Mycobacterial resistance to zinc poisoning requires assembly of P-ATPase-containing membrane metal efflux platforms.

The human pathogen Mycobacterium tuberculosis requires a P1B-ATPase metal exporter, CtpC (Rv3270), for resistance to zinc poisoning. Here, we show that zinc resistance also depends on a chaperone-like protein, PacL1 (Rv3269). PacL1 contains a transmembrane domain, a cytoplasmic region with glutamine/alanine repeats and a C-terminal metal-binding motif (MBM). PacL1 binds Zn2+, but the MBM is required only at high zinc concentrations. PacL1 co-localizes with CtpC in dynamic foci in the mycobacterial plasma membrane, and the two proteins form high molecular weight complexes. Foci formation does not require flotillin nor the PacL1 MBM. However, deletion of the PacL1 Glu/Ala repeats leads to loss of CtpC and sensitivity to zinc. Genes pacL1 and ctpC appear to be in the same operon, and homologous gene pairs are found in the genomes of other bacteria. Furthermore, PacL1 colocalizes and functions redundantly with other PacL orthologs in M. tuberculosis. Overall, our results indicate that PacL proteins may act as scaffolds that assemble P-ATPase-containing metal efflux platforms mediating bacterial resistance to metal poisoning.

Structural insights into ATP hydrolysis by the MoxR ATPase RavA and the LdcI-RavA cage-like complex.

The hexameric MoxR AAA+ ATPase RavA and the decameric lysine decarboxylase LdcI form a 3.3 MDa cage, proposed to assist assembly of specific respiratory complexes in E. coli. Here, we show that inside the LdcI-RavA cage, RavA hexamers adopt an asymmetric spiral conformation in which the nucleotide-free seam is constrained to two opposite orientations. Cryo-EM reconstructions of free RavA reveal two co-existing structural states: an asymmetric spiral, and a flat C2-symmetric closed ring characterised by two nucleotide-free seams. The closed ring RavA state bears close structural similarity to the pseudo two-fold symmetric crystal structure of the AAA+ unfoldase ClpX, suggesting a common ATPase mechanism. Based on these structures, and in light of the current knowledge regarding AAA+ ATPases, we propose different scenarios for the ATP hydrolysis cycle of free RavA and the LdcI-RavA cage-like complex, and extend the comparison to other AAA+ ATPases of clade 7.

Functional characterization of the Ca2+-ATPase SMA1 from Schistosoma mansoni.

Schistosoma mansoni is a parasite that causes bilharzia, a neglected tropical disease affecting hundreds of millions of people each year worldwide. In 2012, S. mansoni had been identified as the only invertebrate possessing two SERCA-type Ca2+-ATPases, SMA1 and SMA2. However, our analysis of recent genomic data shows that the presence of two SERCA pumps is rather frequent in parasitic flatworms. To understand the reasons of this redundancy in S. mansoni, we compared SMA1 and SMA2 at different levels. In terms of sequence and organization, the genes SMA1 and SMA2 are similar, suggesting that they might be the result of a duplication event. At the protein level, SMA1 and SMA2 only slightly differ in length and in the sequence of the nucleotide-binding domain. To get functional information on SMA1, we produced it in an active form in Saccharomyces cerevisiae, as previously done for SMA2. Using phosphorylation assays from ATP, we demonstrated that like SMA2, SMA1 bound calcium in a cooperative mode with an apparent affinity in the micromolar range. We also showed that SMA1 and SMA2 had close sensitivities to cyclopiazonic acid but different sensitivities to thapsigargin, two specific inhibitors of SERCA pumps. On the basis of transcriptomic data available in GeneDB, we hypothesize that SMA1 is a housekeeping Ca2+-ATPase, whereas SMA2 might be required in particular striated-like muscles like those present the tail of the cercariae, the infecting form of the parasite.

From Peptide Aptamers to Inhibitors of FUR, Bacterial Transcriptional Regulator of Iron Homeostasis and Virulence.

FUR (Ferric Uptake Regulator) protein is a global transcriptional regulator that senses iron status and controls the expression of genes involved in iron homeostasis, virulence, and oxidative stress. Ubiquitous in Gram-negative bacteria and absent in eukaryotes, FUR is an attractive antivirulence target since the inactivation of the fur gene in various pathogens attenuates their virulence. The characterization of 13-aa-long anti-FUR linear peptides derived from the variable part of the anti-FUR peptide aptamers, that were previously shown to decrease pathogenic E. coli strain virulence in a fly infection model, is described herein. Modeling, docking, and experimental approaches in vitro (activity and interaction assays, mutations) and in cells (yeast two-hybrid assays) were combined to characterize the interactions of the peptides with FUR, and to understand their mechanism of inhibition. As a result, reliable structure models of two peptide-FUR complexes are given. Inhibition sites are mapped in the groove between the two FUR subunits where DNA should also bind. Another peptide behaves differently and interferes with the dimerization itself. These results define these novel small peptide inhibitors as lead compounds for inhibition of the FUR transcription factor.

Quaternary Structure of Fur Proteins, a New Subfamily of Tetrameric Proteins.

The ferric uptake regulator (Fur) belongs to the family of the DNA-binding metal-responsive transcriptional regulators. Fur is a global regulator found in all proteobacteria. It controls the transcription of a wide variety of genes involved in iron metabolism but also in oxidative stress or virulence factor synthesis. When bound to ferrous iron, Fur can bind to specific DNA sequences, called Fur boxes. This binding triggers the repression or the activation of gene expression, depending on the regulated genes. As a general view, Fur proteins are considered to be dimeric proteins both in solution and when bound to DNA. In this study, we have purified Fur from four pathogenic strains (Pseudomonas aeruginosa, Francisella tularensis, Yersinia pestis, and Legionella pneumophila) and compared them to Fur from Escherichia coli (EcFur), the best characterized of this family. By using a series of "in solution" techniques, including multiangle laser light scattering and small-angle X-ray scattering, as well as cross-linking experiments, we have shown that the Fur proteins can be classified into two groups, according to their quaternary structure. The group of dimers is represented by EcFur and YpFur and the group of very stable tetramers by PaFur, FtFur, and LpFur. Using PaFur as a case study, we also showed that the dissociation of the tetramers into dimers is necessary for binding of Fur to DNA, and that this dissociation requires the combined effect of metal ion binding and DNA proximity.

Biochemical characterization of AtHMA6/PAA1, a chloroplast envelope Cu(I)-ATPase.

Copper is an essential plant micronutrient playing key roles in cellular processes, among them photosynthesis. In Arabidopsis thaliana, copper delivery to chloroplasts, mainly studied by genetic approaches, is thought to involve two P(IB)-type ATPases: AtHMA1 and AtHMA6/PAA1. The lack of biochemical characterization of AtHMA1 and PAA1, and more generally of plant P(IB)-type ATPases, is due to the difficulty of getting high amounts of these membrane proteins in an active form, either from their native environment or after expression in heterologous systems. In this study, we report the first biochemical characterization of PAA1, a plant copper-transporting ATPase. PAA1 produced in Lactococcus lactis is active, forming an aspartyl phosphate intermediate in the presence of ATP and the adequate metal ion. PAA1 can also be phosphorylated using inorganic phosphate in the absence of transition metal. Both phosphorylation types allowed us to demonstrate that PAA1 is activated by monovalent copper ions (and to a lower extent by silver ions) with an apparent affinity in the micromolar range. In agreement with these biochemical data, we also demonstrate that when expressed in yeast, PAA1 induces increased sensitivities to copper and silver. These data provide the first enzymatic characterization of a P(IB-1)-type plant ATPase and clearly identify PAA1 as a high affinity Cu(I) transporter of the chloroplast envelope.

Interplay between glutathione, Atx1 and copper: X-ray absorption spectroscopy determination of Cu(I) environment in an Atx1 dimer.

X-ray absorption techniques have been used to characterise the primary coordination sphere of Cu(I) bound to glutathionate (GS-), to Atx1 and in Cu2I(GS-)2(Atx1)2, a complex recently proposed as the major form of Atx1 in the cytosol. In each complex, Cu(I) was shown to be triply coordinated. When only glutathione is provided, each Cu(I) is triply coordinated by sulphur atoms in the binuclear complex CuI2(GS-)5, involving bridging and terminal thiolates. In the presence of Atx1 and excess of glutathione, under conditions where CuI2(GS-)2(Atx1)2 is formed, each Cu(I) is triply coordinated by sulphur atoms. Given these constraints, there are two different ways for Cu(I) to bridge the Atx1 dimer: either both Cu(I) ions contribute to bridging the dimer, or only one Cu(I) ion is responsible for bridging, the other one being coordinated to two glutathione molecules. These two models are discussed as regards Cu(I) transfer to Ccc2a.

Interplay between glutathione, Atx1 and copper. 1. Copper(I) glutathionate induced dimerization of Atx1.

Copper is both an essential element as a catalytic cofactor and a toxic element because of its redox properties. Once in the cell, Cu(I) binds to glutathione (GSH) and various thiol-rich proteins that sequester and/or exchange copper with other intracellular components. Among them, the Cu(I) chaperone Atx1 is known to deliver Cu(I) to Ccc2, the Golgi Cu-ATPase, in yeast. However, the mechanism for Cu(I) incorporation into Atx1 has not yet been unraveled. We investigated here a possible role of GSH in Cu(I) binding to Atx1. Yeast Atx1 was expressed in Escherichia coli and purified to study its ability to bind Cu(I). We found that with an excess of GSH [at least two GSH/Cu(I)], Atx1 formed a Cu(I)-bridged dimer of high affinity for Cu(I), containing two Cu(I) and two GSH, whereas no dimer was observed in the absence of GSH. The stability constants (log beta) of the Cu(I) complexes measured at pH 6 were 15-16 and 49-50 for CuAtx1 and Cu (2) (I) (GS(-))(2)(Atx1)(2), respectively. Hence, these results suggest that in vivo the high GSH concentration favors Atx1 dimerization and that Cu (2) (I) (GS(-))(2)(Atx1)(2) is the major conformation of Atx1 in the cytosol.

Structural basis for metal binding specificity: the N-terminal cadmium binding domain of the P1-type ATPase CadA.

In bacteria, P1-type ATPases are responsible for resistance to di- and monovalent toxic heavy metals by taking them out of the cell. These ATPases have a cytoplasmic N terminus comprising metal binding domains defined by a betaalphabetabetaalphabeta fold and a CXXC metal binding motif. To check how the structural properties of the metal binding site in the N terminus can influence the metal specificity of the ATPase, the first structure of a Cd(II)-ATPase N terminus was determined by NMR and its coordination sphere was investigated by X-ray absorption spectroscopy. A novel metal binding environment was found, comprising the two conserved Cys residues of the metal binding motif and a Glu in loop 5. A bioinformatic search identifies an ensemble of highly homologous sequences presumably with the same function. Another group of highly homologous sequences is found which can be referred to as zinc-detoxifying P1-type ATPases with the metal binding pattern DCXXC in the N terminus. Because no carboxylate groups participate in Cu(I) or Ag(I) binding sites, we suggest that the acidic residue plays a key role in the coordination properties of divalent cations, hence conferring a function to the N terminus in the metal specificity of the ATPase.