Biophysical characterization and structural elucidation of the spectrin-ankyrin interaction

Organization and membrane integrity in the metazoan cell is maintained through intracellular tethering of membrane proteins to an extensive, underlying, protein network. The principal component of this network, spectrin, is anchored to membrane proteins through the adaptor protein ankyrin. Despite the roles of these membrane skeletal proteins in maintaining cell integrity and polarity, the atomic-level details of their interaction remained unclear. The work presented details the biochemical, biophysical, and structural characterization of the interaction domains of these proteins both individually and as a complex.;To characterize the atomic basis for this interaction, it was first necessary to identify the minimal spectrin and ankyrin binding domains. Previous work had indicated the ankyrin binding region of beta-spectrin to be in the vicinity of repeat 15. Numerous binding assays eventually revealed that tandem beta-spectrin repeats 14 and 15 were necessary to form a stable complex with ankyrin. A simultaneous investigation into the minimal spectrin binding domain of ankyrin was also performed. In the case of ankyrin, previous studies identified two non-overlapping subdomains of ankyrin as possessing binding activity. From a number of in vitro binding assays, it was determined that ZU5-ANK (spanning residues 911-1069) was responsible for high affinity spectrin binding.;After successful characterization of these domains, structural studies were initiated. Crystal structures of the two binding fragments showed that beta-spectrin repeats 14 and 15 maintained a conventional spectrin fold while displaying a large negatively-charged surface patch. ZU5-ANK, on the other hand, exhibited a newly-discovered protein fold and a considerable patch of positively-charged surface residues. Finally, structure determination of the minimal spectrin-ankyrin complex was achieved. Biophysical characterization of clinical, structure-based, and phenotype-inducing mutations verified the structure and provided insight into the nature of a number of clinical mutations.;By focusing on the structure-function relationship of this interaction, a greater understanding of both normal and diseased-state spectrin-ankyrin interactions was provided. While much remains unknown, it is hoped that further studies will continue to detail the roles of spectrin and ankyrin and ultimately advance our knowledge of related diseases such as cardiac arrhythmias, hereditary spherocytosis, elliptocytosis, and malaria.