Katharine A. Michie

Localized Dimerization and Nucleoid Binding Drive Gradient Formation by the Bacterial Cell Division Inhibitor MipZ

Summary Protein gradients play a central role in the spatial organization of cells, but the mechanisms of their formation are incompletely understood. This study analyzes the determinants responsible for establishing bipolar gradients of the ATPase MipZ, a key regulator of division site placement in Caulobacter crescentus. We have solved the crystal structure of MipZ in different nucleotide states, dissected its ATPase cycle, and investigated its interaction with FtsZ, ParB, and the nucleoid. Our results suggest that the polar ParB complexes locally stimulate the formation of ATP-bound MipZ dimers, which are then retained near the cell poles through association with chromosomal DNA. Due to their intrinsic ATPase activity, dimers eventually dissociate into freely diffusible monomers that undergo spontaneous nucleotide exchange and are recaptured by ParB. These findings clarify the molecular function of a conserved gradient-forming system and reveal mechanistic principles that might be commonly used to sustain protein gradients within cells.

Filament structure of bacterial tubulin homologue TubZ

Low copy number plasmids often depend on accurate partitioning systems for their continued survival. Generally, such systems consist of a centromere-like region of DNA, a DNA-binding adaptor, and a polymerizing cytomotive filament. Together these components drive newly replicated plasmids to opposite ends of the dividing cell. The Bacillus thuringiensis plasmid pBToxis relies on a filament of the tubulin/FtsZ-like protein TubZ for its segregation. By combining crystallography and electron microscopy, we have determined the structure of this filament. We explain how GTP hydrolysis weakens the subunit–subunit contact and also shed light on the partitioning of the plasmid–adaptor complex. The double helical superstructure of TubZ filaments is unusual for tubulin-like proteins. Filaments of ParM, the actin-like partitioning protein, are also double helical. We suggest that convergent evolution shapes these different types of cytomotive filaments toward a general mechanism for plasmid separation.

A positively charged channel within the Smc1/Smc3 hinge required for sister chromatid cohesion

Cohesins structural maintenance of chromosome 1 (Smc1) and Smc3 are rod‐shaped proteins with 50‐nm long intra‐molecular coiled‐coil arms with a heterodimerization domain at one end and an ABC‐like nucleotide‐binding domain (NBD) at the other. Heterodimerization creates V‐shaped molecules with a hinge at their centre. Inter‐connection of NBDs by Scc1 creates a tripartite ring within which, it is proposed, sister DNAs are entrapped. To investigate whether cohesins hinge functions as a possible DNA entry gate, we solved the crystal structure of the hinge from Mus musculus, which like its bacterial counterpart is characterized by a pseudo symmetric heterodimeric torus containing a small channel that is positively charged. Mutations in yeast Smc1 and Smc3 that together neutralize the channels charge have little effect on dimerization or association with chromosomes, but are nevertheless lethal. Our finding that neutralization reduces acetylation of Smc3, which normally occurs during replication and is essential for cohesion, suggests that the positively charged channel is involved in a major conformational change during S phase.

The Bacillus subtilis cell division proteins FtsL and DivIC are intrinsically unstable and do not interact with one another in the absence of other septasomal components

The bacterial septum appears to comprise a macromolecular assembly of essential cell division proteins (the ‘septasome’) that are responsible for physically dividing the cell during cytokinesis. FtsL and DivIC are essential components of this division machinery in Bacillus subtilis. We used yeast two‐hybrid analysis as well as a variety of biochemical and biophysical methods to examine the proposed interaction between Bacillus subtilis FtsL and DivIC. We show that FtsL and DivIC are thermodynamically unstable proteins that are likely to be unfolded and therefore targeted for degradation unless stabilized by interactions with other components of the septasome. However, we show that this stabilization does not result from a direct interaction between FtsL and DivIC. We propose that the observed interdepend‐ence of DivIC and FtsL stability is a result of indirect interactions that are mediated by other septasomal proteins.

CetZ tubulin-like proteins control archaeal cell shape

Tubulin is a major component of the eukaryotic cytoskeleton, controlling cell shape, structure and dynamics, whereas its bacterial homologue FtsZ establishes the cytokinetic ring that constricts during cell division. How such different roles of tubulin and FtsZ evolved is unknown. Studying Archaea may provide clues as these organisms share characteristics with Eukarya and Bacteria. Here we report the structure and function of proteins from a distinct family related to tubulin and FtsZ, named CetZ, which co-exists with FtsZ in many archaea. CetZ X-ray crystal structures showed the FtsZ/tubulin superfamily fold, and one crystal form contained sheets of protofilaments, suggesting a structural role. However, inactivation of CetZ proteins in Haloferax volcanii did not affect cell division. Instead, CetZ1 was required for differentiation of the irregular plate-shaped cells into a rod-shaped cell type that was essential for normal swimming motility. CetZ1 formed dynamic cytoskeletal structures in vivo, relating to its capacity to remodel the cell envelope and direct rod formation. CetZ2 was also implicated in H. volcanii cell shape control. Our findings expand the known roles of the FtsZ/tubulin superfamily to include archaeal cell shape dynamics, suggesting that a cytoskeletal role might predate eukaryotic cell evolution, and they support the premise that a major function of the microbial rod shape is to facilitate swimming.

LeoA, B and C from Enterotoxigenic Escherichia coli (ETEC) Are Bacterial Dynamins

Escherichia coli (ETEC) strain H10407 contains a GTPase virulence factor, LeoA, which is encoded on a pathogenicity island and has been shown to enhance toxin release, potentially through vesicle secretion. By sequence comparisons and X-ray structure determination we now identify LeoA as a bacterial dynamin-like protein (DLP). Proteins of the dynamin family remodel membranes and were once thought to be restricted to eukaryotes. In ETEC H10407 LeoA localises to the periplasm where it forms a punctate localisation pattern. Bioinformatic analyses of leoA and the two upstream genes leoB and leoC suggest that LeoA works in concert with a second dynamin-like protein, made up of LeoB and LeoC. Disruption of the leoAB genes leads to a reduction in secretion of periplasmic Tat-GFP and outer membrane OmpA. Our data suggest a role for LeoABC dynamin-like proteins in potentiating virulence through membrane vesicle associated toxin secretion.

A Highly Conserved Yet Flexible Linker Is Part of a Polymorphic Protein-Binding Domain in Myosin-Binding Protein C

The nuclear magnetic resonance (NMR) structure of the tri-helix bundle (THB) of the m-domain plus C2 (ΔmC2) of myosin-binding protein C (MyBP-C) has revealed a highly flexible seven-residue linker between the structured THB and C2. Bioinformatics shows significant patterns of conservation across the THB-linker sequence, with the linker containing a strictly conserved serine in all MyBP-C isoforms. Clinically linked mutations further support the functional significance of the THB-linker region. NMR, small-angle X-ray scattering, and binding studies show the THB-linker plus the first ten residues of C2 undergo dramatic changes when ΔmC2 binds Ca2+-calmodulin, with the linker and C2 N-terminal residues contributing significantly to the affinity. Modeling of all available experimental data indicates that the THB tertiary structure must be disrupted to form the complex. These results are discussed in the context of the THB-linker and the N-terminal residues of C2 forming a polymorphic binding domain that could accommodate multiple binding partners in the dynamic sarcomere.

Clinically Linked Mutations in the Central Domains of Cardiac Myosin-Binding Protein C with Distinct Phenotypes Show Differential Structural Effects

The structural effects of three missense mutations clinically linked to hypertrophic cardiomyopathy (HCM) and located in the central domains of cardiac myosin-binding protein C (cMyBP-C) have been determined using small-angle scattering, infrared spectroscopy, and nuclear magnetic resonance spectroscopy. Bioinformatics and modeling were used to initially predict the expected structural impacts and assess the broader implications for function based on sequence conservation patterns. The experimental results generally affirm the predictions that two of the mutations (D745G, P873H) disrupt domain folding, while the third (R820Q) is likely to be entirely solvent exposed and thus more likely to have its impact through its interactions within the sarcomere. Each of the mutations is associated with distinct disease phenotypes, with respect to severity, stage of onset, and end phase. The results are discussed in terms of understanding key structural features of these domains essential for healthy function and the role they may play in disease development.

Molecular Components of the Bacterial Cytoskeleton

It is only relatively recently that a prokaryotic cytoskeleton akin to that in eukaryotes has been identified, revealing a much higher order of cellular complexity than was previously thought. The proteins that form these bacterial cytoskeletal elements not only carry out similar roles to their eukaryotic counterparts, but they also have related protein folds, suggesting an ancient evolutionary relationship and Katharine A. Michie Medical Research Council Laboratory of Molecular Biology, Hills Rd, Cambridge CB2 0QH, UK kmichie@mrc-lmb.cam.ac.uk 2.