Analysis of the molecular determinants of Apaf-1 function

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Apaf-1 (apoptotic protease activating factor 1) is a key player in the mitochondrial pathway of apoptosis. The decisive death signal in this pathway is the release of cytochrome c from the mitochondrial intermembrane space into the cytosol. Upon binding of cytochrome c Apaf-1 oligomerizes into the heptameric apoptosome complex, which triggers the downstream caspase cascade. We aim to elucidate the molecular determinants of this process and to formulate a complete and coherent model for apoptosome assembly. We will determine the role of nucleotide exchange for the release of the autoinhibition of Apaf-1 by combining the results from mutational studies, nucleotide binding studies, small angle scattering experiments, and X-ray crystallographic analysis of an ATP state of Apaf-1. To provide a quasi atomic model of the apoptosome, we will determine X-ray structures of apoptosomal subcomplexes and combine them with cryoEM studies. Combining X-ray crystallographic and biochemical analyses, we seek to clarify the modes of action of selected protein and small molecule interaction partners of Apaf-1. With the aid of the readily crystallizable murine Apaf-1 ortholog, we will determine the ligand binding sites in Apaf-1. These studies will lay the ground for the use of Apaf-1 as drug target aiming at the therapeutic control of the apoptosis machinery.