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Changes in Apaf-1 conformation that drive apoptosome assembly.

Biochemistry (2013-03-26)
Shujun Yuan, Maya Topf, Thomas F Reubold, Susanne Eschenburg, Christopher W Akey
ABSTRACT

Apoptosome assembly is highly regulated in the intrinsic cell death pathway. To better understand this step, we created an improved model of the human apoptosome using a crystal structure of full length Apaf-1 and a single particle, electron density map at ~9.5 Å resolution. The apoptosome model includes N-terminal domains of Apaf-1, cognate β-propellers, and cytochrome c. A direct comparison of Apaf-1 in the apoptosome and as a monomer reveals conformational changes that occur during the first two steps of assembly. This includes an induced-fit mechanism for cytochrome c binding to regulatory β-propellers, which is dependent on shape and charge complementarity, and a large rotation of the nucleotide binding module during nucleotide exchange. These linked conformational changes create an extended Apaf-1 monomer and drive apoptosome assembly. Moreover, the N-terminal CARD in the inactive Apaf-1 monomer is not shielded from other proteins by β-propellers. Hence, the Apaf-1 CARD may be free to interact with a procaspase-9 CARD either before or during apoptosome assembly. Irrespective of the timing, the end product of assembly is a holo-apoptosome with an acentric CARD-CARD disk and tethered pc-9 catalytic domains. Subsequent activation of pc-9 leads to a proteolytic cascade and cell death.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Cytochrome c from Saccharomyces cerevisiae, ≥85% based on Mol. Wt. 12,588 basis
Sigma-Aldrich
ProteoMass Cytochrome c MALDI-MS Standard, vial of 10 nmol, (M+H+) 12,361.96 Da by calculation
Sigma-Aldrich
Cytochrome c from equine heart, BioUltra, ≥99% (SDS-PAGE), powder, suitable for mammalian cell culture
Sigma-Aldrich
Cytochrome c from equine heart, ≥95% based on Mol. Wt. 12,384 basis
Sigma-Aldrich
Cytochrome c from pigeon breast muscle, ≥95% based on Mol. Wt. 12,173 basis
Sigma-Aldrich
Cytochrome c from bovine heart, ≥95% based on Mol. Wt. 12,327 basis
Sigma-Aldrich
Cytochrome c from bovine heart, ≥95% based on Mol. Wt. 12,327 basis, powder, suitable for mammalian cell culture
Sigma-Aldrich
Cytochrome c from equine heart, BioReagent, suitable for GFC marker
Sigma-Aldrich
Cytochrome c from equine heart, ≥95% (SDS-PAGE)