Protein phosphorylation is a universal regulatory mechanism, controlling virtually all aspects of bacterial physiology and pathogenesis, yet histidine phosphorylation remains among the least understood. The histidine phosphocarrier protein HPr not only drives bacterial glucose transmembrane uptake through the phosphotransferase system (PTS), but also controls key enzymes for central carbon metabolism, including glycogen phosphorylase (GP). Here we report cryoEM structures of multimeric Escherichia coli EcGP and their complexes with EcHPr. The EM maps reveal an unanticipated density at H806 of EcGP, consistent with histidine phosphorylation within a histidine-rich pocket at the N-terminal domain. Enzymatic assays reveal that EcHPr transfers a phosphoryl group to the N1 position of a histidine residue in EcGP. Through an integrative structural, mutational and functional approach, we uncover the molecular basis of EcHPr- EcGP selectivity and define the allosteric mechanism by which EcHPr regulates EcGP. We establish histidine phosphorylation as a mechanism of GP regulation, expanding the traditional paradigm of glycogen metabolism control in bacteria. Glycogen serves as an intracellular energy storage polymer while some bacteria also produce structurally diverse extracellular α-glucans. Here, the authors establish the molecular basis of phosphorylation and allosteric regulation of glycogen phosphorylase by histidine phosphocarrier protein HPr.
Domenico et al. (Mon,) studied this question.