Hybrid Amyloid-Based Redox Hydrogel for Bioelectrocatalytic H2 Oxidation.

An artificial amyloid-based redox hydrogel was designed for mediating electron transfer between a [NiFeSe] hydrogenase and an electrode. Starting from a mutated prion-forming domain of fungal protein HET-s, a hybrid redox protein containing a single benzyl methyl viologen moiety was synthesized. This protein was able to self-assemble into structurally homogenous nanofibrils. Molecular modeling confirmed that the redox groups are aligned along the fibril axis and are tethered to its core by a long, flexible polypeptide chain that allows close encounters between the fibril-bound oxidized or reduced redox groups. Redox hydrogel films capable of immobilizing the hydrogenase under mild conditions at the surface of carbon electrodes were obtained by a simple pH jump. In this way, bioelectrodes for the electrocatalytic oxidation of H2 were fabricated that afforded catalytic current densities of up to 270 µA cm-2 , with an overpotential of 0.33 V, under quiescent conditions at 45 °C.

The carbon monoxide dehydrogenase accessory protein CooJ is a histidine-rich multidomain dimer containing an unexpected Ni(II)-binding site.

Activation of nickel enzymes requires specific accessory proteins organized in multiprotein complexes controlling metal transfer to the active site. Histidine-rich clusters are generally present in at least one of the metallochaperones involved in nickel delivery. The maturation of carbon monoxide dehydrogenase in the proteobacterium Rhodospirillum rubrum requires three accessory proteins, CooC, CooT, and CooJ, dedicated to nickel insertion into the active site, a distorted [NiFe3S4] cluster coordinated to an iron site. Previously, CooJ from R. rubrum (RrCooJ) has been described as a nickel chaperone with 16 histidines and 2 cysteines at its C terminus. Here, the X-ray structure of a truncated version of RrCooJ, combined with small-angle X-ray scattering data and a modeling study of the full-length protein, revealed a homodimer comprising a coiled coil with two independent and highly flexible His tails. Using isothermal calorimetry, we characterized several metal-binding sites (four per dimer) involving the His-rich motifs and having similar metal affinity (KD = 1.6 µm). Remarkably, biophysical approaches, site-directed mutagenesis, and X-ray crystallography uncovered an additional nickel-binding site at the dimer interface, which binds Ni(II) with an affinity of 380 nm Although RrCooJ was initially thought to be a unique protein, a proteome database search identified at least 46 bacterial CooJ homologs. These homologs all possess two spatially separated nickel-binding motifs: a variable C-terminal histidine tail and a strictly conserved H(W/F)X2HX3H motif, identified in this study, suggesting a dual function for CooJ both as a nickel chaperone and as a nickel storage protein.

A Homozygous Ancestral SVA-Insertion-Mediated Deletion in WDR66 Induces Multiple Morphological Abnormalities of the Sperm Flagellum and Male Infertility.

Multiple morphological abnormalities of the sperm flagellum (MMAF) is a severe form of male infertility defined by the presence of a mosaic of anomalies, including short, bent, curled, thick, or absent flagella, resulting from a severe disorganization of the axoneme and of the peri-axonemal structures. Mutations in DNAH1, CFAP43, and CFAP44, three genes encoding axoneme-related proteins, have been described to account for approximately 30% of the MMAF cases reported so far. Here, we searched for pathological copy-number variants in whole-exome sequencing data from a cohort of 78 MMAF-affected subjects to identify additional genes associated with MMAF. In 7 of 78 affected individuals, we identified a homozygous deletion that removes the two penultimate exons of WDR66 (also named CFAP251), a gene coding for an axonemal protein preferentially localized in the testis and described to localize to the calmodulin- and spoke-associated complex at the base of radial spoke 3. Sequence analysis of the breakpoint region revealed in all deleted subjects the presence of a single chimeric SVA (SINE-VNTR-Alu) at the breakpoint site, suggesting that the initial deletion event was potentially mediated by an SVA insertion-recombination mechanism. Study of Trypanosoma WDR66's ortholog (TbWDR66) highlighted high sequence and structural analogy with the human protein and confirmed axonemal localization of the protein. Reproduction of the human deletion in TbWDR66 impaired flagellar movement, thus confirming WDR66 as a gene associated with the MMAF phenotype and highlighting the importance of the WDR66 C-terminal region.

Short oligopeptides with three cysteine residues as models of sulphur-rich Cu(i)- and Hg(ii)-binding sites in proteins.

The essential Cu(i) and the toxic Hg(ii) ions possess similar coordination properties, and therefore, similar cysteine rich proteins participate in the control of their intracellular concentration. In this work we present the metal binding properties of linear and cyclic model peptides incorporating the three-cysteine motifs, CxCxxC or CxCxC, found in metallothioneins. Cu(i) binding to the series of peptides at physiological pH revealed to be rather complicated, with the formation of mixtures of polymetallic species. In contrast, the Hg(ii) complexes display well-defined structures with spectroscopic features characteristic for a HgS2 and HgS3 coordination mode at pH = 2.0 and 7.4, respectively. Stability data reflect a ca. 20 orders of magnitude larger affinity of the peptides for Hg(ii) (log ßpH7.4HgP ≈ 41) than for Cu(i) (log ßpH7.4CuP ≈ 18). The different behaviour with the two metal ions demonstrates that the use of Hg(ii) as a probe for Cu(i), coordinated by thiolate ligands in water, may not always be fully appropriate.

Mercury Trithiolate Binding (HgS3) to a de Novo Designed Cyclic Decapeptide with Three Preoriented Cysteine Side Chains.

Mercury(II) is an unphysiological soft ion with high binding affinity for thiolate ligands. Its toxicity lies in the interactions with low molecular weight thiols including glutathione and cysteine-containing proteins that disrupt the thiol balance and alter vital functions. However, mercury can also be detoxified via interactions with Hg(II)-responsive regulatory proteins such as MerR, which coordinates Hg(II) with three cysteine residues in a trigonal planar fashion (HgS3 coordination). The model cyclodecapeptide P3C, c(GCTCSGCSRP) was designed to promote Hg(II) chelation in a HgS3 coordination environment through the parallel orientation of three cysteine side chains. The binding motif is derived from the dicysteine P2C cyclodecapeptide validated previously as a model for d10 metal transporters containing the binding sequence CxxC. The formation of the mononuclear HgP3C complex with a HgS3 coordination is demonstrated using electrospray ionization mass spectrometry, UV absorption, and 199Hg NMR. Hg LIII-edge extended X-ray absorption fine structure (EXAFS) spectroscopy indicates that the Hg(II) coordination environment is T-shaped with two short Hg-S distances at 2.45 Å and one longer distance at 2.60 Å. The solution structure of the HgP3C complex was refined based on 1H-1H NMR constraints and EXAFS results. The cyclic peptide scaffold has a rectangular shape with the three binding cysteine side chains pointing toward Hg(II). The HgP3CH complex has a p Ka of 4.3, indicating that the HgS3 coordination mode is stable over a large range of pH. This low p Ka value suggests that the preorientation of the three cysteine groups is particularly well-achieved for Hg(II) trithiolate coordination in P3C.

Identification of Two Conserved Residues Involved in Copper Release from Chloroplast PIB-1-ATPases.

Copper is an essential transition metal for living organisms. In the plant model Arabidopsis thaliana, half of the copper content is localized in the chloroplast, and as a cofactor of plastocyanin, copper is essential for photosynthesis. Within the chloroplast, copper delivery to plastocyanin involves two transporters of the PIB-1-ATPases subfamily: HMA6 at the chloroplast envelope and HMA8 in the thylakoid membranes. Both proteins are high affinity copper transporters but share distinct enzymatic properties. In the present work, the comparison of 140 sequences of PIB-1-ATPases revealed a conserved region unusually rich in histidine and cysteine residues in the TMA-L1 region of eukaryotic chloroplast copper ATPases. To evaluate the role of these residues, we mutated them in HMA6 and HMA8. Mutants of interest were selected from phenotypic tests in yeast and produced in Lactococcus lactis for further biochemical characterizations using phosphorylation assays from ATP and Pi Combining functional and structural data, we highlight the importance of the cysteine and the first histidine of the CX3HX2H motif in the process of copper release from HMA6 and HMA8 and propose a copper pathway through the membrane domain of these transporters. Finally, our work suggests a more general role of the histidine residue in the transport of copper by PIB-1-ATPases.

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.

HMA6 and HMA8 are two chloroplast Cu+-ATPases with different enzymatic properties.

Copper (Cu) plays a key role in the photosynthetic process as cofactor of the plastocyanin (PC), an essential component of the chloroplast photosynthetic electron transfer chain. Encoded by the nuclear genome, PC is translocated in its apo-form into the chloroplast and the lumen of thylakoids where it is processed to its mature form and acquires Cu. In Arabidopsis, Cu delivery into the thylakoids involves two transporters of the PIB-1 ATPases family, heavy metal associated protein 6 (HMA6) located at the chloroplast envelope and HMA8 at the thylakoid membrane. To gain further insight into the way Cu is delivered to PC, we analysed the enzymatic properties of HMA8 and compared them with HMA6 ones using in vitro phosphorylation assays and phenotypic tests in yeast. These experiments reveal that HMA6 and HMA8 display different enzymatic properties: HMA8 has a higher apparent affinity for Cu(+) but a slower dephosphorylation kinetics than HMA6. Modelling experiments suggest that these differences could be explained by the electrostatic properties of the Cu(+) releasing cavities of the two transporters and/or by the different nature of their cognate Cu(+) acceptors (metallochaperone/PC).

Structure prediction of GPCRs using piecewise homologs and application to the human CCR5 chemokine receptor: validation through agonist and antagonist docking.

This article describes the construction and validation of a three-dimensional model of the human CC chemokine receptor 5 (CCR5) receptor using multiple homology modeling. A new methodology is presented where we built each secondary structural model of the protein separately from distantly related homologs of known structure. The reliability of our approach for G-protein coupled receptors was assessed through the building of the human C-X-C chemokine receptor type 4 (CXCR4) receptor of known crystal structure. The models are refined using molecular dynamics simulations and energy minimizations using CHARMM, a classical force field for proteins. Finally, docking models of both the natural agonists and the antagonists of the receptors CCR5 and CXCR4 are proposed. This study explores the possible binding process of ligands to the receptor cavity of chemokine receptors at molecular and atomic levels. We proposed few crucial residues in receptors binding to agonist/antagonist for further validation through experimental analysis. In particular, our study provides better understanding of the blockage mechanism of the chemokine receptors CCR5 and CXCR4, and may help the identification of new lead compounds for drug development in HIV infection, inflammatory diseases, and cancer metastasis.

Dynamics and stability of the metal binding domains of the Menkes ATPase and their interaction with metallochaperone HAH1.

Human copper-ATPases ATP7A and ATP7B are essential for intracellular copper homeostasis. The main roles of the Menkes protein, ATP7A, are the delivery of copper to the secretory pathway and the export of excess copper from the enterocytes. The N-terminal domain of membrane protein ATP7A consists of six repetitive sequences of 60-70 amino acids (Mnk1-Mnk6) that fold into individual metal binding domains (MBDs) and bind a single copper ion in the reduced Cu(I) form via two cysteine residues. The structure of each individual MBD is known from nuclear magnetic resonance experiments. Here, we were interested in the stability and dynamics of each isolated MBD in their apo and holo forms and their interactions with the soluble metallochaperone HAH1 that delivers copper to ATP7A. Using molecular dynamics simulations of the MBDs under different conditions, we show that some MBDs (Mnk1 and Mnk5) present large root-mean-square deviations from initial structures or large root-mean-square fluctuations, and great care has to be taken in setting up the simulations. We propose that the first MBD, Mnk1, probably important in the transfer of copper between the metallochaperone and ATPase, could be stabilized by interactions with other MBDs, including a domain located in the loop between Mnk1 and Mnk2. An important result of this work is the apparent direct correlation between the difference in the fluctuations of the metal binding site loop in its apo and holo forms and the measured affinity of the MBD for copper. This difference decreases from Mnk1 to Mnk6, Mnk4, and Mnk2 in this order. The study of the exposure to the solvent of the metal and the residues of the metal binding loop of the MBDs also shows different behavior for each MBD. In particular, copper in serine-rich domain Mnk3 and largely fluctuating domain Mnk5 appears to be more solvent-exposed than in the other MBDs. In the second part of this work, we investigated the importance of electrostatics in the MBD-chaperone interactions using different docking programs. Mnk1 and Mnk4 present a large electrostatic dipole moment and large stabilizing interaction energies with HAH1. Finally, we propose a model structure of ATP7A from Mnk6 (E561) to P1413 based on the crystal structure of LpCopA and docking simulations.

Probing the conformation of the resting state of a bacterial multidrug ABC transporter, BmrA, by a site-directed spin labeling approach.

Previously published 3-D structures of a prototypic ATP-binding cassette (ABC) transporter, MsbA, have been recently corrected revealing large rigid-body motions possibly linked to its catalytic cycle. Here, a closely related multidrug bacterial ABC transporter, BmrA, was studied using site-directed spin labeling by focusing on a region connecting the transmembrane domain and the nucleotide-binding domain (NBD). Electron paramagnetic resonance (EPR) spectra of single spin-labeled cysteine mutants suggests that, in the resting state, this sub-domain essentially adopts a partially extended conformation, which is consistent with the crystal structures of MsbA and Sav1866. Interestingly, one of the single point mutants (Q333C) yielded an immobilized EPR spectrum that could arise from a direct interaction with a vicinal tyrosine residue. Inspection of different BmrA models pointed to Y408, within the NBD, as the putative interacting partner, and its mutation to a Phe residue indeed dramatically modified the EPR spectra of the spin labeled Q333C. Moreover, unlike the Y408F mutation, the Y408A mutation abolished both ATPase activity and drug transport of BmrA, suggesting that a nonpolar bulky residue is required at this position. The spatial proximity of Q333 and Y408 was also confirmed by formation of a disulfide bond when both Q333 and T407 (or S409) were replaced jointly by a cysteine residue. Overall, these results indicate that the two regions surrounding Q333 and Y408 are close together in the 3-D structure of BmrA and that residues within these two sub-domains are essential for proper functioning of this transporter.

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.

The Q-loop disengages from the first intracellular loop during the catalytic cycle of the multidrug ABC transporter BmrA.

The ATP-binding cassette is the most abundant family of transporters including many medically relevant members and gathers both importers and exporters involved in the transport of a wide variety of substrates. Although three high resolution three-dimensional structures have been obtained for a prototypic exporter, MsbA, two have been subjected to much criticism. Here, conformational changes of BmrA, a multidrug bacterial transporter structurally related to MsbA, have been studied. A three-dimensional model of BmrA, based on the "open" conformation of Escherichia coli MsbA, was probed by simultaneously introducing two cysteine residues, one in the first intracellular loop of the transmembrane domain and the other in the Q-loop of the nucleotide-binding domain (NBD). Intramolecular disulfide bonds could be created in the absence of any effectors, which prevented both drug transport and ATPase activity. Interestingly, addition of ATP/Mg plus vanadate strongly prevented this bond formation in a cysteine double mutant, whereas ATP/Mg alone was sufficient when the ATPase-inactive E504Q mutation was also introduced, in agreement with additional BmrA models where the ATP-binding sites are positioned at the NBD/NBD interface. Furthermore, cross-linking between the two cysteine residues could still be achieved in the presence of ATP/Mg plus vanadate when homobifunctional cross-linkers separated by more than 13 Angstrom were added. Altogether, these results give support to the existence, in the resting state, of a monomeric conformation of BmrA similar to that found within the open MsbA dimer and show that a large motion is required between intracellular loop 1 and the nucleotide-binding domain for the proper functioning of a multidrug ATP-binding cassette transporter.

Is the cytoplasmic loop of MerT, the mercuric ion transport protein, involved in mercury transfer to the mercuric reductase?

In MerT, the mercury transporter, a first cysteine pair, located in the first trans-membrane helix, receives mercury from the periplasm. Then, a second cysteine pair, housed in a cytoplasmic loop connecting the second and the third trans-membrane helices, is thought to transfer the metal to another cysteine pair located in the N-terminal extension of the mercuric reductase. We found that a 23-amino acid synthetic peptide corresponding to the cytoplasmic loop can bind one mercury atom per molecule and that this mercury atom can be transferred specifically to MerAa. The solution structure of Hg-bound ppMerT has been solved by 1H NMR spectroscopy.

Molecular dynamics simulations of the transmembrane domain of the oncogenic ErbB2 receptor dimer in a DMPC bilayer.

Molecular dynamics simulations of an atomic model of the transmembrane domain of the oncogenic ErbB2 receptor dimer embedded in an explicit dimyristoylphosphatidylcholine (DMPC) bilayer were performed for more than 4 ns. The oncogenic Glu mutation in the membrane spanning segment plays a major role in tyrosine kinase activity and receptor dimerization, and is thought to be partly responsible for the structure of the transmembrane domain of the active receptor. MD results show that the interactions between the two transmembrane helices are characteristic of a left-handed packing as previously demonstrated from in vacuo simulations. Moreover, MD results reveal the absence of persistent hydrogen bonds between the Glu side chains in a membrane environment, which raise the question of the ability for Glu alone to stabilize the TM domain of the ErbB2 receptor. Interestingly the formation of the alpha-pi motif in the two ErbB2 transmembrane helices confirms the concept of intrinsic sequence-induced conformational flexibility. From a careful analysis of our MD results, we suggest that the left-handed helix-helix packing could be the key to correctly orient the intracellular domain of the activated receptor dimer. The prediction of such interactions from computer simulations represents a new step towards the understanding of signaling mechanisms.