Die P-loop ATPase MipZ - Mechanismus der Bildung eines Proteingradienten in einer prokaryotischen Zelle

Protein gradients have a key role in the spatial regulation of biological processes, thereby contributing to the complexity of both prokaryotic and eukaryotic organisms. In eukaryotes, intracellular protein and protein phosphorylation gradients were shown to be involved in embryonic development, mitotic spindle morphogenesis and cell division. Similarly, phosphorylation gradients are critical for the establishment of asymmetry in prokaryotes. However, well-studied examples for protein concentration gradients are still rare for these organisms. At the scale of a small prokaryotic cell, intracellular concentration gradients have long been assumed to be unsustainable due to the process of diffusion. Nevertheless, steady-state concentration gradients can be maintained in bacteria, as exemplified by the bipolar gradient of the P-loop-ATPase MipZ which is required for proper division site placement in the α-proteobacterium Caulobacter crescentus. MipZ interacts with a kinetochore-like nucleoprotein complex formed by the chromosome segregation protein ParB in the vicinity of the chromosomal origin of replication. Upon entry into S-phase, the two newly duplicated origin regions are partitioned and sequestered to opposite cell poles, resulting in a bipolar distribution of MipZ with a defined concentration minimum at mid-cell. Acting as a direct inhibitor of the essential cell division protein FtsZ, MipZ thus restricts cytokinesis to the cell center. In this study, the mechanism underlying the formation of the MipZ gradient was analyzed. Based on the crystal structures of the apo and ATP-bound protein and by means of mutant variants of MipZ, I dissected the role of nucleotide binding and hydrolysis. Gradient formation is found to rely on nucleotide-regulated alternation of MipZ between a monomeric and dimeric form. MipZ monomers interact with ParB, which results in the recruitment of MipZ to the polar regions of the cell. Upon ATP binding, MipZ dimerizes and is converted into its biologically active form that inhibits FtsZ assembly. Moreover, diffusion of the dimer is decelerated by its association with the nucleoid. The MipZ gradient can thus be envisioned as an asymmetric distribution of dimers that are released from a polar pool and slowly diffuse towards mid-cell. By virtue of the marked differences in the interaction networks and diffusion rates of monomers and dimers, ATP hydrolysis promotes oscillation of MipZ between the polar ParB complexes and pole-distal regions of the nucleoid. The MipZ gradient thus represents the steady-state distribution of molecules in a highly dynamic system, providing a general mechanism for the establishment of protein gradients within the confined space of the bacterial cytoplasm. The generation of a concentration gradient by a P-loop-ATPase of the Mrp/MinD family exemplifies the diverse regulatory strategies and interaction networks that are used by different family members to fulfill their particular function in the cell. Concurrently, the unique features of MipZ identify it as the member of a novel subfamily of Mrp/MinD proteins.