Influence of Lateral Association on Forced Unfolding of Antiparallel Spectrin Heterodimers

Abstract

Protein extensibility appears to be based broadly on conformational changes that can in principle be modulated by protein-protein interactions. Spectrin family proteins, with their extensible three-helix folds, enable evaluation of dimerization effects at the single molecule level by atomic force microscopy. Although some spectrin family members function physiologically only as homodimers (e.g. ␣-actinin) or are strictly monomers (e.g. dystrophin), ␣-and ␤-spectrins are stable as monomeric forms but occur physiologically as ␣,␤-heterodimers bound laterally lengthwise. For short constructs of ␣-and ␤-spectrin, either as monomers or as ␣,␤-dimers, sawtooth patterns in atomic force microscopy-forced extension show that unfolding stochastically extends repeats ϳ4 -5-fold greater in length than native conformations. For both dimers and monomers, distributions of unfolding lengths appear bimodal; major unfolding peaks reflect single repeats, and minor unfolding peaks at twice the length reflect tandem repeats. Cooperative unfolding thus propagates through helical linkers between serial repeats (1, 2). With lateral heterodimers, however, the force distribution is broad and shifted to higher forces. The associated chains in a dimer can stay together and unfold simultaneously in addition to unfolding independently. Weak lateral interactions do not inhibit unfolding, but strong lateral interactions facilitate simultaneous unfolding analogous to serial repeat coupling within spectrin family proteins.As cytoskeletal proteins, spectrin superfamily proteins play important roles in cell organization and membrane mechanics (3, 4). As flexible linkers that bind to actin filaments, these proteins function in both monomeric and associated forms. Erythroid ␣I-and ␤I-spectrin were the first identified spectrins among a still growing superfamily that includes ␣-actinin, dystrophin, utrophin, and many additional proteins that share homologous triple-helical repeat motifs (5). In the red cell, ␣-chains of 20 repeats associate antiparallel along their lengths with ␤-chains of 17 repeats. The association is nucleated near the N terminus of ␤-spectrin (6) that ends with an actin binding domain, which mediates formation of a cross-linked network (7). The ability of spectrin monomers to first dimerize laterally and then associate head-to-head as tetramers that cross-link actin is especially crucial to the resilience of the red cell membrane in circulation. Defects in association, for example, lead to membrane instabilities typical in hereditary pyropoikilocytosis (8). Like red cell spectrin, ␣-actinin, with its four homologous repeats, also associates laterally and cross-links actin, imparting stability to structures that range from focal adhesions to Z-lines of myotubes (5). Dystrophin and utrophin (5), in contrast, are hypothesized to impart cortical stability to diverse membranes as monomeric actin-binding proteins.For all of the spectrin family proteins, length and extensibility are deemed central to function. Surprisingly, perhaps, rec...

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