Nohaile M, Kern D, Wemmer D, Stedman K, Kustu S
Department of Chemistry, University of California, Berkeley 94720, USA.
J Mol Biol. 1997 Oct 17;273(1):299-316. doi: 10.1006/jmbi.1997.1296.
The bacterial enhancer-binding protein NtrC activates transcription when phosphorylated on aspartate 54 in its amino (N)-terminal regulatory domain or when altered by constitutively activating amino acid substitutions. The N-terminal domain of NtrC, which acts positively on the remainder of the protein, is homologous to a large family of signal transduction domains called receiver domains. Phosphorylation of an aspartate residue in a receiver domain modulates the function of a downstream target, but the accompanying structural changes are not clear. In the present work we examine structural and functional differences between the wild-type receiver domain of NtrC and mutant forms carrying constitutively activating substitutions. Combinations of such substitutions resulted in both increased structural changes in the N-terminal domain, monitored by NMR chemical shift differences, and increased transcriptional activation by the full-length protein. Structural changes caused by substitutions outside the active site (D86N and A89T) were not only local but extended over a substantial portion of the N-terminal domain including the region from alpha-helix 3 to beta-strand 5 ("3445 face") and propagating to the active site. Interestingly, the activating substitution of glutamate for aspartate at the site of phosphorylation (D54E) also triggered structural changes in the 3445 face. Thus, the active site and the 3445 face appear to interact. Implications with respect to how phosphorylation may affect the structure of receiver domains and how structural changes may be communicated to the remainder of NtrC are discussed.
细菌增强子结合蛋白NtrC在其氨基（N）末端调节结构域中的天冬氨酸54被磷酸化时，或被组成型激活的氨基酸取代改变时，会激活转录。NtrC的N末端结构域对蛋白质的其余部分起正向作用，它与一大类称为受体结构域的信号转导结构域同源。受体结构域中天冬氨酸残基的磷酸化调节下游靶标的功能，但其伴随的结构变化尚不清楚。在本研究中，我们研究了NtrC野生型受体结构域与携带组成型激活取代的突变形式之间的结构和功能差异。这些取代的组合导致N末端结构域的结构变化增加（通过NMR化学位移差异监测），以及全长蛋白的转录激活增加。活性位点外的取代（D86N和A89T）引起的结构变化不仅是局部的，而且延伸到N末端结构域的很大一部分，包括从α螺旋3到β链5的区域（“3445面”），并传播到活性位点。有趣的是，在磷酸化位点将天冬氨酸替换为谷氨酸的激活取代（D54E）也引发了3445面的结构变化。因此，活性位点和3445面似乎相互作用。本文讨论了磷酸化如何影响受体结构域的结构以及结构变化如何传递到NtrC其余部分的相关问题。
