Rapid binding of copper(I) to folded aporusticyanin

Kinetics of copper uptake in both oxidation states by the folded and unfolded forms of the type 1 copper protein rusticyanin have been studied. The speed of the binding of copper(I) to the folded rusticyanin is fast, and of the same order of magnitude as copper(I) uptake by the unfolded form. Thus, the binding of copper can be subsequent to the protein folding, contrary to previous proposals. Implications for the mechanism of the formation of the active holoprotein in vivo are discussed.     

In-cell NMR in E. coli to monitor maturation steps of hSOD1

In-cell NMR allows characterizing the folding state of a protein as well as posttranslational events at molecular level, in the cellular context. Here, the initial maturation steps of human copper, zinc superoxide dismutase 1 are characterized in the E. coli cytoplasm by in-cell NMR: from the apo protein, which is partially unfolded, to the zinc binding which causes its final quaternary structure. The protein selectively binds only one zinc ion, whereas in vitro also the copper site binds a non-physiological zinc ion. However, no intramolecular disulfide bridge formation occurs, nor copper uptake, suggesting the need of a specific chaperone for those purposes.     

Mechanism of Cu,Zn-superoxide dismutase activation by the human metallochaperone hCCS

The mechanism for copper loading of the antioxidant enzyme copper, zinc superoxide dismutase (SOD1) by its partner metallochaperone protein is not well understood. Here we show the human copper chaperone for Cu,Zn-SOD1 (hCCS) activates either human or yeast enzymes in vitro by direct protein to protein transfer of the copper cofactor. Interestingly, when denatured with organic solvents, the apo-form of human SOD1 cannot be reactivated by added copper ion alone, suggesting an additional function of hCCS such as facilitation of an active folded state of the enzyme. While hCCS can bind several copper ions, metal binding studies in the presence of excess copper scavengers that mimic the intracellular chelation capacity indicate a limiting stoichiometry of one copper and one zinc per hCCS monomer. This protein is active and unlike the yeast protein, is a homodimer regardless of copper occupancy. Matrix-assisted laser desorption ionization-mass spectrometry and metal binding studies suggest that Cu(I) is bound by residues from the first and third domains and no bound copper is detected for the second domain of hCCS in either the full-length or truncated forms of the protein. Copper-induced conformational changes in the essential C-terminal peptide of hCCS are consistent with a "pivot, insert, and release" mechanism that is similar to one proposed for the well characterized metal handling enzyme, mercuric ion reductase.     

Characterization and crystal structure of zinc azurin, a by-product of heterologous expression in Escherichia coli of Pseudomonas aeruginosa copper azurin.

Azurin*, a by-product of heterologous expression of the gene encoding the blue copper protein azurin from Pseudomonas aeruginosa in Escherichia coli, was characterized by chemical analysis and electrospray ionization mass spectrometry, and its structure determined by X-ray crystallography. It was shown that azurin* is native azurin with its copper atom replaced by zinc in the metal binding site. Zinc is probably incorporated in the apo-protein after its expression and transport into the periplasm. Holo-azurin can be reconstituted from azurin* by prolonged exposure of the protein to high copper ion concentrations or unfolding of the protein and refolding in the presence of copper ions. An X-ray crystallographic analysis of azurin* at 0.21-nm resolution revealed that the overall structure of azurin is not perturbed by the metal exchange. However, the geometry of the co-ordination sphere changes from trigonal bipyramidal in the case of copper azurin to distorted tetrahedral for the zinc protein. The copper ligand Met121 is no longer co-ordinated to zinc which adopts a position close to the carbonyl oxygen atom from residue Gly45. The polypeptide structure surrounding the metal site undergoes moderate reorganization upon zinc binding. The largest displacement observed is for the carbonyl oxygen from residue Gly45, which is involved in copper and zinc binding. It moves by 0.03 nm towards the zinc, thereby reducing its distance to the metal from 0.29 nm in the copper protein to 0.23 nm in the derivative.     

Methionine-121 coordination determines metal specificity in unfolded<Emphasis Type="Italic"> Pseudomonas aeruginosa</Emphasis> azurin

Pseudomonas aeruginosa azurin binds copper so tightly that it remains bound even upon polypeptide unfolding. Copper can be substituted with zinc without change in protein structure, and also in this complex the metal remains bound upon protein unfolding. Previous work has shown that native-state copper ligands Cys112 and His117 are two of at least three metal ligands in the unfolded state. In this study we use isothermal titration calorimetry and spectroscopic methods to test if the native-state ligand Met121 remains a metal ligand upon unfolding. From studies on a point-mutated version of azurin (Met121Ala) and a set of model peptides spanning the copper-binding C-terminal part (including Cys112, His117 and Met121), we conclude that Met121 is a metal ligand in unfolded copper-azurin but not in the case of unfolded zinc-azurin. Combination of unfolding and metal-titration data allow for determination of copper (Cu(II) and Cu(I)) and zinc affinities for folded and unfolded azurin polypeptides, respectively.     

Generation of novel copper sites by mutation of the axial ligand of amicyanin. Atomic resolution structures and spectroscopic properties

Amicyanin from Paracoccus denitrificans is a type 1 copper protein with three strong equatorial copper ligands provided by nitrogens of His53 and His95 and the sulfur of Cys92, with an additional weak axial ligand provided by the sulfur of Met98. Met98 was replaced with either Gln or Ala. As isolated, the M98A and M98Q mutant proteins contain zinc in the active site. The zinc is then removed and replaced with copper so that the copper-containing proteins may be studied. Each of the mutant amicyanins exhibits a marked decrease in thermal stability relative to that of native amicyanin, consistent with the weaker affinity for copper. Crystal structures were obtained for the oxidized and reduced forms of M98A and M98Q amicyanins at atomic resolution (相似文献   

The role of solvent exclusion in the interaction between D124 and the metal site in SOD1: implications for ALS

Structural changes in the metal site of the copper–zinc superoxide dismutase (SOD1) are involved in the various mechanisms proposed for the pathogenesis of the SOD1-linked familial form of amyotrophic lateral sclerosis (ALS). Elucidating how the metal site of SOD1 can be disrupted by ALS-linked mutations is important for a better understanding of the pathogenesis of the disease and for developing more efficient treatments. Residue D124, a second-sphere ligand of the copper and zinc ions, is known from experimental studies to be essential for the integrity of the metal-site structure. In this work, we used density functional theory calculations and molecular dynamics simulations to elucidate which factors keep D124 attached to the metal site and how structural changes may disrupt the binding between D124 and the metal first-sphere ligands. The calculations show that D124 is kept attached to the metal site in a kinetic trap. The exclusion of solvent molecules by the electrostatic loop of the protein is found to create the binding of D124 to the metal site. The calculations also indicate that changes in the structure of the electrostatic loop of the protein can weaken the D124–metal site interaction, lowering the affinity of the zinc site for the metal. Destabilization of the electrostatic loop of SOD1 has been previously shown to be a common property of ALS-linked variants of the protein, but its role in the pathogenesis of SOD1-linked ALS has not been elucidated.     

Divalent Metal Ion-Induced Folding Mechanism of RNase H1 from Extreme Halophilic Archaeon Halobacterium sp. NRC-1

RNase H1 from Halobacterium sp. NRC-1 (Halo-RNase H1) is characterized by the abundance of acidic residues on the surface, including bi/quad-aspartate site residues. Halo-RNase H1 exists in partially folded (I) and native (N) states in low-salt and high-salt conditions respectively. Its folding is also induced by divalent metal ions. To understand this unique folding mechanism of Halo-RNase H1, the active site mutant (2A-RNase H1), the bi/quad-aspartate site mutant (6A-RNase H1), and the mutant at both sites (8A-RNase H1) were constructed. The far-UV CD spectra of these mutants suggest that 2A-RNase H1 mainly exists in the I state, 6A-RNase H1 exists both in the I and N states, and 8A-RNase H1 mainly exists in the N state in a low salt-condition. These results suggest that folding of Halo-RNase H1 is induced by binding of divalent metal ions to the bi/quad-aspartate site. To examine whether metal-induced folding is unique to Halo-RNase H1, RNase H2 from the same organism (Halo-RNase H2) was overproduced and purified. Halo-RNase H2 exists in the I and N states in low-salt and high-salt conditions respectively, as does Halo-RNase H1. However, this protein exists in the I state even in the presence of divalent metal ions. Halo-RNase H2 exhibits junction ribonuclease activity only in a high-salt condition. A tertiary model of this protein suggests that this protein does not have a quad-aspartate site. We propose that folding of Halo-RNase H1 is induced by binding of divalent metal ion to the quad-aspartate site in a low-salt condition.     

Zinc binding modulates the entire folding free energy surface of human Cu,Zn superoxide dismutase

Over 100 amino acid replacements in human Cu,Zn superoxide dismutase (SOD) are known to cause amyotrophic lateral sclerosis, a gain-of-function neurodegenerative disease that destroys motor neurons. Supposing that aggregates of partially folded states are primarily responsible for toxicity, we determined the role of the structurally important zinc ion in defining the folding free energy surface of dimeric SOD by comparing the thermodynamic and kinetic folding properties of the zinc-free and zinc-bound forms of the protein. The presence of zinc was found to decrease the free energies of a peptide model of the unfolded monomer, a stable variant of the folded monomeric intermediate, and the folded dimeric species. The unfolded state binds zinc weakly with a micromolar dissociation constant, and the folded monomeric intermediate and the native dimeric form both bind zinc tightly, with subnanomolar dissociation constants. Coupled with the strong driving force for the subunit association reaction, the shift in the populations toward more well-folded states in the presence of zinc decreases the steady-state populations of higher-energy states in SOD under expected in vivo zinc concentrations (approximately nanomolar). The significant decrease in the population of partially folded states is expected to diminish their potential for aggregation and account for the known protective effect of zinc. The ∼ 100-fold increase in the rate of folding of SOD in the presence of micromolar concentrations of zinc demonstrates a significant role for a preorganized zinc-binding loop in the transition-state ensemble for the rate-limiting monomer folding reaction in this β-barrel protein.     

Copper binding before polypeptide folding speeds up formation of active (holo) Pseudomonas aeruginosa azurin.

Cofactors often stabilize the native state of the proteins; however, their effects on folding dynamics remain poorly understood. To uncover the role of one cofactor, we have examined the folding kinetics of Pseudomonas aeruginosa azurin, a small blue-copper protein with a copper cofactor uniquely coordinated to five protein residues. Copper removal produces apo-azurin which adopts a folded structure identical to that of the holo-form. The folding and unfolding kinetics for apo-azurin follow two-state behavior. The extrapolated folding time in water, tau approximately 7 ms, is in good agreement with the topology-based prediction. Copper uptake by folded apo-azurin, to govern active (holo) protein, is slow (tau approximately 14 min, 50:1 copper-to-protein ratio). In contrast, the formation of active (holo) azurin is much faster when copper is allowed to interact with the unfolded polypeptide. Refolding in the presence of 10:1, 50:1, and 100:1 copper:protein ratios yields identical time-trajectories: active azurin forms in two kinetic phases with folding times, extrapolated to water, of tau = 10 +/- 2 ms (major phase) and tau = 190 +/- 30 ms (minor phase), respectively. Correlating copper-binding studies, with a small peptide derived from the metal-binding region of azurin, support that initial cofactor binding is fast (tau approximately 3.7 ms) and thus not rate-limiting. Taken together, introducing copper prior to protein folding does not speed up the polypeptide-folding rate; nevertheless, it results in much faster (> 4000-fold) formation of active (i.e., holo) azurin. Living systems depend on efficient formation of functional biomolecules; attachment of cofactors prior to polypeptide folding appears to be one method to achieve this.     

Solution structures of the reduced and Cu(I) bound forms of the first metal binding sequence of ATP7A associated with Menkes disease

The coding sequence for the first N-terminal copper binding motif of the human Menkes disease protein (MNK1; residues 2-79) was synthesized, cloned, and expressed in bacteria for biochemical and structural studies. MNK1 adopts the betaalphabetabetaalphabeta fold common to all the metal binding sequences (MBS) found in other metal transport systems (e.g., the yeast copper chaperone for superoxide dismutase CCS, the yeast copper chaperone ATX1 bound to Hg(II), and most recently Cu(I), the bacterial copper binding protein, CopZ, and the bacterial Hg(II) binding protein MerP), although substantial differences were found in the metal binding loop. Similar to ATX1, MNK1 binds Cu(I) in a distorted linear bicoordinate geometry. As with MerP, MNK1 has a high affinity for both Hg(II) and Cu(I), although it displays a marked preference for Cu(I). In addition, we found that F71 is a key residue in the compact folding of MNK1, and its mutation to alanine results in an unfolded structure. The homologous residue in MerP has also been mutated with similar results. Finally, to understand the relationship between protein folding and metal affinity and specificity, we expressed a chimeric MBS with the MNK1 protein carrying the binding motif of MerP (CAAC-MNK1); this chimeric protein showed differences in structure and the dynamics of the binding site that may account for metal specificity.     

Analysis of the non-covalent interaction between metal ions and the cysteine-rich domain of protein kinase C eta by electrospray ionization mass spectrometry

Effect of zinc and other metal ions on the folding of the protein kinase C (PKC) surrogate peptide (PKCeta-C1B) was analyzed intact under neutral conditions by electrospray ionization mass spectrometry (ESI-MS). ESI-MS spectrum of 64ZnCl(2)-folded PKCeta-C1B clearly showed that PKCeta-C1B coordinates specifically two atoms of zinc, and that the two thiol protons are lost in each zinc ion coordinate center. 113CdCl(2)-folded PKCeta-C1B also showed stoichiometry of two cadmium atoms that was proved by addition of EDTA. The dissociation constants of zinc- and cadmium-folded PKCeta-C1B in the phorbol 12,13-dibutyrate binding (PDBu) were similar (0.66 and 0.81 nM) with different B(max) values (46.4 and 71.4%). The difference would reflect higher coordination potency of cadmium ion that was demonstrated by ESI-MS when PKCeta-C1B was folded by 1:1 mixture of zinc and cadmium ions. In contrast, 63CuCl(2)-treated PKCeta-C1B did not show any copper-coordinated peak, instead a molecular mass less than 6 mass units smaller than that of apo-PKCeta-C1B was observed. The multiple charge mass envelope of copper-treated PKCeta-C1B shifted to that of the lower mass charge state like zinc-treated PKCeta-C1B. These data suggest that the copper treatment formed three intramolecular S-S bonds to abolish the PDBu binding of PKCeta-C1B.     

Studies of thermally induced denaturation of azurin and azurin derivatives by differential scanning calorimetry: evidence for copper selectivity

Azurin, a blue copper protein from Pseudomonas aeruginosa, and several derivatives of azurin have been studied by differential scanning calorimetry. Two well-separated, irreversible transitions are observed in a scan of apoazurin under a variety of conditions, and they are assigned to distinct steps in the denaturation process. No specific structural component can be assigned to the lower temperature transition, but a "flap" structure which is found near the metal binding site may be involved. Circular dichroic spectra suggest that melting of the beta-sheet structure, the main structural motif in the native protein, occurs during the second transition. With the exceptions of the Ni(II) and p-(hydroxymercuri)benzoate derivatives, the transitions are superposed in the metalated forms, and the enthalpies of denaturation are more endothermic. By comparison with other first-row divalent transition ions and especially Zn(II), the Cu(II) derivative exhibits the most endothermic denaturation process. Along with other data, this suggests that the binding energy is greater for Cu(II). It is postulated that the selectivity for copper over zinc arises because of the irregular binding geometry offered by the folded protein. Denaturation of the Hg(II) derivative is even more endothermic, confirming that the type 1 binding site has a very great affinity for Hg(II). Finally, when substoichiometric amounts of Hg(II) are added to the apoprotein, there is evidence that a novel mercury-bridged dimer of azurin forms.     

Replacement of the axial copper ligand methionine with lysine in amicyanin converts it to a zinc-binding protein that no longer binds copper

The mutation of the axial ligand of the type I copper protein amicyanin from Met to Lys results in a protein that is spectroscopically invisible and redox inactive. M98K amicyanin acts as a competitive inhibitor in the reaction of native amicyanin with methylamine dehydrogenase indicating that the M98K mutation has not affected the affinity for its natural electron donor. The crystal structure of M98K amicyanin reveals that its overall structure is very similar to native amicyanin but that the type I binding site is occupied by zinc. Anomalous difference Fourier maps calculated using the data collected around the absorption edges of copper and zinc confirm the presence of Zn²⁺ at the type I site. The Lys98 NZ donates a hydrogen bond to a well-ordered water molecule at the type I site which enhances the ability of Lys98 to provide a ligand for Zn²⁺. Attempts to reconstitute M98K apoamicyanin with copper resulted in precipitation of the protein. The fact that the M98K mutation generated such a selective zinc-binding protein was surprising as ligation of zinc by Lys is rare and this ligand set is unique for zinc.     

The Folding Free-Energy Surface of HIV-1 Protease: Insights into the Thermodynamic Basis for Resistance to Inhibitors

Spontaneous mutations at numerous sites distant from the active site of human immunodeficiency virus type 1 protease enable resistance to inhibitors while retaining enzymatic activity. As a benchmark for probing the effects of these mutations on the conformational adaptability of this dimeric β-barrel protein, the folding free-energy surface of a pseudo-wild-type variant, HIV-PR^?, was determined by a combination of equilibrium and kinetic experiments on the urea-induced unfolding/refolding reactions. The equilibrium unfolding reaction was well described by a two-state model involving only the native dimeric form and the unfolded monomer. The global analysis of the kinetic folding mechanism reveals the presence of a fully folded monomeric intermediate that associates to form the native dimeric structure. Independent analysis of a stable monomeric version of the protease demonstrated that a small-amplitude fluorescence phase in refolding and unfolding, not included in the global analysis of the dimeric protein, reflects the presence of a transient intermediate in the monomer folding reaction. The partially folded and fully folded monomers are only marginally stable with respect to the unfolded state, and the dimerization reaction provides a modest driving force at micromolar concentrations of protein. The thermodynamic properties of this system are such that mutations can readily shift the equilibrium from the dimeric native state towards weakly folded states that have a lower affinity for inhibitors but that could be induced to bind to their target proteolytic sites. Presumably, subsequent secondary mutations increase the stability of the native dimeric state in these variants and, thereby, optimize the catalytic properties of the resistant human immunodeficiency virus type 1 protease.     

Copper coordination in blue proteins

The spectroscopic and electrochemical properties of blue copper proteins are strikingly different from those of inorganic copper complexes in aqueous solution. Over three decades ago this unusual behavior was ascribed to constrained coordination in the folded protein; consistent with this view, crystal structure determinations of blue proteins have demonstrated that the ligand positions are essentially unchanged on reduction as well as in the apoprotein. Blue copper reduction potentials are tuned to match the particular function of a given protein by exclusion of water from the metal site and strict control of the positions of axial ligands in the folded structure. Extensive experimental work has established that the reorganization energy of a prototypal protein, Pseudomonas aeruginosa azurin, is approximately 0.7 eV, a value that is much lower than those of inorganic copper complexes in aqueous solution. The lowered reorganization energy in the protein, which is attributable to constrained coordination, is critically important for function, since the driving forces for electron transfer often are low (approximately 0.1 eV) between blue copper centers and distant (>10 A) donors and acceptors.     

An NMR view of the unfolding process of rusticyanin: Structural elements that maintain the architecture of a beta-barrel metalloprotein

The unfolding process of the blue copper protein rusticyanin (Rc) as well as its dynamic and D(2)O/H(2)O exchange properties in an incipient unfolded state have been studied by heteronuclear NMR spectroscopy. Titrations of apo, Cu(I), and Cu(II)Rc with guanidinium chloride (GdmCl) show that the copper ion stabilizes the folded species and remains bound in the completely unfolded state. The oxidized state of the copper ion is more efficient than the reduced form in this respect. The long loop of Rc (where the first ligand of the copper ion is located) is one of the most mobile domains of the protein. This region has no defined secondary structure elements and is prone to exchange its amide protons. In contrast, the last loop (including a short alpha-helix) and the last beta-strand (where the other three ligands of the metal ion are located) form the most rigid domain of the protein. The results taken as a whole suggest that the first ligand detaches from the metal ion when the protein unfolds, while the other three ligands remain bound to it. The implications of these findings for the biological folding process of Rc are also discussed.     

Solution structure of Apo Cu,Zn superoxide dismutase: role of metal ions in protein folding

The solution structure of the demetalated copper, zinc superoxide dismutase is obtained for the monomeric Glu133Gln/Phe50Glu/Gly51Glu mutant through NMR spectroscopy. The demetalated protein still has a well-defined tertiary structure; however, two beta-strands containing two copper ligands (His46 and His48, beta4) and one zinc ligand (Asp83, beta5) are shortened, and the sheet formed by these strands and strands beta7 and beta8 moves away from the other strands of the beta-barrel to form an open clam with respect to a closed conformation in the holoprotein. Furthermore, loop IV which contains three zinc ligands (His63, His71, and His80) and loop VII which contributes to the definition of the active cavity channel are severely disordered, and experience extensive mobility as it results from thorough (15)N relaxation measurements. These structural and mobility data, if compared with those of the copper-depleted protein and holoprotein, point out the role of each metal ion in the protein folding, leading to the final tertiary structure of the holoprotein, and provide hints for the mechanisms of metal delivery by metal chaperones.     

Import of small Tim proteins into the mitochondrial intermembrane space

Proteins of the intermembrane space (IMS) of mitochondria are typically synthesized without presequences. Little is known about their topogenesis. We used Tim13, a member of the 'small Tim protein' family, as model protein to investigate the mechanism of translocation into the IMS. Tim13 contains four conserved cysteine residues that bind a zinc ion as cofactor. Import of Tim13 did not depend on the membrane potential or ATP hydrolysis. Upon import into mitochondria Tim13 adopted a stably folded conformation in the IMS. Mutagenesis of the cysteine residues or pretreatment with metal chelators interfered with folding of Tim13 in vitro and impaired its import into mitochondria. Upon depletion of metal ions or modification of cysteine residues, imported Tim13 diffused back out of the IMS. We propose an import pathway in which (1) Tim13 can pass through the TOM complex into and out of the IMS in an unfolded conformation, and (2) cofactor acquisition stabilizes folding on the trans side of the outer membrane and traps Tim13 in the IMS, and drives unidirectional movement of the protein across the outer membrane of mitochondria.