Cell Division Regulators MinC and MinD Form Polymers in the Presence of Nucleotide

The Min system of proteins, comprised of MinC, MinD and MinE, is essential for normal cell division in Escherichia coli. MinC forms an oscillating intracellular gradient near the poles that restricts placement of the division septum at midcell by inhibiting polymerization of FtsZ, the major component of the septal ring. MinC localization is directed by MinD, a Par-like ATPase that binds to the membrane in the presence of ATP. MinE functions as a regulator of MinD by stimulating MinD ATP hydrolysis, which leads to membrane dissociation. Results using purified proteins in vitro show that MinC stabilizes a conformational change in MinD in the presence of ATP characterized by the formation of large oligomers, which are detectable in light scattering and ultracentrifugation assays. Electron microscopy of MinC-MinD complexes formed with ATP show long polymers (300-500 nm) that are approximately 10-20 nm wide. Increasing the concentration of MinC in polymerization assays with MinD and ATP enhances oligomer formation. MinCD complexes assemble in the presence of the ATP analog ATPγS, but not ADP, suggesting that MinD is likely in an ATP-bound conformation. Using site-directed mutagenesis, we constructed a MinD mutant protein defective for ATP-dependent dimerization and a truncated MinD mutant protein lacking the membrane targeting sequence (MTS); both MinD mutant proteins are unable to polymerize with MinC. Additionally, the in vitro activity of MinC to prevent GTP-dependent FtsZ pelleting in sedimentation assays is impaired by MinD and ATP, suggesting polymerization with MinD may modulate MinC function. Finally, addition of MinE to MinC-MinD polymers induces concentration-dependent disassembly manner suggesting MinE competes with MinC for binding to MinD. Results suggest a novel nucleotide-dependent conformation of MinD with MinC and provides mechanistic insight into the functional interactions of the Min proteins.