Phosphorylation of histidine kinase CpxA is not cooperative 1 Structural asymmetry does not indicate hemi-phosphorylation 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 of a single conserved histidine residue in the individual protomers, leading to di-phosphorylation. 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 phosphorylated species: unphosphorylated, monoand di-phosphorylated 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 hemi-phosphorylation. 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. _______________________________________ Two-component systems (TCSs), the prevalent signaling pathways in bacteria, are one of the best studied models of signal transduction schemes (1,2). A conserved phosphotransfer reaction between the sensor histidine kinase (HK) and the response regulator (RR) is used to couple a large variety of inputs and outputs. HKs, usually functioning as homodimers, sense stimuli through their variable sensing domains and modulate phosphorylation levels of their cognate RRs. HKs display multiple enzyme activities, including autokinase, phosphotransferase that is catalyzed in conjunction with the RR protein, and often phosphatase activity toward the phosphorylated cognate RR. The conserved catalytic core of an HK consists of a dimerization and histidine phosphotransfer (DHp) domain that contains the https://www.jbc. org/cgi/doi/10.1074/jbc.RA120.012757 The latest version is at JBC Papers in Press. Published on February 24, 2020 as Manuscript RA120.012757 by gest on M ay 7, 2020 hp://w w w . jb.org/ D ow nladed from Phosphorylation of histidine kinase CpxA is not cooperative 2 conserved histidine residue for receiving and transferring the phosphoryl group, and a catalytic ATP-binding (CA) domain that contains residues critical for kinase activity. Structures of HKs and HK-RR complexes representing different enzymatic states have been captured, providing mechanistic details of HK activities. Signaldependent regulatory mechanisms have been investigated for HKs in response to ligand binding (3-6) or light sensing (7,8). The structures and mechanisms have been extensively reviewed (2,912). Distinct structural features are often linked to individual catalytic functions and biochemical behaviors of HKs. Except for several atypical HKs with unusual oligomeric or monomeric states (13-15), one predominant emerging theme in HK signaling is the symmetry/asymmetry transitions in HK dimer structures. The symmetric or asymmetric HK structures involve similar or different arrangements of structural elements between individual protomers within an HK dimer. Structures with both symmetric and asymmetric conformations have been observed in the periplasmic sensing domains (3-5), the signal-transducing transmembrane helices and HAMP domains (6), and the catalytic core domains (16-20). Transitions between the two conformations have often been associated with signal-dependent switching of HK activities (16-18). Bhate and colleagues (10) analyzed more than 20 HK structures and revealed a general trend of symmetry/asymmetry transitions in different HKs. Symmetry or asymmetry in the catalytic core domains refers to the packing of DHp helices and the relative positioning of the two CA domains. While the phosphatase state of an HK is often symmetric, with two CA domains held at positions unfavorable for phosphorylation, the autokinase state harbors an asymmetric conformation with one CA domain in close proximity of one phosphorylatable histidine whereas the other CA domain is located far from the other histidine. The CA domain that provides the reactive ATP and the histidine receiving the phosphoryl group can be from the same or different protomers, resulting in a cis or a trans phosphorylation mechanism in different HKs. Nevertheless, both mechanisms make use of similar asymmetric conformations that only allow phosphorylation of one histidine at a time (16,17,19,21,22). Such asymmetric structures are often intuitively associated with asymmetric phosphorylation, in which phosphorylation of one protomer hinders phosphorylation of the second protomer, leading to hemi-phosphorylation in the extreme case, with a single phosphorylation event per dimer. For example, observation of asymmetric conformations in CpxA led to implication of hemiphosphorylation despite the experimental observation of a 70% total phosphorylation level, where the phosphorylation exceeding 50% was attributed to subunit exchange of protomers within HK dimers (17). As structural asymmetry is repeatedly demonstrated for the kinase state, is phosphorylation asymmetry also a general trend in HKs? Asymmetric phosphorylation, or negative cooperativity, has been biochemically explored in several HKs (19,23,24). Early studies on NRII indicated that the equilibrium constant for phosphorylation of the first protomer is ~78 fold larger than that of the second protomer (23). Negative cooperativity has been attributed to an exceptionally rapid reverse reaction with transfer of the phosphoryl group from the di-phosphorylated HK to ADP generating the hemi-phosphorylated HK, thus ADP has a large inhibitory effect on diphosphorylation of HK dimers. Hemiphosphorylated HKs have also been observed on native gels in studies of HK853 (19). A complete di-phosphorylation of HK853 dimers was not achieved unless ADP present in the reaction mixture was continuously recycled back to ATP. An even stronger negative cooperativity has been demonstrated for DesK (24). The observed phosphorylation stoichiometry of DesK was ~0.5 and full phosphorylation was not achieved even in the presence of a coupled enzyme system that continuously converted ADP to ATP, indicating an extremely high negative cooperativity. Such extreme negative cooperativity may be uncommon for HKs because full phosphorylation at high ATP levels or in the presence of ATP regeneration has been documented for many HKs (19,23,25,26). On the other hand, analyses of the hybrid HK ShkA indicates no cooperativity in autophosphorylation (25). Biochemical analyses of negative cooperativity of phosphorylation often rely on deriving equilibrium constants by measuring HK phosphorylation levels across a wide range of ATP by gest on M ay 7, 2020 hp://w w w . jb.org/ D ow nladed from Phosphorylation of histidine kinase CpxA is not cooperative 3 concentrations. However, likely due to the spontaneous hydrolysis of ATP, even the “high purity” commercial ATP reagent contains trace amount of contaminating ADP (26-28). The initial amount of ADP, relative to that generated during the phosphorylation reaction, is non-trivial when high concentrations of ATP are used. Under such conditions, the inhibitory effect of the contaminating ADP might not be negligible, as presumed in prior analyses, and could impact interpretation of negative cooperativity. Here we take account of the initial ADP concentration in the kinetic equilibrium modeling and show that the neglect of ADP contamination can lead to significant overestimation of negative cooperativity. We develop a strategy to examine the phosphorylation cooperativity by measuring the un, monoand di-phosphorylated HK dimer species using a covalently linked HK dimer. Applying this strategy to E. coli CpxA, we demonstrate that autophosphorylation of CpxA is not negatively cooperative, or asymmetric, despite the structural asymmetry.