Biophys Chem. 2016 Feb;209:28-40.

ALG-2 divalent-ion affinity: Calorimetric analysis of the des23 versions reveals high-affinity site for Mg(2).

Henzl MT1, Frey BB2, Wolf AJ2.

1 Department of Biochemistry, University of Missouri, Columbia, MO, 65211. Electronic address:
2 Department of Biochemistry, University of Missouri, Columbia, MO, 65211.



ALG-2 (­Apoptosis-Linked Gene 2) is a penta-EF hand protein that has been linked to several vital cellular activities, including ER-to-Golgi transport and endosomal sorting and transport. In fact, there are two ALG-2 isoforms, denoted ALG-2wt and ALG-2DGF122, the result of differential splicing. In the human cell, they are expressed at a ratio of 3:1, respectively. This study examines ALG-2 divalent-ion-binding behavior using titration calorimetry. Des23 versions of the two isoforms were subjected to titrations with Ca2+ or Mg2+, in the absence and presence of chelators, and the resulting data were analyzed globally to extract estimates for the binding parameters. The measurements were performed at 25 °C, in 0.15 M KCl, 0.025 M Hepes, 0.5% Tween20, pH 7.4, conditions similar to those employed in a 45Ca2+ flow-dialysis study on the full-length proteins conducted by Tarabykina et al. [J. Biol. Chem. 275, 10514(2000)]. Consistent with that earlier work, we find that both isoforms contain three Ca2+ sites and that ALG-2des23wt exhibits perceptibly higher Ca2+ affinity. Unexpectedly, the data reveal that both isoforms possess a high-affinity Mg2+-binding site having atypically low affinity for Ca2+. Under intracellular conditions, occupancy of the Mg2+ site should be nearly complete.

KEYWORDS: Apoptosis-Linked Gene 2; Ca(2+)-binding proteins; Mg(2+)-binding proteins; Penta-EF-hand proteins; Titration calorimetry

PMID: 26705706; DOI:10.1016/j.bpc.2015.10.009




ALG-2 (short for Apoptosis-Linked-Gene-2)1-3 is a member of the penta-EF-hand (PEF) family of proteins 4. The protein is expressed in virtually all mammalian cells and is present in the cytoplasm and nucleus. There are two ALG-2 isoforms. The more abundant, ALG-2wt, includes 191 residues. Roughly 30% of the time, however, the codons for G121 and F122 are excised during splicing, resulting in ALG-2DGF122. ALG-2 was discovered two decades ago in a genetic screen to identify gene-products capable of rescuing a T-cell hybridoma line from ligand-induced apoptosis5. In fact, it was the anti-sense transcript of ALG-2 that conferred protection, suggesting that the protein itself should exhibit pro-apoptotic activity. Curiously, ablation of the gene produces no apparent phenotype6. ALG-2-/- mice develop normally and have no obvious immune defect. Its name notwithstanding, ALG-2 can evidently promote either cell-death or cell-proliferation1. It has been implicated in ER-Golgi vesicular transport, endosomal biogenesis and transport, and cell-membrane repair.

Ca2+ binding triggers exposure of an apolar surface on ALG-2, presumably for association with other proteins. Consistent with that hypothesis, a number of candidate ALG-2 target-proteins have been identified – including AIP1/Alix, and annexins VII and XI. The majority of these putative targets possess specific proline-rich regions called ALG-2-binding motifs (ABMs). ALG-2wt and ALG-2ΔGF122 display distinct target-protein specificities. The former associates with either ABM-1 (PPYPXXPGYP) or ABM-2 (PXPGF) motifs; the latter interacts exclusively with ABM-2. In common with other PEF proteins, ALG-2 is dimeric. The dimeric structure (Fig. 1A) enables it to simultaneously bind two target proteins. ALG-2 is proposed to function as a molecular adaptor (Fig. 1B) – either stabilizing a weak protein-protein complex or bridging two distinct proteins, thereby ensuring their proximity.

The Ca2+-binding behavior of ALG-2 was first examined, by 45Ca2+ flow-dialysis, in 2000 7. Both isoforms were found to bind two Ca2+ ions with high affinity (in EF3 and EF1) and a third Ca2+ ion with low affinity (in EF5). Inclusion of Mg2+ had no perceptible impact on binding, implying that the EF-hand sites were Ca2+-specific. As a prelude to examining the thermodynamic linkage between target-peptide association and Ca2+ binding, we have re-examined the divalent-ion-binding behavior of ALG-2, using titration calorimetry. The study was conducted at 25 °C on des23 versions of the two isoforms, lacking the N-terminal 23 residues. The experiments were performed in 0.15 M KCl, 0.025 M Hepes, pH 7.4, containing 0.5% Tween 20. Under these conditions, Ca2+ binding was strongly exothermic (e.g., Fig. 2A). Samples of the proteins were titrated with Ca2+ in the absence and presence of small-molecule chelators (EDTA, EGTA, and NTA). The resulting data were analyzed globally (simultaneously) to extract estimates for the binding constants and -enthalpies (e.g., Fig. 2B). The site-specific Ca2+ association constants determined for ALG-2des23wt were 7.3 ´ 106 (EF3), 5.5 ´ 105 (EF1), and 1.3 ´ 105 M-1 (EF5). The Ca2+-binding behavior of ALG-2des23ΔGF122 was somewhat more complicated, evidently due to cooperative interactions between the two ALG-2 monomers. The average macroscopic association constant for the first two binding events (presumably at the EF3 sites) was 2.3 ´ 106 M-1; the average association constant for events 3 and 4 (presumably in EF1) was 5.4 ´ 105 M-1; and the average value for binding at the EF5 sites was 3.4 ´ 104 M-1.

Because the earlier flow-dialysis study had suggested that the ALG-2 divalent-ion-binding sites were Ca2+-specific, we were surprised to observe a strong interaction with Mg2+ (Fig. 3A) ALG-2des23wt and ALG-2des23ΔGF122 each bind one equivalent of Mg2+ with apparent association constants of 5.1 ´ 104 and 7.0 ´ 104, respectively. These values were obtained by global analysis of Mg2+ titrations conducted in the absence and presence of EDTA (e.g., Fig. 3B). Typically, EF-hand motifs that display high Mg2+-affinity also display very high Ca2+ affinity. In this case, however, the presence of Mg2+ had little impact on the high-affinity Ca2+ binding events. Instead, the perturbation was confined to the latter stages of the titration, when presumably the low-affinity site was filling. This result suggested that high-affinity Mg2+ binding was associated with the C-terminal EF-hand motif, EF5. That hypothesis was confirmed recently by X-ray crystallography on Mg2+-bound ALG-2des23wt 8.

It is conceivable, if not likely, that Mg2+ binding will influence ALG-2 activity. The proposed function of ALG-2 as a molecular adaptor is dependent on its dimeric structure. ALG-2 forms both homo- and heterodimers. Because the two ALG-2 isoforms exhibit disparate target-protein specificities, the homo- and heterodimers will conjoin different target-protein combinations, presumably with distinct biological consequences. Thus, the energetic and kinetic properties of the two dimer populations – largely unknown at present – are relevant to ALG-2 function. For example, how do the relative stabilities of the homo- and heterodimeric complexes compare? And what is the timescale for subunit exchange? In this context, the structural changes that occur upon occupation of EF5 by Mg2+ are confined to the C-terminus and would be predicted to influence ALG-2 dimerization behavior. Specifically, Mg2+ binding provokes a reorientation of the C-terminal helix that results in formation of 1) a hydrogen bond between the main-chain carbonyl of F188 in one subunit and K137 in the opposing subunit and 2) a salt-bridge between the C-terminal carboxylate of one monomer and K137 (Fig. 4A). One would predict that these additional inter-subunit noncovalent interactions would significantly strengthen the dimer forms.

Fig. 4B displays the predicted occupancy of EF5 as a function of the free Mg2+ concentration. The intracellular Mg2+ level is commonly assumed to be on the order of 1 mM. Under these conditions, the EF5 site would be nearly saturated (98%). However, there is evidence that the Mg2+ concentration is, in fact, more variable and responsive to physiological activity. In a study of cultured neurons, for example, the resting-state Mg2+ levels in the nucleus and cytoplasm were found to be 0.11 and 0.68 mM, respectively 9. Upon depolarization, the concentrations shifted to 0.25 and 1.52 mM, respectively. Thus, the extent to which EF5 is occupied by Mg2+ might be influenced by intracellular conditions.

To our knowledge, this study marks the first observation of an EF-hand motif possessing high affinity and specificity for Mg2+. The ion is coordinated by three aspartyl carboxylates, a main-chain carbonyl, and two water molecules 8. An insertion at the C-terminal end of EF5 prevents E180 from participating in the Mg2+ binding. Similar constellations of liganding residues are found in CAPN1 and CAPN8, two isoforms of the large subunit of calpain.  Whether CAPN1 and CAPN8 likewise display high Mg2+ affinity is presently unknown. Conceivably, the ALG-2 EF5 sequence could be used as a template for introducing specific Mg2+-binding activity into other polypeptide systems.

In closing, this unexpected finding emerged from the detailed physical characterization of a protein that had previously undergone 20 years of investigative inquiry, emphasizing the need for broad support of basic scientific research.



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Acknowledgment:  This work was supported by the University of Missouri Department of Biochemistry.



Michael T. Henzl, Ph.D.


Dept. of Biochemistry

University of Missouri

117 Schweitzer Hall

Columbia, MO 65211



Figure 1. (A) Ribbon diagram of ALG-2, emphasizing its dimeric structure. The EF-hand motifs that retain metal-ion-binding activity are denoted – from N- to C-terminal – as EF1, EF3, and EF5. (B) Depiction of the putative molecular adaptor role of ALG-2. The protein could serve either (top) to stabilize weak protein-protein complexes in which both proteins harbor ALG-2 interaction sites or (bottom) to insure the proximity – in a lipid bilayer, for example – of two distinct proteins possessing ALG-2 interaction sites.



Figure 2. Calorimetric analysis of Ca2+ binding by ALG-2. (A) Representative raw calorimetry data, obtained by titrating 60 mM ALG-2des23wt with 2.0 mM Ca2+, in buffer containing 0.5% Tween 20. ALG-2des23DGF122 yielded qualitatively similar data. (B) A subset of the data used in the estimation of ALG-2des23wt (P) Ca2+-binding parameters: 32 mM P (orange); 62 mM P, 100 mM NTA (blue); 120 mM P (cyan); 65 mM P, 100 mM EDTA (green); 62 mM P, 100 mM EGTA (magenta). Vertical offsets have been added to the curves to facilitate their display without overlap.



Figure 3. Calorimetric analysis of Mg2+-binding by ALG-2. (A) Representative raw calorimetry data acquired by titrating 60 mM ALG-2des23wt with 2.0 mM Mg2+, in the presence of 0.5% Tween 20. The behavior of ALG-2des23DGF122 was qualitatively similar. (B) Subset of the data used to determine the Mg2+-binding parameters for ALG-2des23wt (P):  63 mM P (red); 109 mM P (green); 112 mM P, 100 mM EDTA (blue); 189 mM P, 100 mM EDTA (magenta). As in Figure 2, vertical offsets have been added to facilitate display without overlap.



Figure 4. (A) Inter-subunit interactions that result from the binding of Mg2+ in EF5. (B) Mg2+ saturation curves for ALG-2des23wt (green) and ALG-2des23DGF122 (red), as a function of the free Mg2+ concentration, assuming binding constants of 5.1 ´ 104 and 7.0 ´ 104 M-1, respectively.



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