含有Tudor结构域的蛋白（TDRDs）与P元件诱导的弱睾丸（PIWI）蛋白的相互作用在转座子沉默和精子发生中起关键作用。大多数人类TDRDs通过其延伸的都铎（eTudor）结构域以甲基精氨酸依赖性方式识别PIWI蛋白，除了TDRD2，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TDRD2eTudor与不同PIWIL1肽的共结晶，但无济于事。重组抗原结合片段（Fab）已用于结晶一些困难的蛋白质，在过去，所以我们使用噬菌体展示抗体文库产生了针对TDRD2eTudor蛋白的Fab，这些Fab片段之一确实促进了TDRD2和PIWIL1的共结晶。Fab的结构分析,与未甲基化的PIWIL1衍生肽复合的TDRD2eTudor结构域表明，PIWIL1残基G3至R8结合在TDRD2的Tudor核心和SN结构域之间。PIWIL1肽的C端残基未被解析，可能是由于与Fab的重链的空间竞争。我们建议Fab辅助结晶不仅作为单一蛋白质结构研究的工具，还用于分析天然蛋白质复合物共结晶失败时蛋白质与其配体之间的相互作用。
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.
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