%0 Journal Article
%T Molecular basis for defect in Alix-binding by alternatively spliced isoform of ALG-2 (ALG-2¦¤GF122) and structural roles of F122 in target recognition
%A Tatsutoshi Inuzuka
%A Hironori Suzuki
%A Masato Kawasaki
%A Hideki Shibata
%A Soichi Wakatsuki
%A Masatoshi Maki
%J BMC Structural Biology
%D 2010
%I BioMed Central
%R 10.1186/1472-6807-10-25
%X We solved the X-ray crystal structure of the PEF domain of ALG-2¦¤GF122 in the Ca2+-bound form and compared it with that of ALG-2. Deletion of the two residues shortened ¦Á-helix 5 (¦Á5) 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 ¦Á4) and that ¦Á5 inclined away from ¦Á4 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
%U http://www.biomedcentral.com/1472-6807/10/25