Structural asymmetry does not indicate hemiphosphorylation in the bacterial histidine kinase CpxA.

摘要: Histidine protein kinases (HKs) are prevalent prokaryotic sensor that central to phosphotransfer in two-component signal transduction systems, regulating phosphorylation of response regulator proteins determine the output responses. HKs typically exist as dimers and can potentially autophosphorylate at each conserved histidine residue individual protomers, leading diphosphorylation. However, analyses HK biochemical assays vitro suggest negative cooperativity, whereby one protomer dimer inhibits second protomer, ∼50% available sites dimers. This cooperativity is often correlated with an asymmetric domain arrangement, a common structural characteristic autophosphorylation states many structures. In this study, we engineered covalent cytoplasmic domains Escherichia coli CpxA, enabling us quantify species: unphosphorylated, monophosphorylated, diphosphorylated Together mathematical modeling, unambiguously demonstrate no CpxA despite its structures, indicating these arrangements not linked hemiphosphorylation. Furthermore, modeling indicated parameters, most notably minor amounts ADP generated during reactions or present ATP preparations, produce total may be mistakenly attributed cooperativity. study also establishes heterodimer provides robust experimental system for investigating offers useful tool testing how symmetric features influence functions.