Phosphorylation-Competent Metastable State of Escherichia coli Toxin HipA

Abstract

Phosphorylation is a key post-translational modification that alters the functional state of many proteins. The Escherichia coli toxin HipA, which phosphorylates glutamyl-tRNA synthetase and triggers bacterial persistence under stress, becomes inactivated upon autophosphorylation of Ser150. Interestingly, Ser150 is phosphorylation-incompetent in the crystal structure of HipA since it is deeply buried ("in-state"), although in the phosphorylated state it is solvent exposed ("out-state"). To be phosphorylated, a minor population of HipA must exist in the phosphorylation-competent "out-state" (solvent-exposed Ser150), not detected in the crystal structure of unphosphorylated HipA.Here we report a molten-globule-like intermediate of HipA at low urea (∼4 kcal/mol unstable than natively folded HipA). The intermediate is aggregation-prone, consistent with a solvent exposed Ser150 and its two flanking hydrophobic neighbors (Val/Ile) in the "out-state". Molecular dynamics simulations showed the HipA "in−out" pathway to contain multiple free energy minima with an increasing degree of Ser150 solvent exposure with the free energy difference between the "in-state" and the metastable exposed state(s) to be ∼2−2.5 kcal/mol, with unique sets of hydrogen bonds and salt bridges associated with the metastable loop conformations. Together, the data clearly identify the existence of a phosphorylation-competent metastable state of HipA. Our results not only suggest a mechanism of HipA autophosphorylation but also add to a number of recent reports on unrelated protein systems where the common proposed mechanism for phosphorylation of buried residues is their transient exposure even without phosphorylation.

Bacterial persistence in Legionella pneumophila clinical isolates from patients with recurring legionellosis

“…This suggests that intracellular stress triggers the development of the persister subpopulation, which is in agreement with previous studies linking Lp quorum sensing and persister development during amoeba infection cycle ( Personnic et al., 2019a ). Stress-associated persistence has already been described with the HipBA toxin-antitoxin (TA) system in E. coli ( Correia et al., 2006 ; Pandey et al., 2023 ). More specifically, the complex regulatory function of HipBA ends in alarmone (p)ppGpp synthesis, activating a stringent response and resulting in dormancy and persistence.…”