Molecular basis for defect in Alix-binding by alternatively spliced isoform of ALG-2 (ALG-2ΔGF122) and structural roles of F122 in target recognition

We solved the X-ray crystal structure of the PEF domain of ALG-2ΔGF122 in the Ca2+-bound form and compared it with that of ALG-2. Deletion of the two residues shortened α-helix 5 (α5) and changed the configuration of the R125 side chain so that it partially blocked Pocket 1. A wall created by the main chain of 121-GFG-123 and facing the two pockets was destroyed. Surprisingly, however, substitution of F122 with Ala or Gly, but not with Trp, increased the Alix-binding capacity in binding assays. The F122 substitutions exhibited different effects on binding of ALG-2 to other known interacting proteins, including TSG101 (Tumor susceptibility gene 101) and annexin A11. The X-ray crystal structure of the F122A mutant revealed that removal of the bulky F122 side chain not only created an additional open space in Pocket 2 but also abolished inter-helix interactions with W95 and V98 (present in α4) and that α5 inclined away from α4 to expand Pocket 2, suggesting acquirement of more appropriate positioning of the interacting residues to accept Alix.We found that the inability of the two-residue shorter ALG-2 isoform to bind Alix is not due to the absence of bulky side chain of F122 but due to deformation of a main-chain wall facing pockets 1 and 2. Moreover, a residue at the position of F122 contributes to target specificity and a smaller side chain is preferable for Alix binding but not favored to bind annexin A11.ALG-2 (apoptosis-linked gene 2) is a 22-kDa protein of 191 amino acid residues containing five serially repetitive EF-hand-type helix-loop-helix Ca2+-binding motifs (EF1 to EF5) and it belongs to the penta-EF-hand (PEF) family, including the calpain small subunit, sorcin, grancalcin and peflin in mammals [1]. ALG-2 is the most conserved protein among the PEF family and its homologues are widely found in eukaryotes. Despite the original report of a pro-apoptotic function of ALG-2 in T cell hybridomas [2], ALG-2-deficient mice develop normally with no obvious abnorm