Oliveira Marco A S, Gerhardt Edileusa C M, Huergo Luciano F, Souza Emanuel M, Pedrosa Fábio O, Chubatsu Leda S
Department of Biochemistry and Molecular Biology, Universidade Federal do Parana, Curitiba, Brazil.
FEBS J. 2015 Dec;282(24):4797-809. doi: 10.1111/febs.13542. Epub 2015 Oct 24.
Nitrogen metabolism in Proteobacteria is controlled by the Ntr system, in which PII proteins play a pivotal role, controlling the activity of target proteins in response to the metabolic state of the cell. Characterization of the binding of molecular effectors to these proteins can provide information about their regulation. Here, the binding of ATP, ADP and 2-oxoglutarate (2-OG) to the Herbaspirillum seropedicae PII proteins, GlnB and GlnK, was characterized using isothermal titration calorimetry. Results show that these proteins can bind three molecules of ATP, ADP and 2-OG with homotropic negative cooperativity, and 2-OG binding stabilizes the binding of ATP. Results also show that the affinity of uridylylated forms of GlnB and GlnK for nucleotides is significantly lower than that of the nonuridylylated proteins. Furthermore, fluctuations in the intracellular concentration of 2-OG in response to nitrogen availability are shown. Results suggest that under nitrogen-limiting conditions, PII proteins tend to bind ATP and 2-OG. By contrast, after an ammonium shock, a decrease in the 2-OG concentration is observed causing a decrease in the affinity of PII proteins for ATP. This phenomenon may facilitate the exchange of ATP for ADP on the ligand-binding pocket of PII proteins, thus it is likely that under low ammonium, low 2-OG levels would favor the ADP-bound state.
变形菌门中的氮代谢由Ntr系统控制，其中PII蛋白起着关键作用，它根据细胞的代谢状态控制靶蛋白的活性。对分子效应物与这些蛋白结合的特性进行表征可以提供有关其调控的信息。在此，使用等温滴定量热法对ATP、ADP和2-氧代戊二酸（2-OG）与草螺菌属血清型PII蛋白GlnB和GlnK的结合进行了表征。结果表明，这些蛋白能以同促负协同性结合三个分子的ATP、ADP和2-OG，并且2-OG的结合能稳定ATP的结合。结果还表明，尿苷酸化形式的GlnB和GlnK对核苷酸的亲和力明显低于未尿苷酸化的蛋白。此外，还显示了细胞内2-OG浓度随氮供应情况的波动。结果表明，在氮限制条件下，PII蛋白倾向于结合ATP和2-OG。相比之下，铵冲击后，观察到2-OG浓度降低，导致PII蛋白对ATP的亲和力下降。这种现象可能有助于PII蛋白配体结合口袋上的ATP与ADP交换，因此在低铵、低2-OG水平下可能有利于ADP结合状态。
