Hosur M V, Nair B, Satyamurthy P, Misquith S, Surolia A, Kannan K K
Solid State Physics Division, Bhabha Atomic Research Centre, Trombay Bombay, India.
J Mol Biol. 1995 Jul 14;250(3):368-80. doi: 10.1006/jmbi.1995.0383.
Gelonin is a single chain ribosome inactivating protein (RIP) with potential application in the treatment of cancer and AIDS. Diffraction quality crystals grown using PEG3350, belong to the space group P21, with a = 49.4 A, b = 44.9 A, c = 137.4 A and beta = 98.4 degrees, and contain two molecules in the asymmetric unit. Diffraction data collected to 1.8 A resolution has a Rm value of 7.3%. Structure of gelonin has been solved by the molecular replacement method, using ricin A chain as the search model. Crystallographic refinement using X-PLOR resulted in a model for which the r.m.s deviations from ideal bond lengths and bond angles are 0.012 A and 2.7 degrees, respectively. The final R-factor is 18.4% for 39,806 reflections for which I > 1.0 sigma (I). The C alpha atoms of the two molecules in the asymmetric unit superpose to within 0.38 A for 247 atom pairs. The overall fold of gelonin is similar to that of other RIPs such as ricin A chain and alpha-momorcharin, the r.m.s.d. for C alpha superpositions being 1.3 and 1.4 A, respectively. The catalytic residues (Glu166, Arg169 and Tyr113) in the active site form a hydrogen bond scheme similar to that observed in other RIPs. The conformation of Tyr74 in the active site, however, is significantly different from that in alpha-momorcharin. Three well defined water molecules are located in the active site cavity, and one of them, X319, superposes to within 0.2 A of a corresponding water molecule in the structure of alpha-momorcharin. Any of the three could be the substrate water molecule in the hydrolysis reaction catalysed by gelonin. Difference electron density for a N-linked sugar moiety has been observed near only one of the two potential glycosylation sites in the sequence. The amino acid at position 239 has been established as Lys by calculation of omit electron density maps. The two cysteine residues in the sequence, Cys44 and Cys50, form a disulphide bond, and are therefore not available for disulphide conjugation with antibodies. Based on the structure, the region of the molecule that is involved in intradimer interactions is suggested to be suitable for introducing a Cys residue for purposes of conjugation with an antibody to produce useful immunotoxins.
gelonin是一种单链核糖体失活蛋白（RIP），在癌症和艾滋病治疗中具有潜在应用价值。使用PEG3350生长的衍射质量晶体，属于空间群P21，a = 49.4 Å，b = 44.9 Å，c = 137.4 Å，β = 98.4°，不对称单元中含有两个分子。收集到1.8 Å分辨率的衍射数据，Rm值为7.3%。gelonin的结构已通过分子置换法解析，使用蓖麻毒素A链作为搜索模型。使用X-PLOR进行晶体学精修得到一个模型，其与理想键长和键角的均方根偏差分别为0.012 Å和2.7°。对于I > 1.0σ（I）的39,806个反射，最终R因子为18.4%。不对称单元中两个分子的Cα原子对于247个原子对的叠加误差在0.38 Å以内。gelonin的整体折叠结构与其他RIPs如蓖麻毒素A链和α-苦瓜素相似，Cα叠加的均方根偏差分别为1.3 Å和1.4 Å。活性位点中的催化残基（Glu166、Arg169和Tyr113）形成的氢键模式与其他RIPs中观察到的相似。然而，活性位点中Tyr74的构象与α-苦瓜素中的显著不同。活性位点腔中有三个明确的水分子，其中一个，X319，与α-苦瓜素结构中相应水分子的叠加误差在0.2 Å以内。这三个水分子中的任何一个都可能是gelonin催化水解反应中的底物水分子。在序列中仅两个潜在糖基化位点之一附近观察到了N-连接糖部分的差异电子密度。通过计算省略电子密度图，已确定第239位的氨基酸为赖氨酸。序列中的两个半胱氨酸残基，Cys44和Cys50，形成了一个二硫键，因此不能用于与抗体进行二硫键偶联。基于该结构，分子中参与二聚体内相互作用的区域被认为适合引入一个半胱氨酸残基，用于与抗体偶联以产生有用的免疫毒素。
