presenting a Basis for PYRIN/PYRIN Recognition

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Abstract

The PYRIN domain is a conserved sequence motif identified in more than 20 human proteins with putative functions in apoptotic and inflammatory signalling pathways. The three-dimensional structure of the PYRIN domain from human ASC was determined by NMR spectroscopy. The structure determination reveals close structural similarity to death domains, death effector domains, and caspase activation and recruitment domains, although the structural alignment with these other members of the death-domain superfamily differs from previously predicted amino acid sequence alignments. Two highly positively and negatively charged surfaces in the PYRIN domain of ASC result in a strong electrostatic dipole moment that is predicted to be present also in related PYRIN domains. These results suggest that electrostatic interactions play an important role for the binding between PYRIN domains. Consequently, the previously reported binding between the PYRIN domains of ASC and ASC2/POP1 or between the zebrafish PYRIN domains of zAsc and Caspy is proposed to involve interactions between helices 2 and 3 of one PYRIN domain with helices 1 and 4 of the other PYRIN domain, in analogy to previously reported homophilic interactions between caspase activation and recruitment domains.

Introduction

The death domain fold is the unifying structural motif of a superfamily of protein domains comprising the death domain (DD) itself,1 the death effector domain (DED)2 and the caspase recruitment domain (CARD).3 Their names express the prominent roles of these domains in programmed cell death. Domains from all three subfamilies occur as modules in diverse human apoptosis proteins in a variety of domain contexts. They all form α-helical bundles acting as adapters in signalling pathways and recruiting other proteins into signalling complexes.4 Domains from the different death domain subfamilies tend to interact with each other, suggesting that their common fold was frequently reused as a module during the evolution of apoptotic adapter proteins, providing the structural backbone of the signalling pathways that control programmed cell death. Commensurate with their biological importance and despite often poor solubility due to self-association, several structures have been determined for DDs,1., 5., 6., 7., 8., 9. DEDs2., 10. and CARDs.3., 11., 12., 13., 14.

The PYRIN domain, also called DAPIN, PAAD or PYD, is a recently identified domain that has been suggested to present a new member of the DD superfamily.15., 16., 17., 18., 19., 20. No experimentally determined structure of a PYRIN domain has been reported to date. An attempt to solve the structure of the PYRIN domain of CARD7 failed due to limited solubility.17

PYRIN domains are located at the N terminus of proteins that are linked intimately to a variety of human diseases, ranging from cancer to inflammatory syndromes.15., 16., 18., 21., 22. The PYRIN domain was originally found in pyrin, the product of the familial Mediterranean fever (FMF)-associated gene, which is involved in a hereditary hyperinflammatory response syndrome,23 and in ASC/TMS1/PYCARD, which functions as a positive mediator of apoptosis.19., 24. Inflammation and apoptosis upregulate ASC in neutrophils and, depending on the cellular context, it can either inhibit or activate NF-κB.25., 26. ASC contains both a PYRIN and a CARD domain. Homophilic and heterophilic interactions of both domains have been reported to be involved in self-association and filament-like aggregation of ASC in vivo.27 ASC and PYRIN seem to interact via their PYRIN domains,19 while the CARD domain of ASC was shown to bind to the CARD domain of caspase-1.24., 28., 29. The PYRIN domain of ASC was further shown to bind to POP1/ASC2, a small protein consisting of a single PYRIN domain with a high level of amino acid sequence similarity to the PYRIN domain of ASC.30 The interaction between ASC and POP1/ASC2 results in a modulation of NF-κB and pro-caspase-1 regulation.30 Finally, there is evidence that ASC and caspase-1, together with NALP1 (another PYRIN-domain protein) and caspase-5, form a pro-apoptotic complex, named inflammasome, which is essential for innate immunity involving LPS-induced apoptosis.31

Two further human hereditary diseases were recently attributed to the PYRIN-domain protein NALP3/CIAS1/PYPAF1, Muckle–Wells syndrome and familial cold autoinflammatory syndrome.22., 32., 33. CIAS1 assembles with ASC and regulates the activation of NF-κB.34 A homologous protein with identical domain architecture, PYPAF7, also binds ASC, activates caspase-1 and regulates NF-κB dependent transcription.35

Although PYRIN domains occur in more than 20 human proteins, only a few additional PYRIN-domain proteins have been characterized functionally. Almost all of them appear to be involved in apoptosis and inflammation.36., 37., 38., 39., 40. To the best of our knowledge, no mutation analysis is available for any PYRIN domain. Considering that PYRIN-domain proteins interact frequently with other PYRIN-domain proteins, PYRIN/PYRIN interactions are likely an important feature of PYRIN-domain function. Besides the binding between the PYRIN domains of ASC and POP1/ASC2,28 conclusive data on PYRIN/PYRIN interactions come from the zebrafish orthologue of ASC (zAsc) and the caspase Caspy, where the PYRIN domains in both proteins were shown to be required for mutual binding in vitro.41

Here, we present the three-dimensional structure of the PYRIN domain from human ASC. The structure establishes the PYRIN domain fold and corrects previous sequence alignments with other members of the DD superfamily. It suggests a PYRIN/PYRIN interaction mode related to that observed between the CARD domains of Apaf-1 and procaspase-9.12

Section snippets

PYRIN domains belong to the DD superfamily

The structure of the PYRIN domain is composed of six helices that are arranged in the classical DD fold (Figure 1). A search of the Protein Data Bank (PDB) with the program DALI42 yielded the death effector domain (DED) from human FADD2 as the structurally most closely related protein, followed by the procaspase-9 prodomain12 which belongs to the CARD group of proteins, and the tumor necrosis factor receptor-1 (TNFR) death domain.9 All three proteins could be aligned to the PYRIN domain with

Cloning of the ASC PYRIN domain

The ASC PYRIN-domain encoding 272 bp DNA fragment was PCR amplified from Marathon-Ready cDNAs prepared from human lymphocytes (Clontech) using BamHI (ATTCGGATCCATGGGGCGCGCGCGCGACGCCA) and HindIII (GAATAAGCTTCTACTGGTGCGTGGCCGCCT) oligonucleotide primers designed according to the sequence of the human ASC gene (NCBI protein number BAA87339). PCR cycle conditions were: ten seconds denaturation at 95 °C, 30 seconds annealing at 58 °C, and 1.5 minutes of polymerization at 72 °C. The first one minute

Acknowledgements

This work was supported by the Swedish and Australian Research Councils, and by the Karolinska Institute. G.O. thanks the Australian Research Council for a Federation Fellowship.

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