A near atomic structure of the active human apoptosome

Abstract

In response to cell death signals, an active apoptosome is assembled from Apaf-1 and procaspase-9 (pc-9). Here we report a near atomic structure of the active human apoptosome determined by cryo-electron microscopy. The resulting model gives insights into cytochrome c binding, nucleotide exchange and conformational changes that drive assembly. During activation an acentric disk is formed on the central hub of the apoptosome. This disk contains four Apaf-1/pc-9 CARD pairs arranged in a shallow spiral with the fourth pc-9 CARD at lower occupancy. On average, Apaf-1 CARDs recruit 3 to 5 pc-9 molecules to the apoptosome and one catalytic domain may be parked on the hub, when an odd number of zymogens are bound. This suggests a stoichiometry of one or at most, two pc-9 dimers per active apoptosome. Thus, our structure provides a molecular framework to understand the role of the apoptosome in programmed cell death and disease.An adult human loses around 50–70 billion cells every day via a process termed apoptosis. The term arises from the Greek word that describes leaves “falling off” a tree, and the process entails damaged or unwanted cells essentially committing suicide in a controlled manner. As such, apoptosis keeps the number of cells in tissues and organs in check. It also allows components of old cells to be recycled to make new ones.In cells that are targeted to die, a protein called cytochrome c interacts with another protein, named Apaf-1, together with a nucleotide triphosphate molecule. These steps work in concert to trigger the assembly of the apoptosome: a large wheel-like complex that contains seven copies each of Apaf-1 and cytochrome c. The central hub of the wheel then recruits a specific protein-cutting enzyme, which once activated sets in motion a cascade of events that dismantle the cell from the inside out.Cheng et al. now use an electron microscope to reveal the three-dimensional structure of the active human apoptosome, in enough detail to determine the positions of many of the amino acids that make up the complex. The three dimensional model provides new insights into how Apaf-1 changes shape as the complex assembles in the presence of cytochrome c and nucleotide triphosphate. Cheng et al. went on to reveal a disk-like structure made from the parts of four Apaf-1 proteins that interact with the protein-cutting enzymes. This disk forms a spiral that sits atop the central hub of the wheel-like apoptosome. Finally, the findings suggest that, although the wheel like complex has seven spokes, at any one time the active apoptosome may only need two (or at most four) copies of the protein-cutting enzyme to trigger the cascade of events that lead to cell deathIn the future, emerging technologies may provide high enough resolution to visualize fine details of the interactions between cytochrome c and Apaf-1, and reveal more about the disk-like spiral as well. This in turn will give a better understanding of how the apoptosome assembles and how the protein-cutting enzyme becomes activated.