Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
J Biol Chem. 2024 Sep;300(9):107683. doi: 10.1016/j.jbc.2024.107683. Epub 2024 Aug 20.
Y-chromosome-encoded master transcription factor SRY functions in the embryogenesis of therian mammals to initiate male development. Through interactions of its conserved high-mobility group box within a widened DNA minor groove, SRY and related Sox factors induce sharp bends at specific DNA target sites. Here, we present the crystal structure of the SRY high-mobility group domain bound to a DNA site containing consensus element 5'-ATTGTT. The structure contains three complexes in the asymmetric unit; in each complex, SRY forms 10 hydrogen bonds with minor-groove base atoms in 5'-CATTGT/ACAATG-3', shifting the recognition sequence by one base pair (italics). These nucleobase interactions involve conserved residues Arg7, Asn10, and Tyr74 on one side of intercalated Ile13 (the cantilever) and Arg20, Asn32, and Ser36 on the other. Unlike the less-bent NMR structure, DNA bend angles (69-84°) of the distinct box-DNA complexes are similar to those observed in homologous Sox domain-DNA structures. Electrophoretic studies indicate that respective substitutions of Asn32, Ser36, or Tyr74 by Ala exhibit slightly attenuated specific DNA-binding affinity and bend angles (70-73°) relative to WT (79°). By contrast, respective substitutions of Arg7, Asn10, or Arg20 by Ala markedly impaired DNA-binding affinity in association with much smaller DNA bend angles (53-65°). In a rodent cell-based model of the embryonic gonadal ridge, full-length SRY variants bearing these respective Ala substitutions exhibited significantly decreased transcriptional activation of SRY's principal target gene (Sox9). Together, our findings suggest that nucleobase-specific hydrogen bonds by SRY are critical for specific DNA binding, bending, and transcriptional activation.
Y 染色体编码的主要转录因子 SRY 在有胎盘哺乳动物的胚胎发生中发挥作用，启动雄性发育。通过其保守的高迁移率族盒在加宽的 DNA 小沟内的相互作用，SRY 和相关的 Sox 因子在特定的 DNA 靶位点诱导急剧弯曲。在这里，我们呈现了与包含共识元件 5'-ATTGTT 的 DNA 位点结合的 SRY 高迁移率组结构域的晶体结构。该结构在不对称单元中包含三个复合物；在每个复合物中，SRY 与 5'-CATTGT/ACAATG-3' 中小沟碱基原子形成 10 个氢键，将识别序列移动一个碱基对（斜体）。这些核碱基相互作用涉及到跨插入的 Ile13（悬臂）一侧的保守残基 Arg7、Asn10 和 Tyr74，以及另一侧的 Arg20、Asn32 和 Ser36。与弯曲角度较小的 NMR 结构不同，不同的盒-DNA 复合物的 DNA 弯曲角度（69-84°）与在同源 Sox 结构域-DNA 结构中观察到的相似。电泳研究表明，Asn32、Ser36 或 Tyr74 分别被 Ala 取代时，与 WT（79°）相比，特异性 DNA 结合亲和力和弯曲角度（70-73°）略有降低。相比之下，Arg7、Asn10 或 Arg20 分别被 Ala 取代时，与较小的 DNA 弯曲角度（53-65°）相关联，显著损害了 DNA 结合亲和力。在胚胎性腺嵴的啮齿动物细胞模型中，携带这些相应 Ala 取代的全长 SRY 变体表现出 SRY 主要靶基因（Sox9）的转录激活显著降低。总之，我们的发现表明，SRY 的碱基特异性氢键对于特异性 DNA 结合、弯曲和转录激活至关重要。
