J Biol Chem. 2008 May 30;283(22):15390-8. Epub 2008 Mar 24.

Mapping of POP1-binding site on pyrin domain of ASC.External

Srimathi, T., Robbins, S. L., Dubas, R. L., Chang, H., Cheng, H., Roder, H., Park, Y. C.,
--- - Basic Science, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an essential adaptor protein in the formation of a multiprotein complex that activates procaspase-1. ASC is also known as a modulator of NF-kappaB activation pathways. ASC has a bipartite domain structure, consisting of an N-terminal pyrin domain (PYD) and a C-terminal caspase-recruitment domain. The PYD of ASC (ASC_PYD) is known to interact with various PYD-containing intracellular danger signal sensors and PYD-only proteins. Using purified proteins, we characterized the in vitro interaction of ASC_PYD with PYD-only protein 1 (POP1). POP1 specifically interacts with ASC_PYD with a dissociation constant of 4.08 +/- 0.52 microm but does not interact with Cryopyrin. NMR and mutagenesis experiments show that a negative electrostatic potential surface patch (EPSP) on ASC_PYD, consisting of the first (H1) and fourth (H4) helices, is essential in the interaction with POP1. A positive EPSP on POP1, consisting of the second (H2) and third (H3) helices, is a counterpart of this interaction. The interaction between ASC_PYD and POP1 is similar to the interaction between caspase recruitment domains of Apaf-1 and procaspase-9. In addition, we present evidence that conformational changes at the long loop of ASC_PYD between the H2 and H3 helices can affect its interaction with POP1. Based on our observations, we propose that the positive EPSP of ASC_PYD, including the H2 and H3 helices, may be the binding site for Cryopyrin, and the interaction with Cryopyrin may induce the dissociation of POP1 from ASC_PYD.
PMID: 18362139External
Arrow2 In vitro interaction Arrow2 In vivo interaction Arrow2 Characterization Arrow2 Functional role Arrow2 top
In vitro interaction
  DD1 DD2 Reference
Family DD1 DD2 Method Species Region Expression Species Region Expression
PYD ASC Link POP1 In vitro purification protein assembly(Surface plasmon resonance) Not specified 1-96 E.coli Not specified 1-89 E.coli 18362139
(Link: click this icon to show interactions only between the two corresponding DDs)
Arrow2 In vitro interaction Arrow2 In vivo interaction Arrow2 Characterization Arrow2 Functional role Arrow2 top
Characterization
  Stoichiometry Affinity Protein-Protein interface Reference
Family DD1 DD2 Method Mutation Complex structure
PYD ASC Link POP1 Surface plasmon resonance 1:1 4.8μM D6A and L25A, E13A and L25A, K21A, Y36A, D48A and L25A, D54A and L25A (ASC) 18362139
(Link: click this icon to show interactions only between the two corresponding DDs)