Unravelling HetC as a peptidase-based ABC exporter driving functional cell differentiation in the cyanobacterium Nostoc PCC 7120.

The export of peptides or proteins is essential for a variety of important functions in bacteria. Among the diverse protein-translocation systems, peptidase-containing ABC transporters (PCAT) are involved in the maturation and export of quorum-sensing or antimicrobial peptides in Gram-positive bacteria and of toxins in Gram-negative organisms. In the multicellular and diazotrophic cyanobacterium Nostoc PCC 7120, the protein HetC is essential for the differentiation of functional heterocysts, which are micro-oxic and non-dividing cells specialized in atmospheric nitrogen fixation. HetC shows similarities to PCAT systems, but whether it actually acts as a peptidase-based exporter remains to be established. In this study, we show that the N-terminal part of HetC, encompassing the peptidase domain, displays a cysteine-type protease activity. The conserved catalytic residues conserved in this family of proteases are essential for the proteolytic activity of HetC and the differentiation of heterocysts. Furthermore, we show that the catalytic residue of the ATPase domain of HetC is also essential for cell differentiation. Interestingly, HetC has a cyclic nucleotide-binding domain at its N-terminus which can bind ppGpp in vitro and which is required for its function in vivo. Our results indicate that HetC is a peculiar PCAT that might be regulated by ppGpp to potentially facilitate the export of a signaling peptide essential for cell differentiation, thereby broadening the scope of PCAT role in Gram-negative bacteria.IMPORTANCEBacteria have a great capacity to adapt to various environmental and physiological conditions; it is widely accepted that their ability to produce extracellular molecules contributes greatly to their fitness. Exported molecules are used for a variety of purposes ranging from communication to adjust cellular physiology, to the production of toxins that bacteria secrete to fight for their ecological niche. They use export machineries for this purpose, the most common of which energize transport by hydrolysis of adenosine triphosphate. Here, we demonstrate that such a mechanism is involved in cell differentiation in the filamentous cyanobacterium Nostoc PCC 7120. The HetC protein belongs to the ATP-binding cassette transporter superfamily and presumably ensures the maturation of a yet unknown substrate during export. These results open interesting perspectives on cellular signaling pathways involving the export of regulatory peptides, which will broaden our knowledge of how these bacteria use two cell types to conciliate photosynthesis and nitrogen fixation.

The transport activity of the multidrug ABC transporter BmrA does not require a wide separation of the nucleotide-binding domains.

ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins responsible for the translocation of a wide diversity of substrates across biological membranes. Some of them confer multidrug or antimicrobial resistance to cancer cells and pathogenic microorganisms, respectively. Despite a wealth of structural data gained in the last two decades, the molecular mechanism of these multidrug efflux pumps remains elusive, including the extent of separation between the two nucleotide-binding domains (NBDs) during the transport cycle. Based on recent outward-facing structures of BmrA, a homodimeric multidrug ABC transporter from Bacillus subtilis, we introduced a cysteine mutation near the C-terminal end of the NBDs to analyze the impact of disulfide-bond formation on BmrA function. Interestingly, the presence of the disulfide bond between the NBDs did not prevent the ATPase, nor did it affect the transport of Hoechst 33342 and doxorubicin. Yet, the 7-amino-actinomycin D was less efficiently transported, suggesting that a further opening of the transporter might improve its ability to translocate this larger compound. We solved by cryo-EM the apo structures of the cross-linked mutant and the WT protein. Both structures are highly similar, showing an intermediate opening between their NBDs while their C-terminal extremities remain in close proximity. Distance measurements obtained by electron paramagnetic resonance spectroscopy support the intermediate opening found in these 3D structures. Overall, our data suggest that the NBDs of BmrA function with a tweezers-like mechanism distinct from the related lipid A exporter MsbA.

Structure of CYRI-B (FAM49B), a key regulator of cellular actin assembly.

In eukaryotes, numerous fundamental processes are controlled by the WAVE regulatory complex (WRC) that regulates cellular actin polymerization, crucial for cell motility, cell-cell adhesion and epithelial differentiation. Actin assembly is triggered by interaction of the small GTPase Rac1 with CYFIP1, a key component of the WRC. Previously known as FAM49B, CYRI-B is a protein that is highly conserved across the Eukaryota and has recently been revealed to be a key regulator of Rac1 activity. Mutation of CYRI-B or alteration of its expression therefore leads to altered actin nucleation dynamics, with impacts on lamellipodia formation, cell migration and infection by intracellular pathogens. In addition, knockdown of CYRI-B expression in cancer cell lines results in accelerated cell proliferation and invasiveness. Here, the structure of Rhincodon typus (whale shark) CYRI-B is presented, which is the first to be reported of any CYRI family member. Solved by X-ray crystallography, the structure reveals that CYRI-B comprises three distinct α-helical subdomains and is highly structurally related to a conserved domain present in CYFIP proteins. The work presented here establishes a template towards a better understanding of CYRI-B biological function.

Insights into bacterial lipoprotein trafficking from a structure of LolA bound to the LolC periplasmic domain.

In Gram-negative bacteria, outer-membrane lipoproteins are essential for maintaining cellular integrity, transporting nutrients, establishing infections, and promoting the formation of biofilms. The LolCDE ABC transporter, LolA chaperone, and LolB outer-membrane receptor form an essential system for transporting newly matured lipoproteins from the outer leaflet of the cytoplasmic membrane to the innermost leaflet of the outer membrane. Here, we present a crystal structure of LolA in complex with the periplasmic domain of LolC. The structure reveals how a solvent-exposed ß-hairpin loop (termed the "Hook") and trio of surface residues (the "Pad") of LolC are essential for recruiting LolA from the periplasm and priming it to receive lipoproteins. Experiments with purified LolCDE complex demonstrate that association with LolA is independent of nucleotide binding and hydrolysis, and homology models based on the MacB ABC transporter predict that LolA recruitment takes place at a periplasmic site located at least 50 Å from the inner membrane. Implications for the mechanism of lipoprotein extraction and transfer are discussed. The LolA-LolC structure provides atomic details on a key protein interaction within the Lol pathway and constitutes a vital step toward the complete molecular understanding of this important system.

Structure and mechanotransmission mechanism of the MacB ABC transporter superfamily.

MacB is an ABC transporter that collaborates with the MacA adaptor protein and TolC exit duct to drive efflux of antibiotics and enterotoxin STII out of the bacterial cell. Here we present the structure of ATP-bound MacB and reveal precise molecular details of its mechanism. The MacB transmembrane domain lacks a central cavity through which substrates could be passed, but instead conveys conformational changes from one side of the membrane to the other, a process we term mechanotransmission. Comparison of ATP-bound and nucleotide-free states reveals how reversible dimerization of the nucleotide binding domains drives opening and closing of the MacB periplasmic domains via concerted movements of the second transmembrane segment and major coupling helix. We propose that the assembled tripartite pump acts as a molecular bellows to propel substrates through the TolC exit duct, driven by MacB mechanotransmission. Homologs of MacB that do not form tripartite pumps, but share structural features underpinning mechanotransmission, include the LolCDE lipoprotein trafficking complex and FtsEX cell division signaling protein. The MacB architecture serves as the blueprint for understanding the structure and mechanism of an entire ABC transporter superfamily and the many diverse functions it supports.