Explorations of the Apoptotic Protease Activating Factor 1 (Apaf‐1) in its Formation of the Seven‐Spoked Apoptosome Responsible for Programmed Cell Death

Abstract

Multicellular life forms first appeared in the fossil record in a burst of evolution termed during the Cambrian period. With multicellularity came the development and specialization of cells, alongside their coordination in the formation of tissues and organs. An adult human, composed of 30–40 trillion cells, progresses through repeated choreographed rounds of growth and cell division from its microscopic begins as a single celled zygote. Some have argued that this is the most critical stage of one’s life. Equally important for development, relatively overlooked, is the intrinsic process of cell death. For example, the webbing between our fingers and toes disappears around week six of gestation. This macroscopic observation during fetal development is the result of a complex series of molecular events initiated by the apoptotic protease activating factor 1 (Apaf‐1) as it forms the holo‐enzyme the apoptosome. The Blue Valley CAPS 2020 SMART Team (Students Modeling a Research Topic), with support from the CBM at MSOE, modeled the Apaf‐1 using JMol and 3D printing technologies in order to better understand how this molecular machine carries out programmed cell death. Structurally, Apaf‐1 is composed of 1249 amino acid residues that form a C‐terminal regulatory domain, a nucleotide binding and oligomerization domain (NOD), and an N‐terminal caspase recruitment domain (CARD). The regulatory domain is composed of two WD40 b‐propellers that are responsible for binding cytochrome c, which itself is released from the inner membrane of the mitochrondria under conditions of stress. Cytochrome c bonding causes a disruption in a salt bridge between Lys192 and Asp616 leading to changes in the orientation of the propellers. The nucleotide binding and oligomerization domain is composed of a number of helical subdomains. Conformational changes in the regulatory domain stimulates nucleotide exchange, where an ADP molecule bond in Apaf‐1’s inactive and closed monomeric state is exchanged for an ATP molecule leading to a more open monomeric state and the subsequent formation of the heptameric apoptosome. The Arg265 trigger which stabilizes an aspartate triad in the inactive monomeric state becomes compromised after ATP binding leading to conformational changes that open up Apaf‐1. Once the polymeric apoptosome forms, the CARD binds and activates procaspase‐9, which itself cleaves loops in procaspases 3 and 7 which become active and responsible of the apoptosomes proteolytic function. Proteolysis ultimately leads to cell death.