Part I. Crystal Structure of Cytotoxic RC-RNase3 from Rana catesbeiana in Complex with Heparin Part II. Crystallization and Preliminary X-ray Diffraction Analysis of Muscarinic Toxin-Like Proteins,BM14 and BM8 from Bungarus multicinctus

• Main Authors: Chien-chang, Tseng, 曾建璋
• Other Authors: Yuh-Ju, Sun
• Format: Others
• Language: en_US
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/00852193855937445814

碩士 === 國立清華大學 === 生命科學系 === 92 === Crystal Structure of Cytotoxic RC-RNase3 from Rana catesbeiana in Complex with Heparin Abstract The Rana catesbeiana (bullfrog) ribonucleases, which belong to the RNase A superfamily, exert cytotoxicity and lectin activity. RC-RNase3 has a unique base preference for pyrimidine-guanine rather than pyrimidine-adenine in RNase A. The crystal structure of RC-RNase3 in complex with heparin disaccharide was determined to resolution of 1.6Å. Heparin is one type of glycosaminoglycan locating primarily on the surface of cells or in the extra-cellular matrix. The overall structure of RC-RNase3-heparin complex consists of seven β-strands and three α-helices that present a bowl shape. Heparin was found locating between sheet 1 (β1, β2, β4, and β5) and sheet 2 (β3, β6, and β7). One of the sulfate groups in heparin takes place in a very close position to the free sulfate in the retro binding RC-RNase6 and d(CpG) complex. More interestingly, two sulfate groups in heparin are close to the phosphate groups of d(ApCpGpA) in the catalytic binding RC-RNase and d(ApCpGpA) complex. Our results indicate a new binding mode where heparin sharing approximately the same binding site to the catalytic and retro binding modes. Crystallization and Preliminary X-ray Diffraction Analysis of Muscarinic Toxin-Like Protein, BM14 and BM8 from Bungarus multicinctus Abstract Two novel proteins BM14 and BM8 were isolated from Bungarus multicinctus (Taiwan banded krait) venom. BM14 and BM8 are sequence identical except Lys37-Lys38 in BM14 and Glu37-Ala38 in BM8. In contrast to BM8, BM14 exhibited an activity on binding to the M2 muscarinic acetlycholine (mAchR) receptor subtype, indicating Lys37 and Lys38 of BM14 play the crucial roles on binding to M2 mAchR. Although BM14 and BM8 shared a 20-38% sequence identity with snake venom cardiotoxins, they did not show the cytotoxicity. Eight out of ten Cys residues in BM14 and BM8 are located at the conserved positions as those in neurotoxin, cardiotoxin and muscarinic toxin protein sequences, so called the three-finger protein. BM14 and BM8 crystals were grown by the hanging drop vapor diffusion method. The crystals of BM14 diffract to 3.0Å and belong to the tetragonal space group I422 with unit-cell parameters a=81.68Å, b=81.68Å, c=207.30Å, and α=β=γ=90°. The crystals of BM8 diffract to 3.8 Å and belong to the hexagonal space group with unit-cell parameters a=55.37Å, b=55.37Å, c=338.0Å, α=β=90°, and γ=120°.