Dissociation of CED-4 from CED-9 upon EGL-1 binding: Molecular mechanism of linear apoptotic pathway in Caenorhabditis elegans

Abstract Many steps in programmed cell death are evolutionarily conserved across different species. The Caenorhabditis elegans proteins CED-9, CED-4 and EGL-1 involved in apoptosis are respectively homologous to anti-apoptotic Bcl-2 proteins, Apaf-1 and the “BH3-only” pro- apototic proteins in mammals. In the linear apoptotic pathway of C.elegans , EGL-1 binding to CED-9 leads to the release of CED-4 from CED-9/CED-4 complex. The molecular events leading to this process are not clearly elucidated. While the structures of CED-9 apo, CED- 9/EGL-1 and CED-9/CED-4 complexes are known, the CED-9/CED-4/EGL-1 ternary complex structure is not yet determined. In this work, we modeled this ternary complex and performed molecular dynamics simulations of six different systems involving CED-9. CED-9 displays differential dynamics depending upon whether it is bound to CED-4 and/or EGL-1. CED-4 exists as an asymmetric dimer (CED4a and CED4b) in CED-9/CED-4 complex. CED-4a exhibits higher conformational flexibility when simulated without CED-4b. Principal Component Analysis revealed that the direction of CED-4a’s winged-helix domain motion differs in the ternary complex. Upon EGL-1 binding, majority of non-covalent interactions involving CARD domain in the CED-4a-CED-9 interface have weakened and only half of the contacts found in the crystal structure between α/β domain of CED4a and CED-9 are found to be stable. Additional stable contacts in the ternary complex and differential dynamics indicate that winged-helix domain may play a role in CED-4a’s dissociation from CED-9. This study has provided a molecular level understanding of potential intermediate states that are likely to occur at the time of CED-4a’s release from CED-9.