Kinetic analysis of copper transfer from a chaperone to its target protein mediated by complex formation

Chaperone proteins that traffic copper around the cell minimise its toxicity by maintaining it in a tightly bound form. The transfer of copper from chaperones to target proteins is promoted by complex formation, but the kinetic characteristics of transfer have yet to be demonstrated for any chaperone-target protein pair. Here we report studies of copper transfer between the Atx1-type chaperone CopZ from Bacillus subtilis and the soluble domains of its cognate P-type ATPase transporter, CopAab. Transfer of copper from CopZ to CopAab was found to occur rapidly, with a rate constant at 25 °C of ∼267 s−1, many orders of magnitude higher than that for Cu(I) dissociation from CopZ in the absence of CopAab. The data demonstrate that complex formation between CopZ and CopAab, evidence for which is provided by NMR and electrospray ionisation mass spectrometry, dramatically enhances the rate of Cu(I) dissociation from CopZ

The N-terminal domains of Bacillus subtilis CopA do not form a stable complex in the absence of their inter-domain linker

Copper-transporting P-type ATPases, which play important roles in trafficking Cu(I) across membranes for the biogenesis of copper proteins or for copper detoxification, contain a variable number of soluble metal-binding domains at their N-termini. It is increasingly apparent that these play an important role in regulating copper transport in a Cu(I)-responsive manner, but how they do this is unknown. CopA, a Cu(I)-transporter from Bacillus subtilis, contains two N-terminal soluble domains that are closely packed, with inter-domain interactions at two principal regions. Here, we sought to determine the extent to which the domains interact in the absence of their inter-domain covalent linker, and how their Cu(I)-binding properties are affected. Studies of a 1:1 mixture of separate CopAa and CopAb domains showed that the domains do not form a stable complex, with only indirect evidence of a weak interaction between them. Their Cu(I)-binding behaviour was distinct from that of the two domain protein and consistent with a lack of interaction between the domains. Cu(I)-mediated protein association was observed, but this occurred only between domains of the same type. Thus, the inter-domain covalent link between CopAa and CopAb is essential for inter-domain interactions and for Cu(I)-binding behaviour

Three-Dimensional Solution Structure of Saccharomyces cerevisiae Reduced Iso-l-cytochromec

Two-dimensional ^1H NMR spectra of Saccharomyces cerevisiae reduced iso-1-cytochrome c have been used to confirm and slightly extend the assignment available in the literature. 1702 NOESY cross-peaks have been assigned, and their intensities have been measured. Through the program DIANA and related protocols (Güntert, 1992), a solution structure has been obtained by using 1442 meaningful NOEs and 13 hydrogen-bond constraints. The RMSD values with respect to the mean structure for the backbone and all heavy atoms for a family of 20 structures are 0.61 ± 0.09 and 0.98 ± 0.09 Å, the average target function value being as small as 0.57 Å^2. The larger number of slowly exchanging amide NHs observed in this system compared to that observed in the cyanide derivative of oxidized Ala 80 cytochrome c suggests that the oxidized form is much more flexible and that the backbone protons are more solvent accessible. Comparison of the present structure with the crystal structures of reduced yeast cytochrome c and of the complex between cytochrome c peroxidase and oxidized yeast cytochrome c reveals substantial similarity among the backbone conformations but differences in the residues located in the region of protein−protein interaction. Interestingly, in solution the peripheral residues involved in the interaction with cytochrome c peroxidase are on average closer to the position found in the crystal structure of the complex than to the solid state structure of the isolated reduced form

Mass spectrometry of B. subtilis CopZ: Cu(I)-binding and interactions with bacillithiol

CopZ from Bacillus subtilis is a well-studied member of the highly conserved family of Atx1-like copper chaperones. It was previously shown via solution and crystallographic studies to undergo Cu(I)-mediated dimerisation, where the CopZ dimer can bind between one and four Cu(I) ions. However, these studies could not provide information about the changing distribution of species at increasing Cu(I) levels. To address this, electrospray ionisation mass spectrometry using soft ionisation was applied to CopZ under native conditions. Data revealed folded, monomeric CopZ in apo- and Cu(I)-bound forms, along with Cu(I)-bound dimeric forms of CopZ at higher Cu(I) loading. Cu4(CopZ)2 was the major dimeric species at loadings >1 Cu(I)/CopZ, indicating the cooperative formation of the tetranuclear Cu(I)-bound species. As the principal low molecular weight thiol in B. subtilis, bacillithiol (BSH) may play a role in copper homeostasis. Mass spectrometry showed that increasing BSH led to a reduction in Cu(I)-bound dimeric forms, and the formation of S-bacillithiolated apo-CopZ and BSH adducts of Cu(I)-bound forms of CopZ, where BSH likely acts as a Cu(I) ligand. These data, along with the high affinity of BSH for Cu(I), determined here to be β2(BSH) = ∼4 × 1017 M−2, are consistent with a role for BSH alongside CopZ in buffering cellular Cu(I) levels. Here, mass spectrometry provides a high resolution overview of CopZ–Cu(I) speciation that cannot be obtained from less discriminating solution-phase methods, thus illustrating the potential for the wider application of this technique to studies of metal–protein interactions

In-cell NMR: From target structure and dynamics to drug screening

The cellular environment can affect the structure and function of pharmacological targets and the interaction with potential drugs. Such complexity is often overlooked in the first steps of drug design, where compounds are screened and optimized in vitro, leading to high failure rates in the pre-clinical and clinical tests. In-cell NMR spectroscopy has the potential to fill this gap, as it allows structural studies of proteins and nucleic acids directly in living cells, from bacteria to human-derived, providing a unique way to investigate the structure and dynamics of ligand–target interactions in the native cellular context. When applied to drug screening, in-cell NMR provides insights on binding kinetics and affinity toward a cellular target, offering a powerful tool for improving drug potency at an early stage of drug development

Understanding the Molecular Basis of the Multiple Mitochondrial Dysfunctions Syndrome 2: The Disease-Causing His96Arg Mutation of BOLA3

Multiple mitochondrial dysfunctions syndrome type 2 with hyperglycinemia (MMDS2) is a severe disorder of mitochondrial energy metabolism, associated with biallelic mutations in the gene encoding for BOLA3, a protein with a not yet completely understood role in iron-sulfur (Fe-S) cluster biogenesis, but essential for the maturation of mitochondrial [4Fe-4S] proteins. To better understand the role of BOLA3 in MMDS2, we have investigated the impact of the p.His96Arg (c.287A > G) point mutation, which involves a highly conserved residue, previously identified as a [2Fe-2S] cluster ligand in the BOLA3-[2Fe-2S]-GLRX5 heterocomplex, on the structural and functional properties of BOLA3 protein. The His96Arg mutation has been associated with a severe MMDS2 phenotype, characterized by defects in the activity of mitochondrial respiratory complexes and lipoic acid-dependent enzymes. Size exclusion chromatography, NMR, UV-visible, circular dichroism, and EPR spectroscopy characterization have shown that the His96Arg mutation does not impair the interaction of BOLA3 with its protein partner GLRX5, but leads to the formation of an aberrant BOLA3-[2Fe-2S]-GLRX5 heterocomplex, that is not functional anymore in the assembly of a [4Fe-4S] cluster on NFU1. These results allowed us to rationalize the severe phenotype observed in MMDS2 caused by His96Arg mutation

A four-helix bundle stores copper for methane oxidation

Methane-oxidising bacteria (methanotrophs) require large quantities of copper for the membrane-bound (particulate) methane monooxygenase (pMMO). Certain methanotrophs are also able to switch to using the iron-containing soluble MMO (sMMO) to catalyse methane oxidation, with this switchover regulated by copper. MMOs are Nature’s primary biological mechanism for suppressing atmospheric levels of methane, a potent greenhouse gas. Furthermore, methanotrophs and MMOs have enormous potential in bioremediation and for biotransformations producing bulk and fine chemicals, and in bioenergy, particularly considering increased methane availability from renewable sources and hydraulic fracturing of shale rock. We have discovered and characterised a novel copper storage protein (Csp1) from the methanotroph Methylosinus trichosporium OB3b that is exported from the cytosol, and stores copper for pMMO. Csp1 is a tetramer of 4-helix bundles with each monomer binding up to 13 Cu(I) ions in a previously unseen manner via mainly Cys residues that point into the core of the bundle. Csp1 is the first example of a protein that stores a metal within an established protein-folding motif. This work provides a detailed insight into how methanotrophs accumulate copper for the oxidation of methane. Understanding this process is essential if the wide-ranging biotechnological applications of methanotrophs are to be realised. Cytosolic homologues of Csp1 are present in diverse bacteria thus challenging the dogma that such organisms do not use copper in this location