Cu(I)- and proton-binding properties of the first N-terminal soluble domain of Bacillus subtilis CopA

CopA, a P-type ATPase transporter involved in copper detoxification in Bacillus subtilis, contains two soluble Atx1-like domains separated by a short linker at its N-terminus, an arrangement that occurs widely in copper transporters from both prokaryotes and eukaryotes. Both domains were previously found to bind Cu(I) with very high affinity. Above a level of 1 Cu(I) per CopAab, dimerization occurred, leading to a highly luminescent multinuclear Cu(I) species [Singleton C & Le Brun NE (2009) Dalton Trans, 688-696]. To try to understand the contributions of each domain to the complex Cu(I)-binding behaviour of this and related proteins, we purified a wild-type form of the first domain (CopAa). In isolation, the domain bound Cu(I) with very high affinity (K = ～ 1 × 10(18) m(-1) ) and underwent Cu(I)-mediated protein association, resulting in a mixture of dimer and tetramer species. Addition of further Cu(I) up to 1 Cu(I) per CopAa monomer led to a weakly luminescent species, whereas further additions [2 Cu(I) per CopAa monomer] resulted in protein unfolding. Analysis of the MTCAAC binding motif Cys residue acid-base properties revealed pK(a) values of 5.7 and 7.3, consistent with the pH dependence of Cu(I) binding, and with the proposal that low proton affinity is associated with high Cu(I) affinity. Finally, Cu(I) exchange between CopAa and the chelator bathocuproine sulfonate revealed rapid exchange in both directions, demonstrating an interaction between the protein and the chelator that catalyses metal ion transfer. Overall, CopAa exhibits similarities to CopAab in terms of affinity and complexity of Cu(I) binding, but the details of Cu(I) binding are distinct.     

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.     

Structural basis for the function of the N-terminal domain of the ATPase CopA from Bacillus subtilis

The solution structure of the N-terminal region (151 amino acids) of a copper ATPase, CopA, from Bacillus subtilis, is reported here. It consists of two domains, CopAa and CopAb, linked by two amino acids. It is found that the two domains, which had already been separately characterized, interact one to the other through a hydrogen bond network and a few hydrophobic interactions, forming a single rigid body. The two metal binding sites are far from one another, and the short link between the domains prevents them from interacting. This and the surface electrostatic potential suggest that each domain receives copper from the copper chaperone, CopZ, independently and transfers it to the membrane binding site of CopA. The affinity constants of silver(I) and copper(I) are similar for the two sites as monitored by NMR. Because the present construct "domain-short link-domain" is shared also by the last two domains of the eukaryotic copper ATPases and several residues at the interface between the two domains are conserved, the conclusions of the present study have general validity for the understanding of the function of copper ATPases.     

A core mutation affecting the folding properties of a soluble domain of the ATPase protein CopA from Bacillus subtilis

The two N-terminal domains of the P-type copper ATPase, CopAa and CopAb, from Bacillus subtilis differ in their folding capabilities in vitro. Whereas CopAb has the typical betaalphabetabetaalphabeta structure and is a rigid protein, CopAa is found to be largely unfolded. A sequence analysis of the two and of orthologue homologous proteins indicates that Ser46 in CopAa may destabilise the hydrophobic core, as also confirmed through a bioinformatic energy study. CopAb has a Val in the corresponding position. The S46V and S46A mutants are found to be folded, although the latter displays multiple conformations. S46VCopAa, in both apo and copper(I) loaded forms, has very similar structural and dynamic properties with respect to CopAb, besides a different length of strand beta2 and beta4. It is intriguing that the oxygen of Thr16 is found close, though at longer than bonding distance, to copper in both domains, as it also occurs in a human orthologue domain. This study contributes to understanding the behaviour of proteins that do not properly fold in vitro. A possible biological significance of the peculiar folding behaviour of this domain is discussed.     

Solution structure of the N-terminal domain of a potential copper-translocating P-type ATPase from Bacillus subtilis in the apo and Cu(I) loaded states

A putative partner of the already characterized CopZ from Bacillus subtilis was found, both proteins being encoded by genes located in the same operon. This new protein is highly homologous to eukaryotic and prokaryotic P-type ATPases such as CopA, Ccc2 and Menkes proteins. The N-terminal region of this protein contains two soluble domains constituted by amino acid residues 1 to 72 and 73 to 147, respectively, which were expressed both separately and together. In both cases only the 73-147 domain is folded and is stable both in the copper(I)-free and in the copper(I)-bound forms. The folded and unfolded state is monitored through the chemical shift dispersion of 15N-HSQC spectra. In the absence of any structural characterization of CopA-type proteins, we determined the structure of the 73-147 domain in the 1-151 construct in the apo state through 1H, 15N and 13C NMR spectroscopies. The structure of the Cu(I)-loaded 73-147 domain has been also determined in the construct 73-151. About 1300 meaningful NOEs and 90 dihedral angles were used to obtain structures at high resolution both for the Cu(I)-bound and the Cu(I)-free states (backbone RMSD to the mean 0.35(+/-0.06) A and 0.39(+/-0.07) A, respectively). The structural assessment shows that the structures are accurate. The protein has the typical betaalpha(betabeta)alphabeta folding with a cysteine in the C-terminal part of helix alpha1 and the other cysteine in loop 1. The structures are similar to other proteins involved in copper homeostasis. Particularly, between BsCopA and BsCopZ, only the charges located around loop 1 are reversed for BsCopA and BsCopZ, thus suggesting that the two proteins could interact one with the other. The variability in conformation displayed by the N-terminal cysteine of the CXXC motif in a number of structures of copper transporting proteins suggests that this may be the cysteine which binds first to the copper(I) carried by the partner protein.     

Biochemical characterization of CopA,the Escherichia coli Cu(I)-translocating P-type ATPase

Escherichia coli CopA is a copper ion-translocating P-type ATPase that confers copper resistance. CopA formed a phosphorylated intermediate with [gamma-(32)P]ATP. Phosphorylation was inhibited by vanadate and sensitive to KOH and hydroxylamine, consistent with acylphosphate formation on conserved Asp-523. Phosphorylation required a monovalent cation, either Cu(I) or Ag(I). Divalent cations Cu(II), Zn(II), or Co(II) could not substitute, signifying that the substrate of this copper-translocating P-type ATPase is Cu(I) and not Cu(II). CopA purified from dodecylmaltoside-solubilized membranes similarly exhibited Cu(I)/Ag(I)-stimulated ATPase activity, with a K(m) for ATP of 0.5 mm. CopA has two N-terminal Cys(X)(2)Cys sequences, Gly-Leu-Ser-Cys(14)-Gly-His-Cys(17), and Gly-Met-Ser-Cys(110)-Ala-Ser-Cys(113), and a Cys(479)-Pro-Cys(481) motif in membrane-spanning segment six. The requirement of these cysteine residues was investigated by the effect of mutations and deletions. Mutants with substitutions of the N-terminal cysteines or deletion of the first Cys-(X)(2)-Cys motif formed acylphosphate intermediates. From the copper dependence of phosphoenzyme formation, the mutants appear to have 2-3 fold higher affinity for Cu(I) than wild type CopA. In contrast, substitutions in Cys(479) or Cys(481) resulted in loss of copper resistance, transport and phosphoenzyme formation. These results imply that the cysteine residues of the Cys-Pro-Cys motif (but not the N-terminal cysteine residues) are required for CopA function.     

High Cu(I) and low proton affinities of the CXXC motif of Bacillus subtilis CopZ

CopZ, an Atx1-like copper chaperone from the bacterium Bacillus subtilis, functions as part of a complex cellular machinery for Cu(I) trafficking and detoxification, in which it interacts specifically with the transmembrane Cu(I)-transporter CopA. Here we demonstrate that the cysteine residues of the MXCXXC Cu(I)-binding motif of CopZ have low proton affinities, with both exhibiting pK(a) values of 6 or below. Chelator competition experiments demonstrated that the protein binds Cu(I) with extremely high affinity, with a small but significant pH-dependence over the range pH 6.5-8.0. From these data, a pH-corrected formation constant, beta(2)= approximately 6 x 10(22) M(-2), was determined. Rapid exchange of Cu(I) between CopZ and the Cu(I)-chelator BCS (bathocuproine disulfonate) indicated that the mechanism of exchange does not involve simple dissociation of Cu(I) from CopZ (or BCS), but instead proceeds via the formation of a transient Cu(I)-mediated protein-chelator complex. Such a mechanism has similarities to the Cu(I)-exchange pathway that occurs between components of copper-trafficking pathways.     

Isolation of carcinoembryonic antigen N-terminal domains (N-A1) from soluble aggregates

Carcinoembryonic antigen (CEA) was identified as a prominent tumor-associated antigen in human colorectal cancer and it is still intensively investigated. However, its physiological role remains unclear. The CEA molecule is composed of seven highly hydrophobic, immunoglobulin-like domains, six of which contain a single disulphide bridge. The production of recombinant protein containing Ig-like domains in bacterial expression systems often results in partial degradation or insolubility due to aggregation hampering the analysis of their native structure and function. Here, we present a new method of expression and purification of CEA N-terminal domains (N-A1) fused to MBP in Escherichia coli. In order to optimize the expression and purification of CEA N-A1 domains we evaluated bacteria cultivation conditions, the length of N-A1 domains, fusion systems (GST- and MBP-tag), IPTG concentrations and protein purification conditions. We have found that MBP-N-A1 fusion protein digested with TEV protease forms soluble aggregates composed of N-A1 domains and incompletely digested MBP-N-A1 fusion protein. Using 1.25 M guanidinium chloride (GdmCl) as a component of the elution buffer we were able to achieve an almost complete dissociation of the aggregates. The dissociation was monitored by circular dichroism and fluorescence measurements. The CD spectra and Ellman's assay suggest that the conformation of N-A1 domains and their disulphide bonds are correct.     

Fine Structure of Bacillus subtilis : I. Fixation

The fine structure of Bacillus subtilis has been studied by observing sections fixed in KMnO₄, OsO₄, or a combination of both. The majority of examinations were made in samples fixed in 2.0 per cent KMnO₄ in tap water. Samples were embedded in butyl methacrylate for sectioning. In general, KMnO₄ fixation appeared to provide much better definition of the boundaries of various structures than did OsO₄. With either type of fixation, however, the surface structure of the cell appeared to consist of two components: cell wall and cytoplasmic membrane. Each of these, in turn, was observed to have a double aspect. The cell wall appeared to be composed of an outer part, broad and light, and an inner part, thin and dense. The cytoplasmic membrane appeared (at times, under KMnO₄ fixation) as two thin lines. In cells fixed first with OsO₄ solution, and then refixed with a mixture of KMnO₄ and OsO₄ solutions, the features revealed were more or less a mixture of those revealed by each fixation alone. A homogeneous, smooth structure, lacking a vacuole-like space, was identified as the nuclear structure in a form relatively free of artifacts. Two unidentified structures were observed in the cytoplasm when B. subtilis was fixed with KMnO₄. One a tortuous, fine filamentous element associated with a narrow light space, was often found near the ends of cells, or attached to one end of the pre-spore. The other showed a special inner structure somewhat similar to cristae mitochondriales.     

Functional roles of metal binding domains of the Archaeoglobus fulgidus Cu(+)-ATPase CopA

CopA, a thermophilic membrane ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu(+) or Ag(+) [Mandal et al. (2002) J. Biol. Chem. 277, 7201-7208]. This, as other P(IB)-ATPases, is characterized by a putative metal binding sequence (C(380)PC(382)) in its sixth transmembrane fragment and cytoplasmic metal binding sequences in its NH(2)- and COOH-terminal ends (C(27)AMC(30) and C(751)HHC(754)). Using isolated CopA, we have studied the functional role of these three putative metal binding domains. Replacement of transmembrane Cys residues by Ala results in nonfunctional enzymes that are unable to hydrolyze ATP. However, the CPC --> APA substituted enzyme binds ATP, indicating its correct folding and suggesting that enzyme turnover is prevented by the lack of metal binding to the transmembrane site. Replacement of C-terminal Cys by Ala (C(751,754)A) has no significant effect on ATPase activity, enzyme phosphorylation, apparent binding affinities of ligands, or E1-E2 equilibrium. In contrast, replacement of Cys in the N-terminal metal binding domain (N-MBD) (C(27,30)A) leads to 40% reduction in enzyme turnover. The C(27,30)A enzyme binds Cu(+), Ag(+), and ATP with the same high apparent affinities as the wild-type CopA. Evidence that N-MBD disruption has no effect on the E1-E2 equilibrium is provided by the normal interaction of ATP acting with low affinity and the unaffected IC(50) for vanadate inhibition observed in the C(27,30)A-substituted enzyme. However, replacement C(27,30)A slowed the dephosphorylation of the E2P(metal) form of the enzyme, suggesting a reduction in the rate of metal release. Other investigators have shown the Cu-dependent interaction of isolated N-MBDs from the Wilson disease Cu-ATPase with the ATP binding cytoplasmic domain [Tsivkovskii et al. (2001) J. Biol. Chem. 276, 2234-2242]. Therefore, the data suggest a regulatory mechanism in which the Cu-dependent N-MBD/ATP binding domain interaction would accelerate cation release, the enzyme rate-limiting step, and consequently Cu(+) transport.     

Synthesis and copper(II)-binding properties of the N-terminal peptide of human alpha-fetoprotein.

The N-terminal native sequence tripeptide of alpha-fetoprotein, L-threonyl-L-leucyl-L-histidine N-methylamide, was synthesized and its interaction with Cu(II) ions was investigated by potentiometric titration at 25 degrees C in 0.15 M-NaCl and by visible-absorption, e.p.r. and n.m.r. spectroscopy. Analyses of the results in the pH range 4-10 indicated the presence of multiple complex species in solution: MHL, MH-2L, MHL2, ML2 and MH-1L2, where M, H and L represent metal ion, proton and ligand anion respectively. Only the species MH-2L and MH-1L2 are present in significant amounts at physiological pH. The results of the visible-absorption spectroscopy are consistent with the findings of species distribution that MH-2L is the major complex species detected above physiological pH that has the spectral characteristics of lambda max. = 523 nm and epsilon max. = 98 M-1.cm-1. The nine superhyperfine lines in e.p.r. spectra of the major species MH-2L strongly support the co-ordination of four nitrogen atoms by Cu(II). Both 1H- and 13C-n.m.r. studies suggest that the species MH-2L is a square-planar complex. The results from the equilibrium-dialysis experiments showed that this peptide is able to compete with albumin for Cu(II) ions. At equimolar concentrations of albumin and the peptide, about 52% of the Cu(II) was bound to the peptide. The possibility that alpha-fetoprotein plays an important role as the Cu(II)-transport protein in fetal life is discussed.     

Understanding copper trafficking in bacteria: interaction between the copper transport protein CopZ and the N-terminal domain of the copper ATPase CopA from Bacillus subtilis

In this paper the interaction of cytoplasmic CopZ and the N-terminal domain of the CopA ATPase from Bacillus subtilis has been studied by NMR through (15)N-(1)H HSQC experiments in order to understand the role of the two proteins in the whole copper trafficking mechanism of the bacteria. It appears that the two proteins interact in a fashion similar to that of the yeast homologue proteins [Arnesano, F., Banci, L., Bertini, I., Cantini, F., Ciofi-Baffoni, S., Huffman, D. L., and O'Halloran, T. V. (2001) J. Biol. Chem. 276, 41365-41376], although the surface potentials are reversed. A structural model for the interaction is proposed. (15)N mobility studies on the free proteins and on their complex are also reported. From these data, it appears that copper is largely transferred from CopZ to CopA, thus suggesting their possible involvement in a detoxification process. Comparing functional data of homologous proteins of other bacteria, it can be concluded that this class of proteins is involved in copper homeostasis but the specific roles are species dependent.     

One-dimensional Cu(I) camphor hydrazone polymers: Structure and catalytic properties

Complexes of general formula [{CuX}₂(YNC₁₀H₁₄O)] (X = Cl; Y = NHMe, NH₂ or X = Br; Y = NH₂) were synthesised from camphor hydrazone ligands (YNC₁₀H₁₄O) by reaction with the suitable copper(I) halide. Structural analysis by X-rays performed on a red crystal of [{CuCl}₂(Me₂NNC₁₀H₁₄O)] revealed that the complex is a one-dimensional copper polymer formed by two rather different copper units bridged by chloride. One of the copper units displays a tetrahedral geometry while the other is linear. Although the geometries and neighbourhoods of the two copper units are very different the oxidation state of the metal is the same, i.e. Cu(I) as corroborated by magnetic and electrochemical measurements.The ability of [{CuCl}₂(Me₂NNC₁₀H₁₄O)] to promote the activation of 4-pentyn-1-ol towards cyclization was studied under homogeneous or heterogeneous experimental conditions. The best results were obtained under homogeneous conditions at 40 °C.     

Extracellular nucleases of Bacillus subtilis I. Purification and properties

Four Ca²⁺-dependent nucleases, nucleases Bs-10, Bs-IA, Bs-IB and Bs-II, have been purified from a culture fluid of Bacillus subtilis SB19 and their properties investigated. Nucleases Bs-IA, Bs-IB and Bs-II hydrolyze native and denatured DNA and rRNA in the presence of Ca²⁺. Without the addition of Ca²⁺, denatured DNA and rRNA are degraded at reduced rates and little degradation of native DNA is found. The activities of these enzymes are inhibited by EDTA and other agents which bind Ca²⁺. The mode of action of these enzymes on DNA is predominantly exonucleolytic and the major products from DNA are deoxyribonucleoside 3′-monophosphates. However, an endonuclease activity at the level of one-hundredth of the gross nucleolytic activity is detected in the purified preparations of these enzymes. The enzyme nuclease Bs-10 is different from the other three enzymes in that it hydrolyses only denatured DNA and only to a limited extent.     

Peptides mimicking the N-terminal Cu(II)-binding site of bovine serum albumin: synthesis, characterization and coordination with Cu(II) ions

The N-terminal region of bovine serum albumin (Asp-Thr-His-Lys) is known to provide a specific binding site for Cu(II) ions, with the histidine residue thought to be mainly responsible for the specificity. Thiomolybdates have been found to increase the binding affinity of Cu(II) to some serum albumins. As part of a series of studies to study the interactions between Cu(II), thiomolybdates and bovine serum albumin, we have performed the syntheses and characterization of small model peptides such as His-Lys, Thr(Ac)-His-Lys and Thr-His-Lys. Proton NMR spectra have been monitored in H(2)O solution as a function of pH and added Cu(II) concentration. Reliable K(a) values for His-Lys and Thr(Ac)-His-Lys have been established. Probable binding sites of Cu(II) and the relative strengths of binding to these peptides are also discussed.     

Cell wall binding properties of the Bacillus subtilis autolysin(s)

Cell walls isolated from exponentially growing Bacillus subtilis have autolysin(s) attached to them. An autolysin can be released from the walls by incubation at 0 C with 3 m LiCl. The enzyme can reattach to walls when the salt concentration is reduced. The bound enzyme cannot be removed or destroyed by washing the walls with 8 m urea at 0 C. The binding of free enzyme to walls at 0 C can take place normally in the presence of 2 m urea.     

Structure of the Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster.

A 10.5-kilobase PstI endonuclease fragment encoding the entire Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster was cloned in Escherichia coli by transformation of a carB strain to uracil-independent growth. The cloned fragment also complemented E. coli pyrB, pyrC, pyrD, pyrE, and pyrF mutants. From the ability of subclones to complement E. coli pyr mutants, the gene order was deduced to be pyrBCADFE. The B. subtilis pyrB gene was shown to be expressed in E. coli, but synthesis of the enzyme was not repressible by the addition of uracil to the growth medium. The approximate molecular weights of the polypeptides encoded by B. subtilis pyrA, pyrB, pyrC, pyrD, pyrE, and pyrF were found to be 110,000, 36,000, 46,000, 34,000, 25,000, and 27,000, respectively.     

The ATP-dependent CodWX (HslVU) protease in Bacillus subtilis is an N-terminal serine protease

HslVU is a two-component ATP-dependent protease, consisting of HslV peptidase and HslU ATPase. CodW and CodX, encoded by the cod operon in Bacillus subtilis, display 52% identity in their amino acid sequences to HslV and HslU in Escherichia coli, respectively. Here we show that CodW and CodX can function together as a new type of two-component ATP-dependent protease. Remarkably, CodW uses its N-terminal serine hydroxyl group as the catalytic nucleophile, unlike HslV and certain beta-type subunits of the proteasomes, which have N-terminal threonine functioning as an active site residue. The ATP-dependent proteolytic activity of CodWX is strongly inhibited by serine protease inhibitors, unlike that of HslVU. Replacement of the N-terminal serine of CodW by alanine or even threonine completely abolishes the enzyme activity. These results indicate that CodWX in B.subtilis represents the first N-terminal serine protease among all known proteolytic enzymes.     

Proteins of ribosome-bearing and free-membrane domains in Bacillus subtilis

In lysates of Bacillus subtilis a free-membrane fraction without ribosomes can be separated from the denser membrane-ribosome complexes. As determined by one-dimensional sodium dodecyl sulfate gel electrophoresis, these two fractions differ markedly in protein composition; at least six major bands (molecular weights, 130,000, 92,000, 68,000, 64,000, 45,000, and 31,000) are essentially unique to the complexed-membrane fraction (CM proteins), and two are unique to the free-membrane fraction. After growth was slowed, the proportion of the free-membrane fraction increased, but the composition of this fraction was the same, whereas after puromycin treatment, which abruptly increased the proportion of the free-membrane fraction, this fraction contained CM proteins. Thus, it appears that the two fractions recovered from growing cells represent topographically and functionally distinct domains. In addition, the effect of growth rate suggests that formation of the complexed domain is regulated at least roughly in parallel with the formation of ribosomes. The separation of these membrane fractions should facilitate the study of protein secretion, membrane topography, and morphogenesis in bacteria.