Abstract
Human Uba5, which contains an adenylation domain and a C-terminal region, is the smallest ubiquitin-like molecule-activating enzyme. The mechanism through which the enzyme recognizes Ufc1 and catalyzes the formation of the Ufc1-Ufm1 complex remains unknown. In this study, Uba5 residues 364-404 were demonstrated to be necessary for the transthiolation of Ufm1 to Ufc1, and Uba5 381-404 was identified to be the minimal region for Ufc1 recognition. The fusion protein between Uba5 381-404 and Ufc1 was cloned, expressed and purified, and exists as a homodimer in solution. Crystallization was performed at 293 K using PEG 4000 as precipitant; the optimized crystals diffracted to 3.0 Å resolution and had unit-cell parameters a = b = 82.49, c = 62.47 Å, α = β = 90, γ = 120°. With one fusion-protein molecule in the asymmetric unit, the Matthews coefficient and solvent content were calculated to be 2.55 Å(3) Da(-1) and 51.84%, respectively.