Mapping of POP1 -binding Site on Pyrin Domain of ASC.

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 pro-caspase-1. ASC is also known as a modulator of NF-κB 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 (ASC̱PYD) 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 ASC̱PYD with PYD-only protein 1 (POP1). POP1 specifically interacts with ASC̱PYD with a dissociation constant of 4.08 ± 0.52 µM but does not interact with Cryopyrin. NMR and mutagenesis experiments show that a negative electrostatic potential surface patch (EPSP) on ASC̱PYD, 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 ASC̱PYD and POP1 is similar to the interaction between caspase recruitment domains of Apaf-1 and pro- caspase-9. In addition, we present evidence that conformational changes at the long loop of ASC̱PYD between the H2 and H3 helices can affect its interaction with POP1. Based on our observations, we propose that the positive EPSP of ASC̱PYD, 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 ASC̱PYD. [ABSTRACT FROM AUTHOR]