Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.
Department of Chemistry, The University of Chicago, Chicago, IL 60637, USA.
Nucleic Acids Res. 2018 Mar 16;46(5):2624-2635. doi: 10.1093/nar/gkx1292.
Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. In particular, Fab BL3-6 binds to an AAACA RNA pentaloop closed by a GC pair with ∼100 nM affinity. The Fab and hairpin have served as a portable module for RNA crystallization. The potential for general application make it desirable to adjust the properties of this crystallization module in a manner that facilitates its use for RNA structure determination, such as ease of purification, surface entropy or binding affinity. In this work, we used both in vitro RNA selection and phage display selection to alter the epitope and paratope sides of the binding interface, respectively, for improved binding affinity. We identified a 5'-GNGACCC-3' consensus motif in the RNA and S97N mutation in complimentarity determining region L3 of the Fab that independently impart about an order of magnitude improvement in affinity, resulting from new hydrogen bonding interactions. Using a model RNA, these modifications facilitated crystallization under a wider range of conditions and improved diffraction. The improved features of the Fab-RNA module may facilitate its use as an affinity tag for RNA purification and imaging and as a chaperone for RNA crystallography.
抗体片段，如 Fabs，具有增强蛋白质和 RNA 结晶的性质，因此可以促进大分子结构的确定。特别是，Fab BL3-6 以约 100 nM 的亲和力结合到由 GC 对封闭的 AAACA RNA 五聚体环。Fab 和发夹已成为 RNA 结晶的便携式模块。由于其具有广泛的应用潜力，因此需要以一种有利于 RNA 结构确定的方式调整该结晶模块的性质，例如易于纯化、表面熵或结合亲和力。在这项工作中，我们分别使用体外 RNA 选择和噬菌体展示选择来改变结合界面的表位和变构位，以提高结合亲和力。我们在 RNA 中鉴定出一个 5'-GNGACCC-3' 共识基序，并且在 Fab 的互补决定区 L3 中发现 S97N 突变，这两个突变分别独立地提高了亲和力约一个数量级，这是由于新的氢键相互作用所致。使用模型 RNA，这些修饰促进了在更广泛条件下的结晶，并提高了衍射效果。Fab-RNA 模块的改进特性可能使其更容易用作 RNA 纯化和成像的亲和标签，以及 RNA 结晶学的伴侣。
