Cabarca Sindy, Frazão de Souza Maximilia, Albert de Oliveira Andrew, Vignoli Muniz Gabriel S, Lamy M Teresa, Vinicius Dos Reis Caio, Takarada Jessica, Effer Brian, Souza Lucas Santos, Iriarte de la Torre Lilia, Couñago Rafael, Pinto Oliveira Cristiano Luis, Balan Andrea
Programa de Pós-graduação em Genética, Universidade Estadual de Campinas, Campinas, 13083-862, SP, Brazil.
Laboratório de Biologia Estrutural Aplicada LBEA, Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, 05508-000, São Paulo, SP, Brazil.
Curr Res Struct Biol. 2021 Jul 23;3:165-178. doi: 10.1016/j.crstbi.2021.07.001. eCollection 2021.
() has 11 Serine-Threonine Protein Kinases (STPK) that control numerous physiological processes, including cell growth, cell division, metabolic flow, and transcription. PknF is one of the 11 Mtb STPKs that has, among other substrates, two FHA domains (FHA-1 and FHA-2) of the ATP-Binding Cassette (ABC) transporter Rv1747. Phosphorylation in T152 and T210 located in a non-structured linker that connects Rv1747 FHA domains is considerate to be the regulatory mechanism of the transporter. In this work, we resolved the three-dimensional structure of the PknF catalytic domain (cPknF) in complex with the human kinase inhibitor IKK16. cPknF is conserved when compared to other STPKs but shows specific residues in the binding site where the inhibitor is positioned. In addition, using Small Angle X-Ray Scattering analysis we monitored the behavior of the wild type and three FHA-phosphomimetic mutants in solution, and measured the cPknF affinity for these domains. The kinase showed higher affinity for the non-phosphorylated wild type domain and preference for phosphorylation of T152 inducing the rapprochement of the domains and significant structural changes. The results shed some light on the process of regulating the transporter's activity by phosphorylation and arises important questions about evolution and importance of this mechanism for the bacillus.
（某物质）有11种丝氨酸 - 苏氨酸蛋白激酶（STPK），它们控制着众多生理过程，包括细胞生长、细胞分裂、代谢流和转录。PknF是11种结核分枝杆菌STPK之一，在其他底物中，它作用于ATP结合盒（ABC）转运蛋白Rv1747的两个FHA结构域（FHA - 1和FHA - 2）。位于连接Rv1747 FHA结构域的非结构化连接子中的T152和T210位点的磷酸化被认为是该转运蛋白的调节机制。在这项工作中，我们解析了与人类激酶抑制剂IKK16结合的PknF催化结构域（cPknF）的三维结构。与其他STPK相比，cPknF是保守的，但在抑制剂所在的结合位点显示出特定残基。此外，我们使用小角X射线散射分析监测了野生型和三个FHA磷酸模拟突变体在溶液中的行为，并测量了cPknF对这些结构域的亲和力。该激酶对非磷酸化的野生型结构域表现出更高的亲和力，并且倾向于T152的磷酸化，从而诱导结构域靠近并产生显著的结构变化。这些结果为通过磷酸化调节转运蛋白活性的过程提供了一些线索，并引发了关于该机制对杆菌的进化和重要性的重要问题。
