Salar Safoura, Silletti Steve, Schubot Florian D
Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA.
Department of Chemistry and Biochemistry, University of California, San Diego, California, USA.
J Biol Chem. 2025 Apr 23;301(6):108521. doi: 10.1016/j.jbc.2025.108521.
Membrane-embedded signaling histidine kinases (SKs) from two-component and phosphorelay signal transduction systems play central roles in the gene regulation of bacteria, fungi, and plants. The SK GacS is a global regulator of gene expression in the human pathogen Pseudomonas aeruginosa. The interactions between GacS and another SK, RetS, are a model for studying non-canonical crosstalk in multikinase networks. During planktonic growth, RetS inhibits GacS to upregulate expression of virulence factors associated with acute P. aeruginosa infections and repress genes linked to chronic infection. Conversely, GacS activation promotes biofilm formation and chronic infection but suppresses factors required during acute infection. Using a combination of hydrogen-deuterium exchange mass spectrometry (HDX-MS) and mutational analysis in conjunction with functional assays, we show that binding of an extracellular ligand promotes GacS signaling through two mechanisms: (1) by increasing GacS autokinase activity and (2) by decreasing the affinity between GacS and RetS. Intriguingly, RetS binding to the intracellular histidine kinase domain of GacS also triggered conformational changes in the extracellular sensory domain of GacS. This allosteric effect was confirmed in a biochemical assay, showing RetS increases the affinity of a chimeric CitAGacS receptor for citrate by almost tenfold. This finding establishes the first precedent of inside-out cross-membrane signaling in SK systems. Taken together, our data are consistent with a model wherein RetS binding primes GacS for signal sensing during planktonic growth. Binding of the unknown ligand at the onset of biofilm formation causes dissociation of the RetS-GacS complex to lock GacS in a kinase ON conformation.
来自双组分和磷酸转移信号转导系统的膜嵌入信号组氨酸激酶（SKs）在细菌、真菌和植物的基因调控中发挥着核心作用。SK GacS是人类病原体铜绿假单胞菌中基因表达的全局调节因子。GacS与另一种SK RetS之间的相互作用是研究多激酶网络中非典型串扰的模型。在浮游生长期间，RetS抑制GacS，以上调与急性铜绿假单胞菌感染相关的毒力因子的表达，并抑制与慢性感染相关的基因。相反，GacS的激活促进生物膜形成和慢性感染，但抑制急性感染期间所需的因子。通过结合氢-氘交换质谱（HDX-MS）、突变分析和功能测定，我们表明细胞外配体的结合通过两种机制促进GacS信号传导：（1）通过增加GacS自身激酶活性；（2）通过降低GacS与RetS之间的亲和力。有趣的是，RetS与GacS的细胞内组氨酸激酶结构域的结合也引发了GacS细胞外传感结构域的构象变化。这种变构效应在生化测定中得到证实，表明RetS使嵌合CitAGacS受体对柠檬酸盐的亲和力增加了近10倍。这一发现确立了SK系统中由内向外跨膜信号传导的首个先例。综上所述，我们的数据与一个模型一致，即在浮游生长期间，RetS的结合使GacS为信号传感做好准备。在生物膜形成开始时未知配体的结合导致RetS-GacS复合物解离，从而将GacS锁定在激酶激活构象中。
