CueOs, members of the multicopper oxidase family, play a crucial role in bacterial copper detoxification. These enzymes feature a unique methionine-rich (Met-rich) domain, which is essential for the oxidation of Cu+ to Cu2+. Recent studies using CueO from Escherichia coli (EcCueO) suggest that the Met-rich domain facilitates Cu+ recruitment from highly chelated species. To further explore this hypothesis, we produced and characterized a novel CueO from the bacterium Hafnia alvei (HaCueO). HaCueO possesses a significantly larger Met-rich domain than EcCueO, providing new insights into the role of this domain in cuprous oxidase activity. We first showed that HaCueO was as efficient in copper detoxification as EcCueO in vivo. The structures of both wild-type HaCueO and a variant lacking the Met-rich domain were resolved by X-ray crystallography and simulated by molecular dynamics, offering a detailed structural basis for understanding their functions. Cuprous oxidase activity was then quantified either from free electrogenerated Cu+ with CueO immobilized on an electrode or from different Cu+-complexes with CueO in solution. These methods enabled the fine-tuning of Cu+ chelation strength. Consistent with findings for EcCueO, it was confirmed that the Met-rich domain of HaCueO is dispensable for Cu+ oxidation when weakly chelated Cu+ is used. However, its role becomes crucial as chelation strength increases. Comparative analyses of cuprous oxidase activity between HaCueO and EcCueO revealed that HaCueO outperforms EcCueO, demonstrating superior efficiency in oxidizing Cu+ from chelated forms. This enhanced activity correlates with the higher methionine content in HaCueO, which appears to play a pivotal role in facilitating Cu+ oxidation under conditions of stronger chelation.

We provide the first crystallographic structure of a green mononuclear monodomain cupredoxin. Analysis of the structure suggests that the coupled distortion model might not explain the behaviour of some cupredoxins.

The type IX secretion system (T9SS) is a large multi-protein transenvelope complex distributed into the Bacteroidetes phylum and responsible for the secretion of proteins involved in pathogenesis, carbohydrate utilization or gliding motility. In Porphyromonas gingivalis, the two-component system PorY sensor and response regulator PorX participate to T9SS gene regulation. Here, we present the crystal structure of PorXFj, the Flavobacterium johnsoniae PorX homolog. As for PorX, the PorXFj structure is comprised of a CheY-like N-terminal domain and an alkaline phosphatase-like C-terminal domain separated by a three-helix bundle central domain. While not activated and monomeric in solution, PorXFj crystallized as a dimer identical to active PorX. The CheY-like domain of PorXFj is in an active-like conformation, and PorXFj possesses phosphodiesterase activity, in agreement with the observation that the active site of its phosphatase-like domain is highly conserved with PorX.

ABSTRACT Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1 copper center coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in term of copper-binding ligands, copper center geometry, redox potential, as well as biological function. AcoP is a periplasmic protein belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans . AcoP presents original features: highly resistant to acidic pH, it possesses a constrained green-type copper center of high redox potential. To understand the unique properties of AcoP, we undertook structural and biophysical characterization of wild-type AcoP and of two Cu-ligand mutants (H166A and M171A). The crystallographic structure of AcoP at 1.65 Å resolution unveils a typical cupredoxin fold with extended loops, never observed in previously characterized cupredoxins, that might be involved in the interaction of AcoP with its physiological partners. Moreover, the structure shows that the green color of AcoP cannot be attributed to nonclassical copper ligands, its green-colored copper center raising from a long Cu-S (Cys) bond, determined by both X-ray diffraction and EXAFS. The crystal structures of two AcoP mutants confirm that the active center of AcoP is highly constrained. Comparative analysis with other cupredoxins of known structures, suggests that in AcoP the second coordination sphere might be an important determinant of active center rigidity due to the presence of an extensive hydrogen bond network.

Porphyromonas gingivalis, the major human pathogen bacterium associated with periodontal diseases, secretes virulence factors through the Bacteroidetes-specific type IX secretion system (T9SS). Effector proteins of the T9SS are recognized by the complex via their conserved C-terminal domains (CTDs). Among the 18 proteins essential for T9SS function in P. gingivalis, PorN is a periplasmic protein that forms large ring-shaped structures in association with the PorK outer membrane lipoprotein. PorN also mediates contacts with the PorM subunit of the PorLM energetic module, and with the effector's CTD. However, no information is available on the PorN structure and on the implication of PorN domains for T9SS assembly and effector recognition. Here we present the crystal structure of PorN at 2.0-Å resolution, which represents a novel fold with no significant similarity to any known structure. In agreement with in silico analyses, we also found that the N- and C-terminal regions of PorN are intrinsically disordered. Our functional studies showed that the N-terminal disordered region is involved in PorN dimerization while the C-terminal disordered region is involved in the interaction with PorK. Finally, we determined that the folded PorN central domain is involved in the interaction with PorM, as well as with the effector's CTD. Altogether, these results lay the foundations for a more comprehensive model of T9SS architecture and effector transport.

Odorant-binding proteins (OBPs), as they occur in insects, form a distinct class of proteins that apparently has no closely related representatives in other animals. However, ticks, mites, spiders and millipedes contain genes encoding proteins with sequence similarity to insect OBPs. In this work, we have explored the structure and function of such non-insect OBPs in the mite Varroa destructor, a major pest of honey bee. Varroa OBPs present six cysteines paired into three disulphide bridges, but with positions in the sequence and connections different from those of their insect counterparts. VdesOBP1 structure was determined in two closely related crystal forms and appears to be a monomer. Its structure assembles five α-helices linked by three disulphide bridges, one of them exhibiting a different connection as compared to their insect counterparts. Comparison with classical OBPs reveals that the second of the six α-helices is lacking in VdesOBP1. Ligand-binding experiments revealed molecules able to bind only specific OBPs with a moderate affinity, suggesting that either optimal ligands have still to be identified, or post-translational modifications present in the native proteins may be essential for modulating binding activity, or else these OBPs might represent a failed attempt in evolution and are not used by the mites.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The type VI secretion system (T6SS) is a widespread mechanism of protein delivery into target cells, present in more than a quarter of all sequenced Gram-negative bacteria. The T6SS constitutes an important virulence factor, as it is responsible for targeting effectors in both prokaryotic and eukaryotic cells. The T6SS comprises a tail structure tethered to the cell envelope via a trans-envelope complex. In most T6SS, the membrane complex is anchored to the cell wall by the TagL accessory protein. In this study, we report the first crystal structure of a peptidoglycan-binding domain of TagL. The fold is conserved with members of the OmpA/Pal/MotB family, and more importantly, the peptidoglycan binding site is conserved. This structure further exemplifies how proteins involved in anchoring to the cell wall for different cellular functions rely on an interaction network with peptidoglycan strictly conserved.

GldL is an inner-membrane protein that is essential for the function of the type IX secretion system (T9SS) inFlavobacterium johnsoniae. The complex that it forms with GldM is supposed to act as a new rotary motor involved in the gliding motility of the bacterium. In the context of structural studies of GldL to gain information on the assembly and function of the T9SS, two camelid nanobodies were selected, produced and purified. Their interaction with the cytoplasmic domain of GldL was characterized and their crystal structures were solved. These nanobodies will be used as crystallization chaperones to help in the crystallization of the cytoplasmic domain of GldL and could also help to solve the structure of the complex using molecular replacement.

Recently, LDH have been considered as a new kind of gene delivery system [1, 2]. In this work, we report the formation of magnesium-gallium LDH-DNA nanohybrids using the coprecipitation method. This “self assembly” approach enabled the incorporation of long DNA fragments up to 6000–8000 bp. X-ray diffraction analyses indicate a parallel orientation of DNA double helix in the interlamellar space with respect to the hydroxide sheets. The Mg/Ga molar ratio within the hydroxide layers appears to be determined by DNA macromolecules which may interact with charged complexes that form during cation hydrolysis. The presence of DNA macromolecules also inhibits the crystal growth: hydrodynamic diameter measurements revealed homogeneous populations of particles with a mean diameter ranging from 90 to 150 nm, compatible with cell penetration through endocytosis. Concerning the charge surface of this new DNA delivery system, ζ-potential measurements indicate negative values ranging from–20 to - 40 mV which suggest incomplete DNA intercalation. Yet, this small negative surface might be suitable for protecting DNA from extra-cellular degradations without preventing cell penetration.

Producing intact recombinant membrane proteins for structural studies is an inherently challenging task due to their requirement for a cell-lipid environment. Most of the procedures developed involve isolating the protein by solubilization with detergent and further reconstitutions into artificial membranes. These procedures are highly time consuming and suffer from further drawbacks, including low yields and high cost. We describe here an alternative method for rapidly obtaining recombinant cell-surface membrane proteins displayed on extracellular vesicles (EVs) derived from cells in culture. Interaction between these membrane proteins and ligands can be analyzed directly on EVs. Moreover, EVs can also be used for protein structure determination or immunization purposes.

Type IX secretion system (T9SS), exclusively present in the Bacteroidetes phylum, has been studied mainly in Flavobacterium johnsoniae and Porphyromonas gingivalis . Among the 18 genes, essential for T9SS function, a group of four, porK-N ( P. gingivalis ) or gldK-N ( F. johnsoniae ) belongs to a co-transcribed operon that expresses the T9SS core membrane complex. The central component of this complex, PorM (or GldM), is anchored in the inner membrane by a trans-membrane helix and interacts through the outer membrane PorK-N complex. There is a complete lack of available atomic structures for any component of T9SS, including the PorKLMN complex. Here we report the crystal structure of the GldM and PorM periplasmic domains. Dimeric GldM and PorM, each contain four domains of ~180-Å length that span most of the periplasmic space. These and previously reported results allow us to propose a model of the T9SS core membrane complex as well as its functional behavior.

The transport of proteins at the cell surface of Bacteroidetes depends on a secretory apparatus known as type IX secretion system (T9SS). This machine is responsible for the cell surface exposition of various proteins, such as adhesins, required for gliding motility in Flavobacterium, S-layer components in Tannerella forsythia, and tooth tissue-degrading enzymes in the oral pathogen Porphyromonas gingivalis. Although a number of subunits of the T9SS have been identified, we lack details on the architecture of this secretion apparatus. Here we provide evidence that five of the genes encoding the core complex of the T9SS are co-transcribed and that the gene products are distributed in the cell envelope. Protein-protein interaction studies then revealed that these proteins oligomerize and interact through a dense network of contacts.

1. Introduction Olivine phosphates have long been investigated as cathode material in Li-ion batteries. In particular, lithium iron phosphate (LiFePO4) has attracted the attention of many researchers because of its high theoretical capacity of 170 mAh g-1, low cost and high electrochemical/thermal stabilities of the phosphate (PO4 3-) anion[1]. However, the large volume difference during two-phase reaction between Li-rich Li1-aFePO4 (LFP) and Li-poor LibFePO4 (FP) phases leads to lattice mismatch at the LFP/FP boundary which induces a low Li-ion diffusion coefficient of 10-14 cm2 s-1 [2]. Such 1D Li+ diffusivity in the olivine-LFP is much slower (by 5-8 orders of magnitude) as compared with that of LCO (10-9 cm2 s-1) and LMO (10-5 cm2 s-1) cathodes. As a result, this slow lithium ion diffusion and the relatively poor electronic conductivity (10-10 to 10-7 Ω-1 cm-1) of the olivine-LFP in the absence of a doping cation, limit the power capability of the LFP material. To overcome these limitations, we have proposed new concept of three-phase structure containing a core of crystalline LFP, a shell of amorphous LFP, and graphitic carbon derived from Ketjen Black (KB) synthesized using our original in-situ ultracentrifugation process (UC process) [3]. The peculiar core-shell structure of LFP nanoparticle within the graphitic carbon prepared by UC-process improves the electronic conductivity in the whole LFP/graphitic carbon composites, while the amorphous LFP phase at the particle surface (shell) can achieve excellent rate performance owing to its high Li-ion diffusion coefficient. 2. Experimental A precursor solution was prepared by mixing KB and H3PO4 aq. in ultra-pure water. The precursor solution was treated by UC process after an addition of iron acetate and lithium acetate. After drying at 80°C in vacuum for 12 h, the precursor composite powder was obtained. The powder was lastly annealed at 700 ◦C under N2 flow for 5 min and the final product (LFP/ graphitic carbon composite powder) was obtained. The LFP/graphitic carbon composite electrode was electrochemically characterized using a 2032 coin half-cell with Li metal in 1M LiPF6/EC+DEC (vol. 1:1). 3. Results and Discussion The electron microscopy observations show that LFP/graphitic carbon composite has a highly crystalline phase of LFP core of ca.12-15 nm diameter with a distance of d=0.32 nm corresponding to (110) plane of the olivine LFP. This crystalline LFP phase is entirely covered with amorphous LFP. The combination of TEM observation together with XRD, XPS and Mössbauer analysis, supports the hypothesis that amorphous LFP contains Fe3+ defects. The most outer layer (“shell”) is composed of random graphitic carbon fragments/sheets stacked onto each other, derived from KB. The thickness of the shell is about 5 nm. An interlayer distance of d=0.35 nm was measured which is a little larger than that of graphene. This LFP/graphitic carbon composite enabled a 100C rate (36 seconds) discharge with 60 mAh g-1 per composite corresponding to 70% of the capacity obtained at the slowest discharge rate (1C). In the crystalline and amorphous LFP phase, different reaction mechanisms were observed and characterized by electrochemical study with a cavity microelectrode. While the reaction mechanism in the crystalline LFP phase is controlled by Li+ diffusion, the amorphous LFP phase shows a fast, surface-controlled, pseudocapacitive charge-storage mechanism[4]. This pseudocapacitive behavior is extrinsic in origin since it comes from the presence of Fe3+ defects in the structure. These features explain the ultrafast performance of the material which offers interesting opportunities as a positive electrode for assembling high power and high energy hybrid supercapacitors. References 1) A. K. Padhi, et al., J. Electrochem. Soc., 1997, 144, 1188-1194. 2) G. Kobayashi, et al., Adv. Funct. Mater., 2009, 19, 395-403. 3) K. Naoi, et al., Energy Environ. Sci., 2016, 9, 2143-2151. 4) K. Kisu, et al., Electrochem. Commun., 2016, 72, 10-14.

PorM is a membrane protein that is involved in the assembly of the type IX secretion system ( T9SS) in Porphyromonas gingivalis, a major bacterial pathogen that is responsible for periodontal disease in humans. In the context of structural studies of PorM to better understand T9SS assembly, four camelid nanobodies were selected, produced and purified, and their specific interaction with the N-terminal or C-terminal part of the periplasmic domain of PorM was investigated. Diffracting crystals were also obtained, and the structures of the four nanobodies were solved by molecular replacement. Furthermore, two nanobodies were used as crystallization chaperones and turned out to be valuable tools in the structure-determination process of the periplasmic domain of PorM.

1. Introduction Olivine phosphates have long been investigated as cathode material in Li-ion batteries. In particular, lithium iron phosphate (LiFePO4) has attracted the attention of many researchers because of its high theoretical capacity of 170 mAh g-1, low cost and high electrochemical/thermal stabilities of the phosphate (PO4 3-) anion.[1] However, the large volume difference during two-phase reaction between Li-rich Li1-aFePO4 (LFP) and Li-poor LibFePO4 (FP) phases leads to lattice mismatch at the LFP/FP boundary which induces a low Li-ion diffusion coefficient of 10-14 cm2 s-1. [2] Such 1D Li+ diffusivity in the olivine-LFP is much slower (by 5-8 orders of magnitude) as compared with that of LCO (10-9 cm2 s-1) and LMO (10-5 cm2 s-1) cathodes. As a result, this slow lithium ion diffusion and the relatively poor electronic conductivity (10-10 to 10-7 Ω-1 cm-1) of the olivine-LFP in the absence of a doping cation, limit the power capability of the LFP material. To overcome these limitations, we have proposed new concept of three-phase structure containing a core of crystalline LFP, a shell of amorphous LFP, and graphitic carbon derived from Ketjen Black (KB) synthesized using our original in-situ ultracentrifugation process (UC process) [3]. The peculiar core-shell structure of LFP nanoparticle within the graphitic carbon prepared by UC-process improves the electronic conductivity in the whole LFP/graphitic carbon composites, while the amorphous LFP phase at the particle surface (shell) can achieve excellent rate performance owing to its high Li-ion diffusion coefficient. 2. Experimental A precursor solution was prepared by mixing KB and H3PO4 aq. in ultra-pure water. The precursor solution was treated by UC process after an addition of iron acetate and lithium acetate. After drying at 80°C in vacuum for 12 h, the precursor composite powder was obtained. The powder was lastly annealed at 700 ◦C under N2 flow for 5 min and the final product (LFP/ graphitic carbon composite powder) was obtained. The LFP/graphitic carbon composite electrode was electrochemically characterized using a 2032 coin half-cell with Li metal in 1M LiPF6/EC+DEC (vol. 1:1). 3. Results and Discussion The electron microscopy observations show that LFP/graphitic carbon composite has a highly crystalline phase of LFP core of ca.12-15 nm diameter with a distance of d=0.32 nm corresponding to (110) plane of the olivine LFP. This crystalline core, that we called “core 1”, is entirely covered with amorphous layers (referred as “core 2”) in light grey in the TEM image. The combination of TEM observation together with XRD, XPS and Mössbauer analysis, supports the hypothesis that amorphous LFP contains Fe3+ defect. The most outer layer (“shell”) is composed of random carbon (KB) fragments/sheets stacked onto each other. The thickness of the shell is about 5 nm. An interlayer distance of d=0.35 nm was measured which is a little larger than that of graphene. This LFP/graphitic carbon composite enabled a 100C rate (36 seconds) discharge with 60 mAh g-1 per composite corresponding to 70% of the capacity obtained at the slowest discharge rate (1C). An amorphous LFP containing Fe3+ defects enhances lithium ion diffusion coefficient probably by switching from one-dimensional to three-dimensional diffusion. Then, the electron utilized in Fe redox reaction can be fast transferred by the covering graphitic carbon coating the particles. Such an ultrafast charge–discharge performance opens the possibility of using LiFePO4 as a cathode material for ultrafast hybrid capacitor with a stable cycle performance over 2,000 cycles at a 10C rate, maintaining 90% of the initial capacity. References 1) A. K. Padhi, et al., J. Electrochem. Soc., 1997, 144, 1188-1194. 2) G. Kobayashi, et al., Adv. Funct. Mater., 2009, 19, 395-403. 3) K. Naoi, et al., J. Electrochem. Soc., 2015, 162 (6), A1-A7.

Highly dispersed crystalline/amorphous LiFePO4 (LFP) nanoparticles encapsulated within hollow-structured graphitic carbon were synthesized using an in situ ultracentrifugation process.

PorM is a membrane protein involved in the assembly of the type IX secretion system (T9SS) from Porphyromonas gingivalis, a major bacterial pathogen responsible for periodontal disease in humans. The periplasmic domain of PorM was overexpressed in Escherichia coli and purified. A fragment of the purified protein was obtained by limited proteolysis. Crystals of this fragment belonged to the tetragonal space group P4(3)2(1)2. Native and MAD data sets were recorded to 2.85 and 3.1 Å resolution, respectively, using synchrotron radiation.

AbstractThe compounds Y3Fe5O12 and Y3Fe5‐xCrxO12 are synthesized by solid state reaction of stoichiometric mixtures of Y2O3, Fe2O3, and Cr2O3 (air, 1350 °C, 6 h).

β-Barrel pore-forming toxins (β-PFT), a large family of bacterial toxins, are generally secreted as water-soluble monomers and can form oligomeric pores in membranes following proteolytic cleavage and interaction with cell surface receptors. Monalysin has been recently identified as a β-PFT that contributes to the virulence of Pseudomonas entomophila against Drosophila. It is secreted as a pro-protein that becomes active upon cleavage. Here we report the crystal and cryo-electron microscopy structure of the pro-form of Monalysin as well as the crystal structures of the cleaved form and of an inactive mutant lacking the membrane-spanning region. The overall structure of Monalysin displays an elongated shape, which resembles those of β-pore-forming toxins, such as Aerolysin, but is devoid of a receptor-binding domain. X-ray crystallography, cryo-electron microscopy, and light-scattering studies show that pro-Monalysin forms a stable doughnut-like 18-mer complex composed of two disk-shaped nonamers held together by N-terminal swapping of the pro-peptides. This observation is in contrast with the monomeric pro-form of the other β-PFTs that are receptor-dependent for membrane interaction. The membrane-spanning region of pro-Monalysin is fully buried in the center of the doughnut, suggesting that upon cleavage of pro-peptides, the two disk-shaped nonamers can, and have to, dissociate to leave the transmembrane segments free to deploy and lead to pore formation. In contrast with other toxins, the delivery of 18 subunits at once, nearby the cell surface, may be used to bypass the requirement of receptor-dependent concentration to reach the threshold for oligomerization into the pore-forming complex. Background: Monalysin is a β-barrel pore-forming toxin secreted by Pseudomonas entomophila. Results: Monalysin structure belongs to the Aerolysin fold family but is devoid of receptor-binding domain; pro-Monalysin forms a stable 18-mer complex. Conclusion: The delivery of 18 subunits may bypass the requirement of receptor-dependent concentration. Significance: The functional mechanism of Monalysin differs from that of described Aerolysin family members.