Structural and biochemical characterization of the cytosolic wheat cyclophilin TaCypA-1 = 빵밀유래 시토졸 시클로필린TaCypA-1의 구조 및 생화학적 특성

Abstract

Cyclophilins belong to a family of proteins that bind to the immunosuppressive drug cyclosporin A (CsA). Several members of this protein family catalyze the cis-trans isomerization of peptide bonds preceding prolyl residues. The present study describes the biochemical and structural characteristics of a cytosolic cyclophilin (TaCypA-1) cloned from wheat (Triticum aestivum L.). Purified TaCypA-1 expressed in Escherichia coli showed peptidyl-prolyl cis-trans isomerase activity, which was inhibited by CsA with an inhibition constant of 78.3nM. The specific activity and catalytic efficiency (k cat/K m) of the purified TaCypA-1 were 99.06 ± 0.13nmols -1mg-1 and 2.32 × 105 M -1s-1, respectively. The structures of apo TaCypA-1 and the TaCypA-1-CsA complex were determined at 1.25 and 1.20A resolution, respectively, using X-ray diffraction. Binding of CsA to the active site of TaCypA-1 did not result in any significant conformational change in the apo TaCypA-1 structure. This is consistent with the crystal structure of the human cyclophilin D-CsA complex reported at 0.96A resolution. The TaCypA-1 structure revealed the presence of a divergent loop of seven amino acids 48KSGKPLH54 which is a characteristic feature of plant cyclophilins. This study is the first to elucidate the structure of an enzymatically active plant cyclophilin which shows peptidyl-prolyl cis-trans isomerase activity and the presence of a divergent loop.