The Actin-binding Site of Adducin Is Regulated by Intramolecular Interactions that Occur Within a Natively Unfolded Domain

Abstract

Adducin is an actin-binding protein with convergent functions in cell motility, including binding of calmodulin, capping of the barbed end of actin filaments, and recruitment of spectrin to the fast growing end of actin filaments. This protein consists of an unfolded C-terminal tail domain containing a positively charged phosphorylation site domain (PSD) that binds to actin, an alpha helical neck domain responsible for oligomerization, and an N-terminal globular head domain with unknown function. We recently established that MARCKS, a protein with a homologous PSD, forms intramolecular interactions that regulate the accessibility of this domain to its binding partners. Our data establish that the PSD of adducin is similarly regulated. Salt bridge formation between the PSD and a negatively charged region within the tail domain stabilize this interaction. The result is a unique configuration in which the tail is looped over upon itself without canonical structural elements so as to sterically regulate binding events at the PSD. Moreover, our data show that oligomerization of adducin results in activation of the PSD. Oligomerization is shown to be mediated either by self-association (occurring in the alpha helical neck domain) or by association with spectrin. Thus, these results explain at a structural level earlier observations showing that spectrin-adducin interactions affect actin-binding function. Finally, a polymorphism of adducin that has been associated with adverse cardiovascular outcomes in humans is shown to affect the actin-binding function of adducin by altering adducin oligomerization, and, therefore, by changing the availability of the PSD.