LIG_EF_ALG2_ABM_1

The penta-EF-hand (PEF) protein family consists of Ca2+-binding proteins that contain five serially repetitive EF-hand motifs. The EF hand has a helix-loop-helix structure, which is comparable to the spread thumb and forefinger of a human hand. Ca2+ ions are coordinated into this hand by residues within the loop. Members of the PEF protein family share three common features. They dimerize through unpaired C-terminal EF5 hands, possess hydrophobic glycine/proline-rich N-terminal domains and are translocated to the membrane in a Ca2+-dependent manner (Maki,2002).
The most conserved protein within the PEF family is the apoptosis-linked Ca2+-binding protein ALG-2, a gene product of the programmed cell death 6 (PDCD6) gene. This 22 kDa protein and its homologues are found in protists, plants, Fungi, Nematoda, TAXON:7215 and Vertebrata. It contains five EF-hands (EF1-EF5) with eight alpha helices in the C-terminal region. Its N-terminal region contains a glycine/proline-rich hydrophobic region of 23 residues. ALG-2 forms homodimers via paired EF5 hands that are positioned in anti-parallel orientation, with each monomer having one peptide-binding site (2ZN8, 2ZNE) (Suzuki,2008).
ALG-2 interacts, in a Ca2+-dependent manner, with a variety of proteins containing regions that are rich in proline, glycine and tyrosine residues (Shibata,2008, Osugi,2012). Known interactors are involved in membrane trafficking, cell death, and regulation of actin cytoskeleton assembly (Shibata,2010). These include ALIX (Q8WUM4) (Vito,1999), TSG101 (Q99816) (Okumura,2009), Annexin A7 (P20073) and A11 (P50995) (Satoh,2002), PLSCR3 (Q9NRY6) and Sec31A (O94979) (Shibata,2008). To date, two ALG-2-binding motifs (ABM-1 and ABM-2) located in proline-rich regions have been characterized, and instances of one or both of these have been validated in several ALG-2 binding partners (Maki,2011). In addition, several proteins that interact with ALG-2 and contain proline-rich regions lack a motif similar to ABM-1 or ABM-2, such as PLSCR4 (Q9NRQ2), while others do not even contain a proline-rich region, indicating that ALG-2 might contain multiple binding surfaces for a wide range of proteins (Shibata,2008).
For ABM-1 (LIG_EF_ALG2_ABM_1), the structural basis for Ca2+-dependent binding to ALG-2 has been elucidated (Suzuki,2008). On its surface, ALG-2 contains a groove with two hydrophobic pockets involved in binding of ABM-1 peptides. The first pocket is formed by residues of the alpha5-alpha6 loop, residues of EF5 and residues of the other subunit in the ALG-2 dimer, while the second pocket is formed by residues of alpha3, alpha4, alpha5 and alpha7 (Maki,2011). Binding of Ca2+ to EF3 induces conformational changes in the loop connecting EF3 and EF4 (between alpha5 and alpha6), making the first pocket accessible as a primary binding site for the motif. The critical residue in this mechanism is Arg125, which blocks the pocket when ALG-2 is in the Ca2+-free state (2ZN9, 2ZND) (Suzuki,2008).
For Ca2+-dependent binding of ABM-2 (LIG_EF_ALG2_ABM_2) however, no structural details are available. In contrast to ABM-1, which can only bind the longer isoform of ALG-2, ABM-2 can bind both the longer ALG-2 isoform and a shorter isoform that lacks the Gly121 and Phe122 residues (characterized in human and mouse) (3AAJ) (Inuzuka,2010). The absence of the Phe122 residue affects the size of the second pocket, which might change its binding specificity. In addition, binding of metal ions to the shorter isoform does not change the configuration of the Arg123 residue (corresponding to Arg125 in the longer isoform) and the first pocket remains closed, suggesting that ABM-2 peptides do not use this pocket for binding ALG-2 (Maki,2011, Takahashi,2012).
It should be noted that for proteins containing both an ABM-1 and ABM-2 motif, the contribution of each motif to binding to ALG-2 is not fully understood yet. For instance, deletion of ABM-1 in CHERP (Q8IWX8) only slightly weakens binding to ALG-2, while deletion of ABM-2 has a more profound effect. Deletion of both motifs however, nearly abrogates binding (Sasaki-Osugi,2013). Similarly, PLSCR3 lacking its ABM-1 can bind both the longer and shorter isoform of ALG-2, while deletion of ABM-2 still allows binding to only the longer isoform. Binding to either isoform is lost upon deletion of both motifs (Shibata,2008). For Sec31A, loss of ABM-1 has no obvious effect, while loss of ABM-2 results in a weak interaction, although similar to the ALIX ABM-1 used as a positive control in the experiment (Shibata,2010). In addition, although Annexin A7 and A11 do not have a characterized ABM-2, they contain a sequence that resembles the ABM-2 sequences in CHERP, and they were predicted to have a low- and high-affinity binding site for ALG-2 (Satoh,2002). However, in contrast to what is expected from ABM-2-containing proteins, Annexin A7 and A11 do not interact with the shorter isoform of ALG-2, indicating that this ABM-2-like sequence might not be the second ALG-2-binding site in these proteins. The two distinct motifs might be used to discriminate between the different ALG-2 isoforms or they might function as a low- and high-affinity (ABM-1 and -2, respectively) binding site for ALG-2, acting cooperatively to mediate tight binding to ALG-2 in a subset of ALG-2 binding partners.