Dynamics and Assembly of ASB-Containing E3 Ubiquitin Ligases

Abstract

E3 Ubiquitin Ligases (UBLs) facilitate the highly-specific covalent attachment of activated ubiquitin to bound substrate proteins through an isopeptide bond on an exposed lysine residue. Ubiquitin signaling regulates cellular protein degradation which is essential for proper cell functioning. The multi-subunit Cullin-RING ligase (CRL) represents the most prominent form of E3 Ligases, and it is responsible for a substantial percentage of ubiquitin-directed protein degradation through the K48 ubiquitin linkage. CRLs share a common structure, composed (in order of arrangement) of a substrate receptor with a suppressor of cytokine signaling (SOCS) domain, one or two adapter proteins, a Cullin protein, and a RING-box (RBX) protein that recruits E2 enzymes charged with activated ubiquitin. Among the substrate receptors belonging to this class of UBLs, all 18 proteins in the Ankyrin Repeat and SOCS-box (ASB) family of proteins associate with Cullin 5 and RBX2 through the Elongin B and C (EB/C) adapter proteins. ASB9 has previously been shown to tightly and specifically bind to Creatine Kinase (CK). I am building the entire seven-protein ASB9-containing E3 ligase complex in order to characterize its dynamics. I have expressed and purified all components of the ASB9 UBL, along with the necessary Ubiquitin and Nedd8 conjugating enzymes. I have obtained crystals of CK-ASB9-EB/C for structural determination, and I performed hydrogen-deuterium exchange (HDX) on the same complex to characterize how protein complex formation influences the dynamics of each subunit in the complex. CK binding to ASB9 reduced exchange across both of the first two Ankyrin repeats, while the removal of the SOCS box increased deuterium exchange in the final Ankyrin repeats in ASB9. This demonstrates the complex relationship between the components of the E3 ligase and the sophisticated substrate recognition by ASB proteins.