Structure and Function of the ASB-Containing E3 Ligases

Abstract

Proteins have half-lives ranging from only a few minutes to several hours, and the metabolism of proteins is regulated by covalent attachment of Ubiquitin and subsequent degradation. The Cullin-RING E3 ligase, composed of 7 individual proteins, catalyzes the ubiquitin transfer to a specific target by means of a recognition protein that binds to that substrate. Ankyrin repeat and SOCS box-containing protein, ASB9, has been identified as a substrate recognition subunit of a Cullin-RING E3 ligase that controls proteasomal degradation of Creatine Kinase (CK). CK is an enzyme with a role in the ATP pathway for providing energy to muscle tissue, but it also is a marker for inflammation, muscle damage, and potentially cancer. Previous work in our lab demonstrated a one-to-one interaction between ASB9 and a dimer of CK, in which the N-terminal ankyrin repeat interacts with the dimer and the disordered N-terminus interacts with one active site of CK. Hydrogen deuterium exchange mass spectrometry (HDXMS) was used to identify the binding interface between ASB9 and the bound portion of the CK dimer. Previously obtained crystal structures depict the 35-294 region of ASB9 with the adapter proteins that link it to the rest of the E3 ligase. While these structures contain the ankyrin repeat and SOCS Box domains, the structure of the N-terminal interaction with CK remains unknown. Our current efforts aim to crystallize ASB9 with the CK dimer as well as the Elongin B and C adapter proteins, which we found to be necessary for the stability of ASB9. We plan to then form the entire Cullin-RING E3 Ligase in order to study the dynamic states and conformational changes that occur to bring ubiquitin within proximity of CK for catalysis.