Structural studies of the Spectrin-binding domain of Ankyrin.

摘要

Organization of the metazoan cell membrane is maintained through an intracellular network of cytoskeletal and membrane proteins. Spectrin and ankyrin, are both recognized as principal components of this protein network. Ankyrin functions as an adapter protein, tethering membrane proteins to the cytoskeletal spectrin network. Despite the critical roles of these proteins in normal animal life, the atomic level details of how this spectrin/ankyrin interaction is regulated are unclear. The work presented here further elucidates the biochemical, biophysical, and structural basis of the interactions between ankyrin and spectrin, with the main focus on the ankyrin subdomains. Previous structural work on ankyrin has focused on its minimal spectrin binding domain, ZU5A. However, other subdomains beyond ZU5A exist, and these domains are speculated to form an auto-regulatory protein supramodule. To clarify what roles each of these subdomains may play, different ankyrin fragments were characterized both biophysically and crystallographically. Crystallization of the first ZU5 tandem structure, ZU5A-ZU5B of erythrocyte ankyrin (Ankyrin-R) reveals that the newly recognized ZU5B domain also folds into the unique ZU5 topology. Biophysical and biochemical characterization showed that ZU5B does not affect the ZU5A interaction with spectrin. Analysis of all the known ZU5 structures demonstrates that the ZU5 domain represents a compact and versatile protein interaction module, and this idea has lead into the finding of a new protein family, ZUNGA. The ankyrin supramodule was isolated and its interaction with spectrin was characterized and analyzed. Finally, the role of ZU5B domain was explored though attempts at identifying and characterizing interactions with other known proteins. By focusing on elucidating the structure-function relationship of ankyrin subdomains, a greater understanding of how ankyrin function is regulated by these subdomains was provided. While much remains unknown, it is hoped that further studies will continue to uncover both structural and biophysical details on how ankyrin and spectrin interact with each other, thereby providing deeper understanding of our knowledge of the metazoan cytoskeletal network and ultimately blood, cardiac and neurological diseases related to ankyrin and spectrin.