Li Biaoru, Phillips Nelson B, Jancso-Radek Agnes, Ittah Varda, Singh Rupinder, Jones David N, Haas Elisha, Weiss Michael A
Department of Biochemistry, Case School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4935, USA.
J Mol Biol. 2006 Jul 7;360(2):310-28. doi: 10.1016/j.jmb.2006.04.048. Epub 2006 May 9.
Sex-reversal mutations in human SRY cluster within its high-mobility group box, a conserved motif of DNA bending. A classical substitution at the crux of this angular domain (M64I) has been reported to impair DNA bending but not DNA binding, implying that sharp bending is required for transcriptional activation and testis determination. Surprisingly, we report that this defect was an inadvertent consequence of protein truncation: in the intact protein, sharp DNA bending is restored by the basic tail of the high-mobility group box. Structural coupling between box and tail is tuned to the native DNA bend angle, damping conformational fluctuations and enabling bidirectional induced fit within the bent complex. M64I-associated sex reversal is instead caused by the impaired function of a flanking non-classical nuclear localization signal (NLS). Similar impairment is caused by M64A, suggesting that mislocalization is due to loss of an M64-specific function and not gain of a non-native I64-specific function. Transcriptional activity, attenuated by mislocalization, is rescued by fusion of a heterologous NLS. In a male embryonic gonadal cell line, M64I and M64A SRY-NLS fusion proteins exhibit native transcriptional activation of Sox9, a key step in testicular differentiation. Our results suggest that male development is robust to subtle alterations in SRY-DNA architecture but depends critically on nuclear localization. The previously unsuspected role of M64 within a non-classical NLS may contribute to its invariance among SOX-related and LEF-1-related transcription factors.
人类SRY基因的性别反转突变发生在其高迁移率族框内，这是一个保守的DNA弯曲基序。据报道，在这个角状结构域的关键位置有一个经典的取代突变（M64I），它会损害DNA弯曲但不影响DNA结合，这意味着急剧弯曲对于转录激活和睾丸决定是必需的。令人惊讶的是，我们发现这个缺陷是蛋白质截短的意外结果：在完整蛋白质中，高迁移率族框的碱性尾巴恢复了急剧的DNA弯曲。框与尾巴之间的结构偶联被调整到天然DNA弯曲角度，抑制构象波动，并在弯曲复合物中实现双向诱导契合。相反，M64I相关的性别反转是由侧翼非经典核定位信号（NLS）功能受损引起的。M64A也会导致类似的功能受损，这表明错误定位是由于M64特异性功能的丧失，而不是非天然I64特异性功能的获得。因错误定位而减弱的转录活性可通过融合异源NLS来挽救。在雄性胚胎性腺细胞系中，M64I和M64A SRY-NLS融合蛋白表现出Sox9的天然转录激活，这是睾丸分化的关键步骤。我们的结果表明，男性发育对SRY-DNA结构的细微改变具有较强的耐受性，但关键取决于核定位。M64在非经典NLS中的先前未被怀疑的作用可能有助于其在SOX相关和LEF-1相关转录因子中的不变性。
