Grafting the ALFA tag for structural studies of aquaporin Z

IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Lauren Stover , Hanieh Bahramimoghaddam , Lie Wang , Samantha Schrecke , Gaya P. Yadav , Ming Zhou , Arthur Laganowsky
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引用次数: 0

Abstract

Aquaporin Z (AqpZ), a bacterial water channel, forms a tetrameric complex and, like many other membrane proteins, activity is regulated by lipids. Various methods have been developed to facilitate structure determination of membrane proteins, such as the use of antibodies. Here, we graft onto AqpZ the ALFA tag (AqpZ-ALFA), an alpha helical epitope, to make use of the high-affinity anti-ALFA nanobody (nB). Native mass spectrometry reveals the AqpZ-ALFA fusion forms a stable, 1:1 complex with nB. Single-particle cryogenic electron microscopy studies reveal the octameric (AqpZ-ALFA)4(nB)4 complex forms a dimeric assembly and the structure was determined to 1.9 Å resolution. Dimerization of the octamer is mediated through stacking of the symmetrically bound nBs. Tube-like density is also observed, revealing a potential cardiolipin binding site. Grafting of the ALFA tag, or other epitope, along with binding and association of nBs to promote larger complexes will have applications in structural studies and protein engineering.

Abstract Image

接枝 ALFA 标签用于水蒸发蛋白 Z 的结构研究
水蒸发蛋白 Z(AqpZ)是一种细菌水通道,形成四聚体复合物,与许多其他膜蛋白一样,其活性受脂类调节。为了便于确定膜蛋白的结构,人们开发了多种方法,如使用抗体。在这里,我们在 AqpZ 上嫁接了 ALFA 标签(AqpZ-ALFA),这是一个阿尔法螺旋表位,从而利用了高亲和力的抗 ALFA 纳米抗体(nB)。本征质谱显示,AqpZ-ALFA 融合体与 nB 形成了稳定的 1:1 复合物。单颗粒低温电子显微镜研究显示,八聚体(AqpZ-ALFA)4(nB)4 复合物形成二聚体组装,其结构的分辨率达到 1.9 Å。八聚体的二聚化是通过对称结合的 nBs 的堆积来介导的。还观察到管状密度,揭示了一个潜在的心磷脂结合位点。嫁接 ALFA 标记或其他表位,以及 nBs 的结合和关联以促进更大的复合物,将在结构研究和蛋白质工程中得到应用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Journal of Structural Biology: X
Journal of Structural Biology: X Biochemistry, Genetics and Molecular Biology-Structural Biology
CiteScore
6.50
自引率
0.00%
发文量
20
审稿时长
62 days
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