Crystallization and preliminary X-ray crystallographic studies of HsIU mutant in Escherichia coli

Abstract

Hs1UV is an ATP dependent protease in E. coli like proteases La (Lon) and Ti (C1pAP/C1pXP).(1) It is composed of two multimeric components, 19 kDa Hs1V and 50 kDa Hs1U proteins.(2) While Hs1U itself has an ATPase activity, Hs1V has a weak peptidase activity so that it slowly degrades certain hydrophobic peptides, such as N-carbobenzoxy-Gly-Gly-Leu-7-amino-4-methyl-coumarin and polypeptides such as insulin B-chain and casein.(3,4) But the proteolytic activity of Hs1V is increased significantly (up to 150 fold) by associating with Hs1U in the presence of ATP.(3) The primary amino acid sequence of Hs1V is similar to certain beta-type subunits of the 20S proteasomes of archaebacterium Thermoplasma acidophilum with 18% identity.(5) While beta-type subunits of the 20S proteasomes show 72-point symmetry, Hs1V is a dimer of hexamers with 62 point symmetry. The crystal structure of Hs1V solved at 3.8 Angstrom resolution shows that in spite of the different symmetry, the folds and the contacts between subunits are conserved, compared with beta-type subunits of the 20S proteasomes. (6) In the case of Hs1U, it is 50% identical to the C1pX protein of E. coli in amino acid sequence. According to the analysis of Hs1U using electron microscopy, Hs1Us make ring-shaped forms in the presence of ATP or AMPPNP (ATP analogue). This ring is composed of 6 or 7 Hs1U molecules to form hexameric or heptameric rings.(7) Hs1U contains two Cys residues, Cys261 and Cys287. It has been suggested that Cys261 is involved in oligomerization and that Cys287 is related to the ATPase function.(8) In order to reveal the three-dimensional structure, and the mechanism of oligomerization between Hs1Us, and between Hs1U and Hs1V, the HS1U(C261V) was crystallized and studied with X-ray crystallographic method.