Creatine kinases (CKs) regulate ATP levels at sites of fluctuating energy demands. Reduced CK activity has been implicated in diseases such as heart failure and multiple sclerosis, however little is known about CK regulation. Ankyrin repeat and SOCS box-containing protein, ASB9, (part of an E3 ubiquitin ligase) has been shown to bind to CK and promote proteasomal degradation of CK. We hypothesize that ASB9 and its splice variants control the levels and activity of CK at sites where energy is critically needed. We have focused on the interaction between CKB and splice variant 2 of ASB9 that contains only the ankyrin repeat domain (ARD). Our results show that the ASB9-ARD binds CKB with very high affinity (nM) and a stoichiometry of one ASB9 to one CK dimer. Comparison of binding data from an N-terminal truncation mutant of ASB9 suggests that residues 1-35 of ASB9 contribute to the binding affinity. In addition, the N-terminus of ASB9 seems to be responsible for inhibiting the enzymatic activity of CK when bound to ASB9. Hydrogen deuterium exchange mass spectrometry (HDXMS) data shows that only one region in CKB (residues 182 - 203) which is right in front of the active site, is protected upon binding of ASB9 when comparing the deuterium incorporation of CKB peptides alone and in complex with ASB9. A model of the CK-ASB9 interaction generated using computational docking agrees with the binding and HDXMS data. Analysis of the docked model showed that residue D32 in the ASB9 N-terminus binds to the ATP binding site in CK and probably plays an important role in the CK-ASB9 interaction. Interestingly, a D32A mutant of ASB9 binds CK, however has reduced affinity and has no effect on CK activity revealing the importance of this residue. Overall, the N-terminus of ASB9 contributes to the binding of CK, and plays a role in directly inhibiting the CK activity by altering regions near the active site of the enzyme. We are further characterizing the N-terminus of ASB9 to narrow down the residues that directly bind CK and determine the inhibitory kinetic constants of the interaction. The goal of this project is provide essential information for the development of inhibitors that would interrupt the CK-ASB9 interaction, resulting in increased amounts of active CK to treat diseases in which there is a an increased energy demand and decreased CK function.