分辨率为3.0埃的自身免疫性抗聚（dT）免疫球蛋白Fab片段的结晶与结构测定

Crystallization and structure determination of an autoimmune anti-poly(dT) immunoglobulin Fab fragment at 3.0 A resolution.

作者信息

Cygler M, Boodhoo A, Lee J S, Anderson W F

出版信息

J Biol Chem. 1987 Jan 15;262(2):643-8.

Abstract

HED10 is an autoimmune antibody (IgG) which shows considerable specificity for the single-stranded DNA poly(dT). Production of Fab fragments by papain digestion resulted in heterogeneity as judged by isoelectric focusing gels, which had a marked negative effect on crystallization. However, a single species of Fab with a pI of 7.6 could be isolated in good yield by DEAE-cellulose chromatography, and good crystals were produced by the hanging drop vapor diffusion method. The space group was P21 with cell dimensions, a = 64.2, b = 90.0, c = 42.3 A, and beta = 96.7 degrees. These crystals diffract to about 2.2 A resolution. The structure of Fab HED10 was solved by the molecular replacement method using the known structure of McPC603 and is refined to R = 27.2% at 3.0 A resolution. Fab HED10 is more extended than McPC603 and has an elbow angle (between the variable and constant domains) of 162 degrees, very similar to that observed in Fab KOL. The majority of the hypervariable regions are visible in the model.

摘要

HED10是一种自身免疫抗体（IgG），对单链DNA多聚（dT）具有相当高的特异性。用木瓜蛋白酶消化产生的Fab片段，通过等电聚焦凝胶判断会导致异质性，这对结晶有显著的负面影响。然而，通过DEAE - 纤维素色谱法可以高产率地分离出一种pI为7.6的单一Fab物种，并且通过悬滴气相扩散法产生了良好的晶体。空间群为P21，晶胞参数为a = 64.2，b = 90.0，c = 42.3 Å，β = 96.7°。这些晶体的衍射分辨率约为2.2 Å。Fab HED10的结构通过分子置换法利用McPC603的已知结构解析得到，并在3.0 Å分辨率下精修至R = 27.2%。Fab HED10比McPC603更伸展，其肘部角度（可变区和恒定区之间）为162°，与在Fab KOL中观察到的非常相似。模型中可见大部分高变区。