Structural analyses unravel the molecular mechanism of cyclic di-GMP regulation of bacterial chemotaxis via a PilZ adaptor protein.

The bacterial second messenger cyclic di-GMP (c-di-GMP) has emerged as a prominent mediator of bacterial physiology, motility, and pathogenicity. c-di-GMP often regulates the function of its protein targets through a unique mechanism that involves a discrete PilZ adaptor protein. However, the molecular mechanism for PilZ protein-mediated protein regulation is unclear. Here, we present the structure of the PilZ adaptor protein MapZ cocrystallized in complex with c-di-GMP and its protein target CheR1, a chemotaxis-regulating methyltransferase in This cocrystal structure, together with the structure of free CheR1, revealed that the binding of c-di-GMP induces dramatic structural changes in MapZ that are crucial for CheR1 binding. Importantly, we found that restructuring and repositioning of two C-terminal helices enable MapZ to disrupt the CheR1 active site by dislodging a structural domain. The crystallographic observations are reinforced by protein-protein binding and single cell-based flagellar motor switching analyses. Our studies further suggest that the regulation of chemotaxis by c-di-GMP through MapZ orthologs/homologs is widespread in proteobacteria and that the use of allosterically regulated C-terminal motifs could be a common mechanism for PilZ adaptor proteins. Together, the findings provide detailed structural insights into how c-di-GMP controls the activity of an enzyme target indirectly through a PilZ adaptor protein.