Characterization, crystallization and preliminary X-ray crystallographic analysis of the Uba5 fragment necessary for high-efficiency activation of Ufm1.

Abstract

Uba5 is the smallest ubiquitin-like molecule-activating enzyme and contains an adenylation domain and a C-terminal region. This enzyme only exists in multicellular organisms. The mechanism through which the enzyme recognizes and activates ubiquitin-fold modifier 1 (Ufm1) remains unknown. In this study, Uba5 adenylation domains with different C-terminal region lengths were cloned, expressed and purified. The results of an in vitro truncation assay suggest that Uba5 residues 57-363 comprise the minimal fragment required for the high-efficiency activation of Ufm1. Crystallization of Uba5 residues 57-363 was performed at 277 K using PEG 3350 as the precipitant, and crystals optimized by microseeding diffracted to 2.95 Å resolution, with unit-cell parameters a=b=97.66, c=144.83 Å, α=β=90, γ=120°. There is one molecule in the asymmetric unit; the Matthews coefficient and the solvent content were calculated to be 2.93 Å3 Da(-1) and 58.1%, respectively.