THERMODYNAMICS OF HHR23A UBIQUITIN-ASSOCIATED (UBA) DOMAINS

Ubiquitin-associated (UBA) domains have been discovered in several proteins within the cell, and at least one domain plays an important functional role in cellular phenomena such as NF-κB signaling. Mutations within the UBA(2) domain of the ubiquitin-binding scaffold protein p62 are frequently observed in patients suffering from Paget’s disease of bone. The human DNA excision repair protein HHR23A contains the UBA(1) and UBA(2) domains, and nuclear magnetic resonance (NMR) structures depict their compact three-helix bundles. Moreover, the HHR23A UBA(1) and UBA(2) only share approximately 20% primary sequence identity. The p62 UBA(2) domain has been shown to contain quaternary structure in the mM concentration range, and has been hypothesized to play an important functional role within the cell via ubiquitination. Herein, the HHR23A UBA domain thermodynamics are determined using far UV CD guanidine hydrochloride titrations, and a UBA(1) quaternary structure investigation is carried out using matrix-assisted laser desorption/ionization mass spectrometry, far UV CD thermal denaturation, and gel filtration size exclusion chromatography (SEC) to elucidate a possible shared property between the p62 and HHR23A UBA domains, which may suggest potential competition between ubiquitin binding and UBA domain dimerization. Our results demonstrate that a C26A mutation in the UBA(2) domain enhances its stability, and that no change in the UBA(1) equilibrium constant for unfolding is observed as a function of protein concentration, indicating no HHR23A UBA(1) dimerization in the uM concentration range. Furthermore, the HHR23A UBA(1) domain interacts with ubiquitin, yet these HHR23A UBA domains do not interact with each other, indicating a different mechanism other than HHR23A UBA domain homodimerization is involved