División de Genética, Universidad de Alicante, Alicante, Spain.
PLoS One. 2012;7(4):e35845. doi: 10.1371/journal.pone.0035845. Epub 2012 Apr 30.
PipX provides a functional link between the cyanobacterial global transcriptional regulator NtcA and the signal transduction protein P(II), a protein found in all three domains of life as integrators of signals of the nitrogen and carbon balance. PipX, which is toxic in the absence of P(II), can form alternative complexes with NtcA and P(II) and these interactions are respectively stimulated and inhibited by 2-oxoglutarate, providing a mechanism by which P(II) can modulate expression at the NtcA regulon. Structural information on PipX-NtcA complexes suggests that PipX coactivates NtcA controlled genes by stabilizing the active conformation of NtcA bound to 2-oxoglutarate and by possibly helping recruit RNA polymerase. To get insights into PipX functions, we perform here a mutational analysis of pipX informed by the structures of PipX-P(II) and PipX-NtcA complexes and evaluate the impact of point mutations on toxicity and gene expression. Two amino acid substitutions (Y32A and E4A) were of particular interest, since they increased PipX toxicity and activated NtcA dependent genes in vivo at lower 2-oxoglutarate levels than wild type PipX. While both mutations impaired complex formation with P(II), only Y32A had a negative impact on PipX-NtcA interactions.
PipX 在蓝细菌全局转录调节剂 NtcA 和信号转导蛋白 P(II)之间提供了功能联系，P(II)作为氮和碳平衡信号的整合因子存在于所有三个生命领域中。在没有 P(II)的情况下，有毒的 PipX 可以与 NtcA 和 P(II)形成替代复合物，这些相互作用分别被 2-氧戊二酸刺激和抑制，提供了 P(II)可以调节 NtcA 调节子表达的机制。关于 PipX-NtcA 复合物的结构信息表明，PipX 通过稳定与 2-氧戊二酸结合的 NtcA 的活性构象，并可能有助于招募 RNA 聚合酶，来共同激活 NtcA 控制的基因。为了深入了解 PipX 的功能，我们根据 PipX-P(II)和 PipX-NtcA 复合物的结构对 pipX 进行了突变分析，并评估了点突变对毒性和基因表达的影响。两个氨基酸取代（Y32A 和 E4A）特别有趣，因为它们在低于野生型 PipX 的 2-氧戊二酸水平下增加了 PipX 的毒性并激活了 NtcA 依赖性基因。虽然这两种突变都损害了与 P(II)的复合物形成，但只有 Y32A 对 PipX-NtcA 相互作用有负面影响。
