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ALG-2, a 22-kDa penta-EF-hand protein, is involved in cell death, signal transduction, membrane trafficking, etc., by interacting with various proteins in mammalian cells in a Ca2+-dependent manner. Most known ALG-2-interacting proteins contain proline-rich regions in which either PPYPXnYP (type 1 motif) or PXPGF (type 2 motif) is commonly found. Previous X-ray crystal structural analysis of the complex between ALG-2 and an ALIX peptide revealed that the peptide binds to the two hydrophobic pockets. In the present study, we resolved the crystal structure of the complex between ALG-2 and a peptide of Sec31A (outer shell component of coat complex II, COPII; containing the type 2 motif) and found that the peptide binds to the third hydrophobic pocket (Pocket 3). While amino acid substitution of Phe85, a Pocket 3 residue, with Ala abrogated the interaction with Sec31A, it did not affect the interaction with ALIX. On the other hand, amino acid substitution of Tyr180, a Pocket 1 residue, with Ala caused loss of binding to ALIX, but maintained binding to Sec31A. We conclude that ALG-2 recognizes two types of motifs at different hydrophobic surfaces. Furthermore, based on the results of serial mutational analysis of the ALG-2-binding sites in Sec31A, the type 2 motif was newly defined. [ABSTRACT FROM AUTHOR]

Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X] was originally identified as a protein that interacts with ALG-2, a member of the penta-EF-hand Ca2+-binding protein family. ALG-2 binds to its C-terminal proline-rich region that contains four tandem repeats of PXY (where X represents an uncharged amino acid). Recent X-ray crystal structural analyses of the Ca2+-free and Ca2+-bound forms of ALG-2, as well as the complex with an Alix oligopeptide, have revealed a mechanism of Ca2+-dependent binding of ALG-2 to its target protein. Binding of Ca2+ to EF3 (third EF-hand) enables the side chain of Arg125, present in the loop connecting EF3 and EF4 (fourth EF-hand), to move sufficiently to make a primary hydrophobic pocket accessible to the critical PPYP (Pro-Pro-Tyr-Pro) motif in Alix, which partially overlaps with the GPP (Gly-Pro-Pro) motif for binding to Cep55 (centrosome protein of 55 kDa). The fact that ALG-2 forms a homodimer and each monomer has one peptide-binding site indicates the possibility that ALG-2 bridges two interacting proteins, including Alix and Tsg101 (tumour susceptibility gene 101), and functions as a Ca2+-dependent adaptor protein. [ABSTRACT FROM AUTHOR]