Phosphorylation-Competent Metastable State of Escherichia coli Toxin HipA

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 (similar to 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 similar to 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.

Automated summary

Phosphorylation is a key post-translational modification that alters the functional state of many proteins. In the case of the Escherichia coli toxin HipA, autophosphorylation of Ser150 inactivates its ability to phosphorylate glutamyl-tRNA synthetase and trigger bacterial persistence. However, Ser150 is buried in the crystal structure of HipA and is not phosphorylation-competent. A molten-globule-like intermediate of HipA with a solvent-exposed Ser150 has been identified, suggesting a phosphorylation-competent metastable state. This study provides insights into the mechanism of HipA autophosphorylation and sheds light on the transient exposure of buried residues for phosphorylation.