Role of Unusual P Loop Ejection and Autophosphorylation in HipA-Mediated Persistence and Multidrug Tolerance

SummaryHipA is a bacterial serine/threonine protein kinase that phosphorylates targets, bringing about persistence and multidrug tolerance. Autophosphorylation of residue Ser150 is a critical regulatory mechanism of HipA function. Intriguingly, Ser150 is not located on the activation loop, as are other kinases; instead, it is in the protein core, where it forms part of the ATP-binding “P loop motif.” How this buried residue is phosphorylated and regulates kinase activity is unclear. Here, we report multiple structures that reveal the P loop motif's exhibition of a remarkable “in-out” conformational equilibrium, which allows access to Ser150 and its intermolecular autophosphorylation. Phosphorylated Ser150 stabilizes the “out state,” which inactivates the kinase by disrupting the ATP-binding pocket. Thus, our data reveal a mechanism of protein kinase regulation that is vital for multidrug tolerance and persistence, as kinase inactivation provides the critical first step in allowing dormant cells to revert to the growth phenotype and to reinfect the host.

Graphical AbstractFigure optionsDownload full-size imageDownload as PowerPoint slideHighlights► Dormant bacterial cells called persisters are the origin of multidrug tolerance (MDT) ► HipA is an MDT kinase that is regulated by autophosphorylation of residue S150 ► S150 is the phosphorylated target but is buried in the hydrophobic core ► When ejected, S150 is phosphorylated and destroys the catalytic P loop structure