Initiation and propagation of spectrin heterodimer assembly involves distinct energetic processes.

Red cell spectrin alpha and beta subunits consist primarily of many tandem homologous motifs with very similar three- helix-bundle structures and similar dimer interfaces. Although misassembled homodimers can form under some conditions, correctly aligned heterodimers consistently assemble provided a small "dimer initiation" site near the actin binding domain is present. The dimer initiation site has been characterized to some extent, but little is known about the subsequent, low-affinity lateral interactions of the remaining motifs along the length of this ropelike molecule or the forces involved in these two steps of the dimerization process. In this study, we used isothermal titration calorimetry to deduce the mechanism and energetics of the two heterodimer assembly phases. The high-affinity initiation of dimerization is primarily enthalpically driven, which is consistent with initial alignment and docking of specific complementary alpha and beta motifs in the dimer initiation site driven by long-range electrostatic interactions followed by tight binding stabilized by hydrogen bonds and other hydrophilic interactions. In contrast, the subsequent weak lateral associations of additional motifs are primarily entropically driven, suggesting binding primarily involves weak hydrophobic interactions. Although initial docking is largely electrostatic, the only lateral interaction within the first four pairs of motifs that involves a net change in protons is the interaction of the alpha18 and beta4 repeats. This substoichiometric uptake of protons could be due to a pKa shift of a histidine in the alpha18 motif located near the dimer interface in a proposed homology-based model. On the basis of this analysis of heterodimer thermodynamics, a detailed model of spectrin dimer assembly is proposed.