Biochim Biophys Acta. 2012 Aug 19. pii: S1570-9639(12)00186-0. doi: 10.1016/j.bbapap.2012.08.013.

Formation of the death domain complex between FADD and RIP1 proteins in vitro.External

Park, Y. H., Jeong, M. S., Park, H. H., Jang, S. B.,
--- - Department of Molecular Biology, College of Natural Sciences, Pusan National University, Jangjeon-dong, Geumjeong-gu, Busan 609-735, Republic of Korea.
Fas-associated death domain (FADD) protein is an adapter molecule that bridges the interactions between membrane death receptors and initiator caspases. The death receptors contain an intracellular death domain (DD) which is essential to the transduction of the apoptotic signal. The kinase receptor-interacting protein 1 (RIP1) is crucial to programmed necrosis. The cell type interplay between FADD and RIP1, which mediates both necrosis and NF-kappaB activation, has been evaluated in other studies, but the mechanism of the interaction of the FADD and RIP1 proteins remain poorly understood. Here, we provided evidence indicating that the DD of human FADD binds to the DD of RIP1 in vitro. We developed a molecular docking model using homology modeling based on the structures of FADD and RIP1. In addition, we found that two structure-based mutants (G109A and R114A) of the FADD DD were able to bind to the RIP1 DD, and two mutations (Q169A and N171A) of FADD DD and four mutations (G595, K596, E620, and D622) of RIP1 DD disrupted the FADD-RIP1 interaction. Six mutations (Q169A, N171A, G595, K596, E620, and D622) lowered the stability of the FADD-RIP1 complex and induced aggregation that structurally destabilized the complex, thus disrupting the interaction.
PMID: 22922561External
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
DD FADD Link RIP1 GST fusion protein pull-down Human 93-184 E.coli Not specified 583-663 E.coli 22922561
DD FADD Link RIP1 In vitro purification protein assembly(Size-exclusion chromatography) Human 93-184 E.coli Not specified 583-663 E.coli 22922561
DD FADD Link RIP1 In vitro purification protein assembly(Surface plasmon resonance) Human 93-184 E.coli Not specified 583-663 E.coli 22922561
(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
DD FADD Link RIP1 GST fusion protein pull-down Q169A, N171A (FADD) G595A, K596A, E620A, D622A (RIP1) 22922561
DD FADD Link RIP1 Surface plasmon resonance 323nM 22922561
(Link: click this icon to show interactions only between the two corresponding DDs)