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

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