Structure of apoptosis-linked protein ALG-2: Insights into Ca2+-induced changes in penta-EF-hand proteins

Background: The Ca2+ binding apoptosis-linked gene-2 (ALG-2) protein acts a s a proapoptotic factor in a variety of cell fines and is required either d ownstream or independently of caspases for apoptosis to occur. ALG-2 belong s to the penta-EF-hand (PEF) protein family and has two high-affinity and o ne low-affinity Ca2+ binding sites. Like other PEF proteins, its N terminus contains a Gly/Pro-rich segment. Ca2+ binding is required for the interact ion with the target protein, ALG-2 interacting protein 1 (AlP1). Results: We present the 2.3 Angstrom resolution crystal structure of Ca2+-l oaded desl-20ALG-2 (aa 21-191), which was obtained by limited proteolysis o f recombinant ALG-2 with elastase. The molecule contains eight alpha helice s that fold into five EF-hands, and, similar to other members of this prote in family, the molecule forms dimers. Ca2+ ions bind to EF1, EF3, and, surp risingly, to EF5. In the related proteins calpain and grancalcin, the EF5 d oes not bind Ca2+ and is thought to primarily facilitate dimerization. Most importantly, the conformation of des1-20ALG-2 is significantly different f rom that of calpain and grancalcin. This difference can be described as a r igid body rotation of EF1-2 relative to EF4-5 and the dimer interface, with a hinge within the EF3 loop. An electron density, which is interpreted as a hydrophobic Gly/Pro-rich decapeptide that is possibly derived from the cl eaved N terminus, was found in a hydrophobic cleft between these two halves of the molecule. Conclusions: A different relative orientation of the N- and C-terminal halv es of des1-20ALG-2 in the presence of Ca2+ and the peptide as compared to o ther Ca2+-loaded PEF proteins changes substantially the shape of the molecu le, exposing a hydrophobic patch on the surface for peptide binding and a l arge cleft near the dimer interface. We postulate that the binding of a Gly /Pro-rich peptide in the presence of Ca2+ induces a conformational rearrang ement in ALG-2, and that this mechanism is common to other PEF proteins.