The mechanisms of Z-ring assembly and regulation in bacteria are poorly understood, particularly in non-model organisms. Actinobacteria, a large bacterial phylum that includes the pathogen Mycobacterium tuberculosis, lack the canonical FtsZ-membrane anchors and Z-ring regulators described for E. coli. Here we investigate the physiological function of Corynebacterium glutamicum SepF, the only cell division-associated protein from Actinobacteria known to interact with the conserved C-terminal tail of FtsZ. We show an essential interdependence of FtsZ and SepF for formation of a functional Z-ring in C. glutamicum. The crystal structure of the SepF-FtsZ complex reveals a hydrophobic FtsZ-binding pocket, which defines the SepF homodimer as the functional unit, and suggests a reversible oligomerization interface. FtsZ filaments and lipid membranes have opposing effects on SepF polymerization, indicating that SepF has multiple roles at the cell division site, involving FtsZ bundling, Z-ring tethering and membrane reshaping activities that are needed for proper Z-ring assembly and function.

Once translocated into the cytosol of target cells, the catalytic domain (AC) of the adenylate cyclase toxin (CyaA), a major virulence factor of Bordetella pertussis, is potently activated by binding calmodulin (CaM) to produce supraphysiological levels of cAMP, inducing cell death. Using a combination of small-angle X-ray scattering (SAXS), hydrogen/deuterium exchange mass spectrometry (HDX-MS), and synchrotron radiation circular dichroism (SR-CD), we show that, in the absence of CaM, AC exhibits significant structural disorder, and a 75-residue-long stretch within AC undergoes a disorder-to-order transition upon CaM binding. Beyond this local folding, CaM binding induces long-range allosteric effects that stabilize the distant catalytic site, whilst preserving catalytic loop flexibility. We propose that the high enzymatic activity of AC is due to a tight balance between the CaM-induced decrease of structural flexibility around the catalytic site and the preservation of catalytic loop flexibility, allowing for fast substrate binding and product release. The CaM-induced dampening of AC conformational disorder is likely relevant to other CaM-activated enzymes.

The adenylate cyclase toxin CyaA is involved in the early stages of infection by Bordetella pertussis, the causative agent of whooping cough. CyaA intoxicates target cells by a direct translocation of its catalytic domain (AC) across the plasma membrane and produces supraphysiological levels of cAMP, leading to cell death. The molecular process of AC translocation remains largely unknown, however. We have previously shown that deletion of residues 375–485 of CyaA selectively abrogates AC translocation into eukaryotic cells. We further identified within this “translocation region” (TR), P454 (residues 454–484), a peptide that exhibits membrane-active properties, i.e., is able to bind and permeabilize lipid vesicles. Here, we analyze various sequences from CyaA predicted to be amphipatic and show that although several of these peptides can bind membranes and adopt a helical conformation, only the P454 peptide is able to permeabilize membranes. We further characterize the contributions of the two arginine residues of P454 to membrane partitioning and permeabilization by analyzing the peptide variants in which these residues are substituted by different amino acids (e.g., A, K, Q, and E). Our data shows that both arginine residues significantly contribute, although diversely, to the membrane-active properties of P454, i.e., interactions with both neutral and anionic lipids, helix formation in membranes, and disruption of lipid bilayer integrity. These results are discussed in the context of the translocation process of the full-length CyaA toxin.

The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells are still a matter of intense research. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. The CyaA translocation region contains a segment, P454 (residues 454–484), which exhibits membrane‐active properties related to antimicrobial peptides. Herein, the results show that this peptide is able to translocate across membranes and to interact with calmodulin (CaM). Structural and biophysical analyses reveal the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrates that these residues play a crucial role in CyaA translocation into target cells. In addition, calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. It is proposed that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of CyaA by the CaM:P454 interaction in the cytosol may assist the entry of the N‐terminal catalytic domain by converting the stochastic motion of the polypeptide chain through the membrane into an efficient vectorial chain translocation into host cells.

The adenylate cyclase (CyaA) toxin is a major virulence factor of Bordetella pertussis, the causative agent of whooping cough. CyaA is synthetized as a pro‐toxin, pro‐CyaA, and converted into its cytotoxic form upon acylation of two lysines. After secretion, CyaA invades eukaryotic cells and produces cAMP, leading to host defense subversion. To gain further insights into the effect of acylation, we compared the functional and structural properties of pro‐CyaA and CyaA proteins. HDX‐MS results show that the refolding process of both proteins upon progressive urea removal is initiated by calcium binding to the C‐terminal RTX domain. We further identified a critical hydrophobic segment, distal from the acylation region, that folds at higher urea concentration in CyaA than in pro‐CyaA. Once refolded into monomers, CyaA is more compact and stable than pro‐CyaA, due to a complex set of interactions between domains. Our HDX‐MS data provide direct evidence that the presence of acyl chains in CyaA induces a significant stabilization of the apolar segments of the hydrophobic domain and of most of the acylation region. We propose a refolding model dependent on calcium and driven by local and distal acylation‐dependent interactions within CyaA. Therefore, CyaA acylation is not only critical for cell intoxication, but also for protein refolding into its active conformation. Our data shed light on the complex relationship between post‐translational modifications, structural disorder and protein folding. Coupling calcium‐binding and acylation‐driven folding is likely pertinent for other repeat‐in‐toxin cytolysins produced by many Gram‐negative bacterial pathogens.—O'Brien, D. P., Cannella, S. E., Voegele, A., Raoux‐Barbot, D., Davi, M., Douché, T., Matondo, M., Brier, S., Ladant, D., Chenal, A. Post‐translational acylation controls the folding and functions of the CyaA RTX toxin. FASEB J. 33, 10065–10076 (2019). http://www.fasebj.org

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Bacterial kinesin light chain is a TPR domain-containing protein encoded by the bklc gene, which co-localizes with the bacterial tubulin (btub) genes in a conserved operon in Prosthecobacter. Btub heterodimers show high structural homology with eukaryotic tubulin and assemble into head-to-tail protofilaments. Intriguingly, Bklc is homologous to the light chain of the microtubule motor kinesin and could thus represent an additional eukaryotic-like cytoskeletal element in bacteria. Using biochemical characterization as well as cryo-electron tomography we show here that Bklc interacts specifically with Btub protofilaments, as well as lipid vesicles and could thus play a role in anchoring the Btub filaments to the membrane protrusions in Prosthecobacter where they specifically localize in vivo. This work sheds new light into possible ways in which the microtubule cytoskeleton may have evolved linking precursors of microtubules to the membrane via the kinesin moiety that in today’s eukaryotic cytoskeleton links vesicle-packaged cargo to microtubules.