Ankyrin adaptors together with their spectrin partners coordinate diverse ion channels and cell adhesion molecules within plasma membrane domains and thereby promote physiological activities including fast signaling in the heart and nervous system. Human ankyrins contain three members: ankyrin-R/B/G (AnkR/B/G), encoded by ANK1/2/3, respectively. They all consist of a highly similar N-terminal membrane binding domain composed of 24 ankyrin repeats (ANK repeats), a middle spectrin binding domain comprised of two ZU5 domains and a UPA domain followed by a death domain (DD), and a variable C-terminal regulatory domain. The membrane binding domain and spectrin binding domain are highly conserved among all ankyrins (across isoforms and species), indicating that the two regions are es...[ Read more ]

Ankyrin adaptors together with their spectrin partners coordinate diverse ion channels and cell adhesion molecules within plasma membrane domains and thereby promote physiological activities including fast signaling in the heart and nervous system. Human ankyrins contain three members: ankyrin-R/B/G (AnkR/B/G), encoded by ANK1/2/3, respectively. They all consist of a highly similar N-terminal membrane binding domain composed of 24 ankyrin repeats (ANK repeats), a middle spectrin binding domain comprised of two ZU5 domains and a UPA domain followed by a death domain (DD), and a variable C-terminal regulatory domain. The membrane binding domain and spectrin binding domain are highly conserved among all ankyrins (across isoforms and species), indicating that the two regions are essential for ankyrin’s functions throughout the evolution. Ankyrins specifically bind to unstructured sequences of membrane targets through their 24 ANK repeats, and directly bind to spectrin via the first ZU5 domain in the middle region, although the molecular mechanisms for the facile and independent evolution of these interactions has not been resolved. As an adaptor protein linking membrane proteins to the underlying cytoskeleton, dysfunctions of ankyrins are closely related to serious human diseases. For example, loss-of-function mutations of ankyrins can cause hemolytic anemia, various cardiac diseases including several cardiac arrhythmia syndromes and sinus node dysfunctions, bipolar disorder and autism spectrum disorders, although the underlying molecular bases are poorly understood.

In this dissertation, I focus on ankyrin biology mainly from a biochemical and structural view and try to answer these fundamental questions in the ankyrin field by solving atomic structures of ankyrin different domains. Here I will describe three crystal structures of ankyrins combined with detailed biochemical analyses, providing the first structural blueprint for ankyrins. The high resolution structure of the ankyrin-B spectrin binding domain, which is arranged into ZU5-ZU5-UPA-DD (ZZUD) tandem, determined here reveals that the ZU5-ZU5-UPA (ZZU) domains form a tightly packed structural supramodule, whereas DD is freely accessible. Further structural analysis indicates that the ZZU supramodule of ankyrins has two additional surfaces that may bind to targets other than spectrin. The structure of the ankyrin ZZUD provides mechanistic explanations to many disease-causing mutations identified in ankyrin-B&R, and provides a “structural map” for further investigation of the spectrin binding domain of ankyrins. The other two structures of 24 ANK repeats in complex with an inhibitory segment from the C-terminal regulatory domain of ankyrin-R and part of the ANK repeats with a sodium channel Nav1.2 peptide, respectively, show that N-terminal 24 ANK repeats from ankyrins form a superhelical solenoid with an extended, extremely conserved inner groove, which contains multiple quasi-independent target binding sites that can accommodate target proteins with very diverse sequences via combinatorial usage of these sites. These structures establish a framework for understanding the evolution of ankyrins’ membrane targets, with implications for other proteins with extended ANK repeat domains. In summary, the work described in this dissertation deepened the understanding of ankyrin biology from a structural angle and provided new mechanisms governing ankyrin’s functions in diverse membrane micro-domains, and laid down a molecular foundation for further research in the ankyrin field.