Three‐Dimensional structure of schistosoma japonicum glutathione s‐transferase fused with a six‐amino acid conserved neutralizing epitope of gp41 from hiv

Abstract

The 3-dimensional crystal structure of glutathione S-transferase (GST) of Schistosomajaponicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV-I) (Muster T et al., 1993, J Virol626642-6647) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the p gene class (Ji X, Zhang P, Armstrong RN, Gilliland GL, 1992, Biochemistry 31:10169-10184) was used as a molecular replacement model. The structure consists of a 4-stranded @-sheet and 3 a-helices in domain 1 and 5 a-helices in domain 2. The space group of the Sj GST crystal is P4,2,2, with unit cell dimensions of a = b = 94.7 A, and c = 58.1 A. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2-fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the C-terminus of Sj GST forms a loop stabilized by symmetry-related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes p , a , and a. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed.

In Vitro Recognition of Specific DNA Targets by AlcR, a Zinc Binuclear Cluster Activator Different from the Other Proteins of This Class

“…Until now, to our knowledge, it had not yet been described that such an approach would introduce a serious bias related to the quaternary structure of the heterologous carrier protein. For example, the GST protein is a dimer in solution (23,24), and ␤-galactosidase is a tetramer (27,28). Therefore, these proteins fused to a DNA-binding domain impose a conformation resulting from their quaternary structure for selecting the DNA targets.…”

“…2,3 Surprisingly, the interactions between the AlcR fusion proteins and guanines within the motif appeared to be different in both types of targets (14,15). Since GST (the fusion protein carrier) is known to be dimeric in solution (23,24) and hence could introduce a bias in the DNA binding specificity, we decided to perform in parallel the same binding experiments with another bacterially synthesized AlcR protein consisting of six histidine residues fused to the C terminus of the truncated AlcR protein (residues 1-197) and purified on a nickel column as described under "Experimental Procedures." Such chimeric proteins are also widely used.…”

Proximity-induced activation of human Cdc34 through heterologous dimerization

“…Taken together, these results demonstrate that GST-Cdc34 is an extensively elongated dimer. On the basis of the three-dimensional structure of the Schistosoma japonicum GST, the C-terminal ends of each subunit protrude in the same direction (16), suggesting that the Cdc34-moeities are placed in close proximity in a GST-Cdc34 dimeric complex.…”

Crystallization of an intact GST-estrogen receptor hormone binding domain fusion protein

“…Since the three-dimensional structures of GST from S. japonicum (GST used to make the fusion GST-ERHBD protein; Lim et al, 1994) and the RXRot receptor ligand binding domain (Bourget et al, 1995) are known, we attempted to model a GST-ERHBD fusion protein based on the two known homologous structures (Fig. 4).…”