Molecular basis for defect in Alix-binding by alternatively spliced isoform of ALG-2 (ALG-2(Delta GF122)) and structural roles of F122 in target recognition

作者：Inuzuka Tatsutoshi; Suzuki Hironori; Kawasaki Masato; Shibata Hideki; Wakatsuki Soichi; Maki Masatoshi^*

来源：BMC Structural Biology, 2010, 10: 25.

DOI：10.1186/1472-6807-10-25

Background: ALG-2 (a gene product of PDCD6) belongs to the penta-EF-hand (PEF) protein family and Ca2+-dependently interacts with various intracellular proteins including mammalian Alix, an adaptor protein in the ESCRT system. Our previous X-ray crystal structural analyses revealed that binding of Ca2+ to EF3 enables the side chain of R125 to move enough to make a primary hydrophobic pocket (Pocket 1) accessible to a short fragment of Alix. The side chain of F122, facing a secondary hydrophobic pocket (Pocket 2), interacts with the Alix peptide. An alternatively spliced shorter isoform, designated ALG-2(Delta GF122), lacks Gly(121)Phe(122) and does not bind Alix, but the structural basis of the incompetence has remained to be elucidated.
Results: We solved the X-ray crystal structure of the PEF domain of ALG-2(Delta GF122) in the Ca2+-bound form and compared it with that of ALG-2. Deletion of the two residues shortened alpha-helix 5 (alpha 5) 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 alpha 4) and that alpha 5 inclined away from alpha 4 to expand Pocket 2, suggesting acquirement of more appropriate positioning of the interacting residues to accept Alix.
Conclusions: 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.

• 出版日期2010-8-6