Mechanistic studies of ankyrin autoinhibition and ankyrin-spectrin complex formation

Abstract

Ankyrins coordinate with spectrins in excitable or mechano-resistant tissues/cells to organize various functional membrane domains, and thereby to promote structural stabilities and to regulate physiological activities including fast signaling in heart and brain. Consistent with their critical roles, ankyrin and spectrin are closely related to many types of severe human diseases. Mammalian ankyrins contain three members: ankyrin-R/B/G (AnkR/B/G), encoded by ANK1/2/3 respectively. Each ankyrin contains an N-terminal membrane-binding domain (MBD) composed of 24 ankyrin repeats (ANK repeats), a central spectrin-binding domain (SBD) composed of a ZU5_N-ZU5_C-UPA supramodule, and a C-terminal death domain (DD); other regions of ankyrins are intrinsically disordered. Ankyrins coordinate numerous membrane targets through its MBD, and bind to spectrins through its SBD, thus acting as a major connector between membranes and cytoskeletons. The MBD and SBD of ankyrins are evolutionally highly conserved, indicating essential roles of these two regions for ankyrins’ functions. The interactions between MBD and membrane proteins, as well as SBD with different β-spectrin subtypes, must be regulated to achieve spatial-temporal specific functions in different physiological contexts. However, very little is known regarding this aspect. Here I present my thesis work based on a biochemical and structural approach trying to elucidate the mechanism underlying how ankyrins regulate their interactions with diverse membrane targets by autoinhibition and how they form complex with different β-spectrin subtypes. I systemically studied the autoinhibition on ankyrin-B/G MBD, and identified 3 discontinuous autoinhibitory segments from the disordered linkers and tails of ankyrin-B/G. They inhibit ankyrin’s MBD in a combinatorial and quasi-independent way, suggesting a mechanistic basis for differential regulations towards membrane targets. Parallely, I identified an extension sequence of ankyrin-B/G SBD, which vastly strengthened ankyrin-spectrin interactions. Structural and biochemical data demonstrated a direct involvement of UPA domain in spectrin binding. The crystal structures of AnkB/β4-, AnkG/β4- and AnkG/β2-spectrin complexes revealed the interaction details of different ankyrin-spectrin pairs and shed lights on isoform specificities between different ankyrin subtypes. Our data provided explanation towards the functional implication of Hereditary Spherocytosis-causing mutations on SBD, and laid the foundation for future studies on the regulation of ankyrin-spectrin interactions by mechanical forces.

Date of Award	2017
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology