Ligation events influence ALG-2 dimerization

ALG-2 dimerization was studied using Förster resonance-energy-transfer. D162C variants of ALG-2des23 were covalently modified with Alexa Fluor 488 and Alexa Fluor 647. When samples of the two labeled protein-preparations are combined, the sensitized emission from AF647 serves as a sensitive probe of dimer formation. At 25 °C, in the absence of divalent ions, the wild-type homodimer, ΔGF122 homodimer, and heterodimer display dissociation constants of 7.1, 26, and 4.5 nM, respectively. At 35 °C, subunit interaction is weaker, indicating that dimer formation is exothermic. Binding of Mg²⁺ in the C-terminal EF-hand (EF5) dramatically enhances ALG-2 dimer stability. Although occupation of EF5 by Ca²⁺ likewise has a stabilizing effect, its direct influence on dimer stability would be negligible at cytosolic Ca²⁺ levels. However, dimer stability is substantially increased by the Ca²⁺-dependent binding of ALG-2 target-peptides, suggesting that the occupation-status of the target-protein binding site is communicated to the dimer interface. Tween 20 is commonly used to improve ALG-2 solubility, the micelles ostensibly acting as target-protein surrogates. Paradoxically, however, the detergent markedly destabilizes ALG-2 dimers, particularly in the presence of Ca²⁺.