The interaction of Tudor domain-containing proteins (TDRDs) with P-element-induced wimpy testis (PIWI) proteins plays critical roles in transposon silencing and spermatogenesis. Most human TDRDs recognize PIWI proteins in a methylarginine-dependent manner via their extended Tudor (eTudor) domains, except TDRD2, which prefers an unmethylated PIWI protein. In order to illustrate the recognition of unmethylated PIWI proteins by TDRD2, we extensively tried co-crystallization of the TDRD2 eTudor with different PIWIL1 peptides, but to no avail. Recombinant antigen-binding fragments (Fabs) have been used to crystallize some difficult proteins in the past, so we generated Fab against the TDRD2 eTudor protein using a phage-display antibody library, and one of these Fab fragments indeed facilitated the co-crystallization of TDRD2 and PIWIL1. Structural analysis of Fab, the TDRD2 eTudor domain in complex with an unmethylated PIWIL1-derived peptide revealed that the PIWIL1 residues G3 through R8 bound between the Tudor core and SN domain of TDRD2. The C-terminal residues of the PIWIL1 peptide were not resolved, presumably due to steric competition with the heavy chain of the Fab. We propose Fab-assisted crystallization as a tool not only for structural studies of single proteins, but also for analysis of interactions between proteins and their ligands in cases where co-crystallization of native protein complexes fails.

中文翻译：

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TDRD2 和 PIWIL1 的 Fab 使能共结晶研究的教训

含有 Tudor 结构域的蛋白 (TDRD) 与 P 元件诱导的睾丸萎缩 (PIWI) 蛋白的相互作用在转座子沉默和精子发生中起着关键作用。大多数人类 TDRD 以甲基精氨酸依赖性方式通过其扩展的 Tudor (eTudor) 域识别 PIWI 蛋白，但 TDRD2 除外，它更喜欢未甲基化的 PIWI 蛋白。为了说明 TDRD2 对未甲基化 PIWI 蛋白的识别，我们广泛尝试了 TDRD2 eTudor 与不同 PIWIL1 肽的共结晶，但无济于事。重组抗原结合片段 (Fab) 过去曾用于结晶一些困难的蛋白质，因此我们使用噬菌体展示抗体库生成了针对 TDRD2 eTudor 蛋白的 Fab，其中一个 Fab 片段确实促进了共结晶TDRD2 和 PIWIL1。Fab 结构分析，TDRD2 eTudor 结构域与未甲基化的 PIWIL1 衍生肽复合，表明 PIWIL1 残基 G3 到 R8 结合在 TDRD2 的 Tudor 核心和 SN 结构域之间。PIWIL1 肽的 C 端残基未解析，可能是由于与 Fab 重链的空间竞争。我们建议将 Fab 辅助结晶作为一种工具，不仅用于单个蛋白质的结构研究，而且还用于在天然蛋白质复合物的共结晶失败的情况下分析蛋白质与其配体之间的相互作用。可能是由于与 Fab 重链的空间竞争。我们建议将 Fab 辅助结晶作为一种工具，不仅用于单个蛋白质的结构研究，而且还用于在天然蛋白质复合物的共结晶失败的情况下分析蛋白质与其配体之间的相互作用。可能是由于与 Fab 重链的空间竞争。我们建议将 Fab 辅助结晶作为一种工具，不仅用于单个蛋白质的结构研究，而且还用于在天然蛋白质复合物的共结晶失败的情况下分析蛋白质与其配体之间的相互作用。