Zinc fingers and thiol-disulfide oxidoreductase activities of chaperone DnaJ.

Chaperone DnaJ is a homodimer with each subunit containing 10 cysteine residues and two Zn(II) ions, which have been identified to form two zinc fingers, C(144)DVC(147)Zn(II)C(197)NKC(200) (Zn1) and C(161)PTC(164)Zn(II)C(183)PHC(186) (Zn2), with C(265) and C(323) in reduced form. Guanidine hydrochloride at 6.4 M destroys only Zn1, which does not reform after refolding. p-Hydroxymercuriphenylsulfonate acid, but not ethylenediaminetetraacetic acid (EDTA) even at high concentrations, can remove two Zn(II) ions from DnaJ, but only Zn2 can be reconstituted. After removal of Zn(II) ions, only C(144) and C(147) in Zn1 are oxidation-resistant, and the other six cysteines are easily oxidizable. DnaJ shows reductase activity and oxidase activity but little, if any, isomerase activity. The reductase activity is reversibly inhibited by EDTA. Zn2 is important for the enzymatic activity, and only -C(183)PHC(186)- among the four motifs of -CXXC- functions as the active site of the enzyme. A C-terminal (Q(181)-R(376)) fragment shows a zinc finger of C(183)PHC(186)Zn(II)C(197)NKC(200) and full enzymatic activity of DnaJ. The N-terminal half sequence (M(1)-Q(180)) and Zn1 are not required for the enzymatic activity but are important for the chaperone activity of DnaJ.     

The C-terminal (331-376) sequence of Escherichia coli DnaJ is essential for dimerization and chaperone activity: a small angle X-ray scattering study in solution

DnaJ, an Escherichia coli Hsp40 protein composed of 376 amino acid residues, is a chaperone with thioldisulfide oxidoreductase activity. We present here for the first time a small angle x-ray scattering study of intact DnaJ and a truncated version, DnaJ (1-330), in solution. The molecular weight of DnaJ and DnaJ (1-330) determined by both small angle x-ray scattering and size-exclusion chromatography provide direct evidence that DnaJ is a homodimer and DnaJ (1-330) is a monomer. The restored models show that DnaJ is a distorted omega-shaped dimeric molecule with the C terminus of each subunit forming the central part of the omega, whereas DnaJ (1-330) exists as a monomer. This indicates that the deletion of the C-terminal 46 residues of DnaJ impairs the association sites, although it does not cause significant conformational changes. Biochemical studies reveal that DnaJ (1-330), while fully retaining its thiol-disulfide oxidoreductase activity, is structurally less stable, and its peptide binding capacity is severely impaired relative to that of the intact molecule. Together, our results reveal that the C-terminal (331-376) residues are directly involved in dimerization, and the dimeric structure of DnaJ is necessary for its chaperone activity but not required for the thiol-disulfide oxidoreductase activity.     

Roles of two conserved amino acid residues in the active site of galactose-1-phosphate uridylyltransferase: an essential serine and a nonessential cysteine

Galactose-1-phosphate uridylyltransferase (GalT) catalyzes the reversible transformation of uridine 5'-diphosphate glucose (UDPGlc) and galactose-1-phosphate into uridine 5'-diphosphate galactose (UDPGal) and glucose-1-phosphate through a double displacement mechanism, with the intermediate formation of a covalent uridylyl-enzyme (UMP-enzyme). The covalent linkage is a phosphoramidate formed between the UMP moiety and the His 166 N(epsilon)(2) of GalT, with His 166 N(delta1) retaining a proton throughout the catalytic cycle. Cys 160 and Ser 161 in Escherichia coli GalT are engaged in hydrogen bonding with the peripheral phosphoryl oxygen atoms of the substrate in the crystalline UMP-enzyme and in the crystalline complex of H166G-GalT with UDPGlc [Wedekind, J. E., Frey, P. A., and Rayment, I. (1996) Biochemistry 35, 11560-11569; Thoden, J. B., Ruzicka, F. J., Frey, P. A., Rayment, I., and Holden, H. M. (1997) Biochemistry 36, 1212-1222]. Site-directed mutagenesis, thermodynamic, transient kinetic, and steady-state kinetic studies have been performed to investigate the roles of Cys 160 and Ser 161 in catalysis. The absence of the thiol group of Cys 160 in the variants C160S and C160A did not seriously alter the enzymatic activity. However, the variant S161A displayed 7000-fold less activity than wild-type GalT. The low activity of S161A was directly related to impaired uridylylation rate constant (3.7 x 10(-)(2) s(-)(1)) and de-uridylylation rate constant (0.5 x 10(-)(2) s(-)(1)) resulting from a higher kinetic barrier for uridylyl-group transfer by the variant S161A as compared with the wild-type GalT. Equilibrium uridylylation studies showed that neither Cys 160 nor Ser 161 was involved in stabilizing the uridylyl-enzyme intermediate. The results lead to the conclusion that the conserved Cys 160 does not play a critical role in catalysis. Ser 161 is most likely involved in donating a hydrogen bond to the beta-phosphoryl group of a substrate, thereby providing proper orientation for nucleophilic catalysis.     

Kinetic properties and metal content of the metallo-beta-lactamase CcrA harboring selective amino acid substitutions.

The crystal structure of the metallo-beta-lactamase CcrA3 indicates that the active site of this enzyme contains a binuclear zinc center. To aid in assessing the involvement of specific residues in beta-lactam hydrolysis and susceptibility to inhibitors, individual substitutions of selected amino acids were generated. Substitution of the zinc-ligating residue Cys181 with Ser (C181S) resulted in a significant reduction in hydrolytic activity; kcat values decreased 2-4 orders of magnitude for all substrates. Replacement of His99 with Asn (H99N) significantly reduced the hydrolytic activity for penicillin and imipenem. Replacement of Asp103 with Asn (D103N) showed reduced hydrolytic activity for cephaloridine and imipenem. Deletion of amino acids 46-51 dramatically reduced both the hydrolytic activity and affinity for all beta-lactams. The metal binding capacity of each mutant enzyme was examined using nondenaturing electrospray ionization mass spectrometry. Two zinc ions were observed for the wild-type enzyme and most of the mutant enzymes. However, for the H99N, C181S, and D103N enzymes, three different zinc content patterns were observed. These enzymes contained two zinc molecules, one zinc molecule, and a mixture of one or two zinc molecules/enzyme molecule, respectively. Two enzymes with substitutions of Cys104 or Cys104 and Cys155 were also composed of mixed enzyme populations.     

Structural effects of the active site mutation cysteine to serine in Bacillus cereus zinc-beta-lactamase

Beta-lactamases are involved in bacterial resistance. Members of the metallo-enzyme class are now found in many pathogenic bacteria and are becoming thus of major clinical importance. Despite the availability of Zn-beta-lactamase X-ray structures their mechanism of action is still unclear. One puzzling observation is the presence of one or two zincs in the active site. To aid in assessing the role of zinc content in beta-lactam hydrolysis, the replacement by Ser of the zinc-liganding residue Cys168 in the Zn-beta-lactamase from Bacillus cereus strain 569/H/9 was carried out: the mutant enzyme (C168S) is inactive in the mono-Zn form, but active in the di-Zn form. The structure of the mono-Zn form of the C168S mutant has been determined at 1.85 A resolution. Ser168 occupies the same position as Cys168 in the wild-type enzyme. The protein residues mostly affected by the mutation are Asp90-Arg91 and His210. A critical factor for the activity of the mono-Zn species is the distance between Asp90 and the Zn ion, which is controlled by Arg91: a slight movement of Asp90 impairs catalysis. The evolution of a large superfamily including Zn-beta-lactamases suggests that they may not all share the same mechanism.     

Cysteine-to-serine mutants of the human copper chaperone for superoxide dismutase reveal a copper cluster at a domain III dimer interface

Cysteine-to-serine mutants of a maltose binding protein fusion with the human copper chaperone for superoxide dismutase (hCCS) were studied with respect to (i) their ability to transfer Cu to E,Zn superoxide dismutase (SOD) and (ii) their Zn and Cu binding and X-ray absorption spectroscopic (XAS) properties. Previous work has established that Cu(I) binds to four cysteine residues, two of which, C22 and C25, reside within an Atox1-like N-terminal domain (DI) and two of which, C244 and C246, reside in a short unstructured polypeptide chain at the C-terminus (DIII). The wild-type (WT) protein shows an extended X-ray absorption fine structure (EXAFS) spectrum characteristic of cluster formation, but it is not known how such a cluster is formed. Cys to Ser mutagenesis was used to investigate the Cu binding in more detail. Single Cys to Ser mutations, as represented by C22S and C244S, did little to affect the metal binding ratios of hCCS. Both mutants still showed approximately 2 Cu(I) ions and 1 Zn ion per protein. The double mutants C22/24S and C244/246S, on the other hand, showed Cu binding stoichiometries close to 1:1. The Zn-EXAFS of WT CCS showed a 3-4 histidine ligand environment that is consistent with Zn binding in the SOD-like domain II of CCS. The Zn environment remained unchanged between wild type and all of the mutant CCS proteins. Single Cys to Ser mutations displayed lower activity than WT protein, although close to full activity could be rescued by increasing the CCS:SOD ratios to 8:1 in the assay mixture. The structure of the Cu centers of the single mutants as revealed by EXAFS was also similar to that of WT protein, with clear indications of a Cu cluster. On the other hand, the double mutants showed a greater degree of perturbation. The DI C22/25S mutant was 70% active and formed a cluster with a more intense Cu-Cu interaction. The DIII C244/246S mutant retained only a fraction (16%) of activity and did not form a cluster. The results suggest the formation of a DIII-DIII cluster within a dimeric or tetrameric protein and further suggest that this cluster may be an important element of the copper transfer machinery.     

Effect of pH on the active site of an Arg121Cys mutant of the metallo-beta-lactamase from Bacillus cereus: implications for the enzyme mechanism

The zinc-dependent metallo-beta-lactamases are a group of bacterial enzymes that pose a threat to the future efficacy of present-day antibiotics. Their mechanism is poorly understood, and there are no clinically useful inhibitors. While most members of the group contain two tightly bound zinc ions in their active sites, the Bacillus cereus enzyme has a much lower affinity for its second zinc (Zn2), thought to be due to the presence of Arg121 immediately beneath the floor of the active site (cf. Cys/Ser/His121 in the bizinc enzymes). Crystal structures of the Arg121Cys mutant of the B. cereus 569/H/9 enzyme were solved at pH 7.0, 5.0, and 4.5, each in the presence of either 20 microM or 20 mM Zn(2+) to generate the mono- and bizinc forms, respectively. Surprisingly, the structure of the active site was unaffected by the mutation; a network of ordered water molecules replaced the interactions made by the arginine side chain, and the occupancy of Zn2 appeared minimally changed. As the pH was lowered, Zn2 moved away from one of its ligands, Asp120, but was "tracked" by two others, Cys221 and His263. Furthermore, the hydroxide ion (and proposed nucleophile for beta-lactam hydrolysis) was bound to Zn1 at pH 5 and above but absent at pH 4.5. This provides experimental evidence for an earlier proposed mechanism in which protonation of Asp120 and the Zn1-bound hydroxide are the two events that lead to the loss of activity at low pH.     

Zn(II) coordination domain mutants of T4 gene 32 protein.

Gene 32 protein (g32P), the replication accessory single-stranded nucleic acid binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol of protein. Zinc coordination provides structural stability to the DNA-binding core domain of the molecule, termed g32P-(A+B) (residues 22-253). Optical absorption studies with the Co(II)-substituted protein and 113Cd NMR spectroscopy of 113Cd(II)-substituted g32P-(A+B) show that the metal coordination sphere in g32P is characterized by approximately tetrahedral ligand symmetry and ligation by the Cys-S- atoms of Cys77, Cys87, and Cys90. These studies predicted the involvement of a fourth protein-derived non-thiol ligand to complete the tetrahedral complex, postulated to be His81 on the basis of primary structure prediction and modeling [Giedroc, D.P., Johnson, B.A., Armitage, I.M., & Coleman, J.E. (1989) Biochemistry 28, 2410-2418]. To test this model, we have employed site-directed mutagenesis to substitute each of the two histidine residues in g32P (His64 and His81), accompanied by purification and structural characterization of these single-site mutant proteins. We show that g32P's containing any of three substitutions at residue 64 (H64Q, H64N, and H64L) are isolated from Escherichia coli in a Zn(II)-free form [less than or equal to 0.03 g.atom Zn(II)]. All derivatives show extremely weak affinity for the ssDNA homopolymer poly(dT). All are characterized by a far-UV-CD spectrum reduced in negative intensity relative to the wild-type protein. These structural features parallel those found for the known metal ligand mutant Cys87----Ser87 (C87S) g32P. In contrast, g32P-(A+B) containing a substitution of His81 with glutamine (H81Q), alanine (H81A) or cysteine (H81C), contains stoichiometric Zn(II) as isolated and binds to polynucleotides with an affinity comparable to the wild-type g32P-(A+B). Spin-echo 1H NMR spectra recorded for wild-type and H81Q g32P-(A+B) as a function of pH allow the assignment of His81 ring proteins to delta = 6.81 and 6.57 ppm, respectively, at pH 7.8, corresponding to the C and D histidyl protons of 1H-His-g32P-(A+B) [Pan, T., Giedroc, D.P., & Coleman, J.E. (1989) Biochemistry 28, 8828-8832]. These resonances shift downfield as the pH is reduced from 7.8 to 6.6 without metal dissociation, a result incompatible with His81 donating a ligand to the Zn(II) in wild-type g32P. Likewise, Cys81 in Zn(II) H81C g32P is readily reactive with 5,5'-dithiobis(2-nitrobenzoic acid), unlike metal ligands Cys77, Cys87, and Cys90.(ABSTRACT TRUNCATED AT 400 WORDS)     

The roles of the two zinc binding sites in DnaJ

All type I DnaJ (Hsp40) homologues share the presence of two highly conserved zinc centers. To elucidate their function, we constructed DnaJ mutants that separately replaced cysteines of either zinc center I or zinc center II with serine residues. We found that in the absence of zinc center I, the autonomous, DnaK-independent chaperone activity of DnaJ is dramatically reduced. Surprisingly, this only slightly impaired the in vivo function of DnaJ, and its ability to function as a co-chaperone in the DnaK/DnaJ/GrpE foldase machine. The DnaJ zinc center II, on the other hand, was found to be absolutely essential for the in vivo and in vitro function of DnaJ. This did not seem to be caused by a lack of substrate binding affinity or an inability to work as an ATPase-stimulating factor. Rather it appears that zinc center II mutant proteins lack a necessary additional interaction site with DnaK, which seems to be crucial for locking-in substrate proteins onto DnaK. These findings led us to a model in which ATP hydrolysis in DnaK is only the first step in converting DnaK into its high affinity binding state. Additional interactions between DnaK and DnaJ are required to make the DnaK/DnaJ/GrpE foldase machinery catalytically active.     

Characterization of catalytic centre mutants of macrophage migration inhibitory factor (MIF) and comparison to Cys81Ser MIF.

Macrophage migration inhibitory factor (MIF) displays both cytokine and enzyme activities, but its molecular mode of action is still unclear. MIF contains three cysteine residues and we showed recently that the conserved Cys57-Ala-Leu-Cys60 (CALC) motif is critical for the oxidoreductase and macrophage-activating activities of MIF. Here we probed further the role of this catalytic centre by expression, purification, and characterization of the cysteine-->serine mutants Cys60Ser, Cys57Ser/Cys60Ser, and Cys81Ser of human MIF and of mutants Ala58Gly/Leu59Pro and Ala58Gly/Leu59His, containing a thioredoxin (Trx)-like and protein disulphide isomerase (PDI)-like dipeptide, respectively. The catalytic centre mutants formed inclusion bodies and the resultant mutant proteins Cys57Ser/Cys60Ser, Ala58Gly/Leu59Pro, and Als58Gly/Leu59His were only soluble in organic solvent or 6 m GdmHCl when reconstituted at concentrations above 1 microgram.mL-1. This made it necessary to devise new purification methods. By contrast, mutant Cys81Ser was soluble. Effects of pH, solvent, and ionic strength conditions on the conformation of the mutants were analysed by far-UV CD spectropolarimetry and mutant stability was examined by denaturant-induced unfolding. The mutants, except for mutant Cys81Ser, showed a close conformational similarity to wild-type (wt) MIF, and stabilization of the mutants was due mainly to acid pH conditions. Intramolecular disulphide bond formation at the CALC region was confirmed by near-UV CD of mutant Cys60Ser. Mutant Cys81Ser was not involved in disulphide bond formation, yet had decreased stability. Analysis in the oxidoreductase and a MIF-specific cytokine assay revealed that only substitution of the active site residues led to inactivation of MIF. Mutant Cys60Ser had no enzyme and markedly reduced cytokine activity, whereas mutant Cys81Ser was active in both tests. The Trx-like variant showed significant enzyme activity but was less active than wtMIF; PDI-like MIF was enzymatically inactive. However, both variants had full cytokine activity. Together with the low but nonzero cytokine activity of mutant Cys60Ser, this indicated that the cytokine activity of MIF may not be tightly regulated by redox effects or that a distinguishable receptor mechanism exists. This study provides evidence for a role of the CALC motif in the oxidoreductase and cytokine activities of MIF, and suggests that Cys81 could mediate conformational effects. Availability and characterization of the mutants should greatly aid in the further elucidation of the mechanism of action of the unusual cytokine MIF.     

Significance of metal ions in galactose-1-phosphate uridylyltransferase: an essential structural zinc and a nonessential structural iron.

Galactose-1-phosphate uridylyltransferase (GalT) catalyzes the reversible transformation of UDP-glucose and galactose-1-phosphate (Gal-1-P) into UDP-galactose and glucose-1-phosphate (Glc-1-P) by a double displacement mechanism, with the intermediate formation of a covalent uridylyl-enzyme (UMP-enzyme). GalT is a metalloenzyme containing 1.2 mol of zinc and 0.7 mol of iron/mol of subunits [Ruzicka, F. J., Wedekind, J. E., Kim, J., Rayment, I., and Frey, P. A. (1995) Biochemistry 34, 5610-5617]. The zinc site lies 8 A from His 166 in active site, and the iron site lies 30 A from the active site [Wedekind,J. E., Frey, P. A., & Rayment, I. (1995) Biochemistry 34, 11049-11061]. Zinc is coordinated in tetrahedral geometry by Cys 52, Cys 55, His 115, and His 164. His 164 is part of the highly conserved active-site triad His 164-Pro 165-His 166, in which His 166 is the nucleophilic catalyst. Iron is coordinated in square pyramidal geometry with His 296, His 298, and Glu 182 in bidentate coordination providing the base ligands and His 281 providing the axial ligand. In the present study, site-directed mutagenesis, kinetic, and metal analysis studies show that C52S-, C55S-, and H164N-GalT are 3000-, 600-, and 10000-fold less active than wild-type. None of the variants formed the UMP-enzyme in detectable amounts upon reaction with UDP-Glc in the absence of Gal-1-P. Their zinc content was very low, and the zinc + iron content was about 50% of that for wild-type GalT. Mutation of His 115 to Asn 115 resulted in decreased activity to 2.9% of wild-type, with retention of zinc and iron. In contrast to the zinc-binding site, Glu 182 in the iron site is not important for enzymatic activity. The variant E182A-GalT displayed about half the activity of wild-type GalT, and all of the active sites underwent uridylylation to the UMP-enzyme, similar to wild-type GalT, upon reaction with UDP-Glc. Metal analysis showed that while E182A-GalT contained 0.9 equiv of zinc/subunit, it contained no iron. The residual zinc can be removed by dialysis with 1,10-phenanthroline, with the loss in activity being proportional to the amount of residual zinc. It is concluded that the presence of zinc is essential for maintaining GalT function, whereas the presence of iron is not essential.     

Modulation of zinc- and cobalt-binding affinities through changes in the stability of the zinc ribbon protein L36

Cysteine-rich Zn(II)-binding sites in proteins serve two distinct functions: to template or stabilize specific protein folds, and to facilitate chemical reactions such as alkyl transfers. We are interested how the protein environment controls metal site properties, specifically, how naturally occurring tetrahedral Zn(II) sites are affected by the surrounding protein. We have studied the Co(II)- and Zn(II)-binding of a series of derivatives of L36, a small zinc ribbon protein containing a (Cys)(3)His metal coordination site. UV-vis spectroscopy was used to monitor metal binding by peptides at pH 6.0. For all derivatives, the following trends were observed: (1) Zn(II) binds tighter than Co(II), with an average K (A) (Zn) /K (A) (Co) of 2.8(+/-2.0)x10(3); (2) mutation of the metal-binding ligand His32 to Cys decreases the affinity of L36 derivatives for both metals; (3) a Tyr24 to Trp mutation in the beta-sheet hydrophobic cluster increases K (A) (Zn) and K (A) (Co) ; (4) mutation in the beta-hairpin turn, His20 to Asn generating an Asn-Gly turn, also increases K (A) (Zn) and K (A) (Co) ; (5) the combination of His20 to Asn and Tyr24 to Trp mutations also increases K (A) (Zn) and K (A) (Co) , but the increments versus C(3)H are less than those of the single mutations. Furthermore, circular dichroism, size-exclusion chromatography, and 1D and 2D (1)H NMR experiments show that the mutations do not change the overall fold or association state of the proteins. L36, displaying Co(II)- and Zn(II)-binding sensitivity to various sequence mutations without undergoing a change in protein structure, can therefore serve as a useful model system for future structure/reactivity studies.     

Computer simulation of Zn(II) speciation and effect of Gd(III) on Zn(II) speciation in human blood plasma

The speciation and distribution of Zn(II) and the effect of Gd(III) on Zn(II) speciation in human blood plasma were studied by computer simulation. The results show that, in normal blood plasma, the most predominant species of Zn(II) are [Zn(HSA)] (58.2%), [Zn(IgG)](20.1%), [Zn(Tf)] (10.4%), ternary complexes of [Zn(Cit)(Cys)] (6.6%) and of [Zn(Cys)(His)H] (1.6%), and the binary complex of [Zn(Cys)₂H] (1.2%). When zinc is deficient, the distribution of Zn(II) species is similar to that in normal blood plasma. Then, the distribution changes with increasing zinc(II) total concentration. Overloading Zn(II) is initially mainly bound to human serum albumin (HSA). As the available amount of HSA is exceeded, phosphate metal and carbonate metal species are established. Gd(III) entering human blood plasma predominantly competes for phosphate and carbonate to form precipitate species. However, Zn(II) complexes with phosphate and carbonate are negligible in normal blood plasma, so Gd(III) only have a little effect on zinc(II) species in human blood plasma at a concentration above 1.0×10⁻⁴ M.     

Nitric oxide inhibits the cochaperone activity of the RING finger-like protein DnaJ.

As a consequence of bacterial infection and the ensuing inflammation, expression of the inducible NO synthase results in prolonged synthesis of NO in high concentrations, which among other functions, contributes to the innate defense against the infectious agent. Here we show that NO inhibits the ability of the bacterial cochaperone DnaJ containing a RING finger-like domain to cooperate with the Hsp70 chaperone DnaK in mediating correct folding of denatured rhodanese. This inhibition is accompanied by S-nitrosation of DnaJ as well as by Zn2+ release from the protein. In contrast, NO has no effect on the activity of GroEL, a bacterial chaperone without zinc sulfur clusters. Escherichia coli cells lacking the chaperone trigger factor and thus relying on the DnaJ/DnaK system are more susceptible toward NO-mediated cytostasis than are wild-type bacteria. Our studies identify the cochaperone DnaJ as a molecular target for NO. Thus, an encounter of bacterial cells with NO can impair the protein folding activity of the bacterial chaperone system, thereby increasing bacterial susceptibility toward the defensive attack by the host.     

Identification of the zinc ligands in cobalamin-independent methionine synthase (MetE) from Escherichia coli

Cobalamin-independent methionine synthase (MetE) from Escherichia coli catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to form tetrahydrofolate and methionine. It contains 1 equiv of zinc that is essential for its catalytic activity. Extended X-ray absorption fine structure analysis of the zinc-binding site has suggested tetrahedral coordination with two sulfur (cysteine) and one nitrogen or oxygen ligands provided by the enzyme and an exchangeable oxygen or nitrogen ligand that is replaced by the homocysteine thiol group in the enzyme-substrate complex [González, J. C., Peariso, K., Penner-Hahn, J. E., and Matthews, R. G. (1996) Biochemistry 35, 12228-34]. Sequence alignment of MetE homologues shows that His641, Cys643, and Cys726 are the only conserved residues. We report here the construction, expression, and purification of the His641Gln, Cys643Ser, and Cys726Ser mutants of MetE. Each mutant displays significantly impaired activity and contains less than 1 equiv of zinc upon purification. Furthermore, each mutant binds zinc with lower binding affinity (K(a) approximately 10(14) M(-)(1)) compared to the wild-type enzyme (K(a) > 10(16) M(-)(1)). All the MetE mutants are able to bind homocysteine. X-ray absorption spectroscopy analysis of the zinc-binding sites in the mutants indicates that the four-coordinate zinc site is preserved but that the ligand sets are changed. Our results demonstrate that Cys643 and Cys726 are two of the zinc ligands in MetE from E. coli and suggest that His641 is a third endogenous ligand. The effects of the mutations on the specific activities of the mutant proteins suggest that zinc and homocysteine binding alone are not sufficient for activity; the chemical nature of the ligands is also a determining factor for catalytic activity in agreement with model studies of the alkylation of zinc-thiolate complexes.     

Redox-regulated cochaperone activity of the human DnaJ homolog Hdj2

The human DnaJ homolog Hdj2 is a cochaperone containing a cysteine-rich zinc finger domain. We identified a specific interaction of Hdj2 with the cellular redox enzyme thioredoxin using a yeast two-hybrid assay and a coimmunoprecipitation assay, thereby investigating how the redox environment of the cell regulates Hdj2 function. In reconstitution experiments with Hsc70, we found that treatment with H2O2 caused the oxidative inactivation of Hdj2 cochaperone activity. Hdj2 inactivation paralleled the oxidation of cysteine thiols and concomitant release of coordinated zinc, suggesting a role of cysteine residues in the zinc finger domain of Hdj2 as a redox sensor of chaperone-mediated protein-folding machinery. H2O2-induced negative regulation of Hdj2 cochaperone activity was also confirmed in mammalian cells using luciferase as a foreign reporter cotransfected with Hsc70 and Hdj2. The in vivo oxidation of cysteine residues in Hdj2 was detected only in thioredoxin-knockdown cells, implying that thioredoxin is involved in the in vivo reduction. The oxidative inactivation of Hdj2 was reversible. Wild-type thioredoxin notably recovered the oxidatively inactivated Hdj2 activity accompanied by the reincorporation of zinc, whereas the catalytically inactive mutant thioredoxin (Cys32Ser/Cys35Ser) did not. Taken together, we propose that oxidation and reduction reversibly regulate Hdj2 function in response to the redox states of the cell.     

Evidence for catalytic cysteine-histidine dyad in chalcone synthase

Chalcone and stilbene synthases (CHS and STS) catalyze condensation reactions of p-coumaroyl-CoA and three C(2)-units from malonyl-CoA, but catalyze different cyclization reactions to produce naringenin chalcone and resveratrol, respectively. Condensing activities of wild-type CHS and STS as well as STS-C60S mutant were inhibited by iodoacetamide (Idm) and diethyl pyrophosphate (DPC). DPC also inhibited malonyl-CoA decarboxylation activity of wild-type and C164S mutants of CHS and STS. Meanwhile, Idm treatment enhanced (two- to fourfold) malonyl decarboxylase activity of wild-type enzymes and STS-C60S, whereas this priming effect was not observed with C164S mutants of CHS and STS, indicating that the cysteine residue being modified by Idm is the catalytic Cys164 of CHS and STS. DPC inhibition of decarboxylation activity of wild-type CHS was pH-independent in the range of pH 5.8 to 7.8; however, its inhibitory effect on CHS-C164S increased as pH increased from 6.2 to 7.4 with a midpoint of 6.4. Based on the 3-D structure of CHS and the observed shift in microscopic pK(a), it was concluded that the histidine residue being modified by DPC in CHS is likely the catalytic His303 and that His303 forms an ionic pair (catalytic dyad) with Cys164 in wild-type CHS. In addition, our results showed that Cys60 in STS is not essential for the activity and only a single cysteine (Cys164) participates in the catalysis as in CHS.     

Mutation of surface-exposed histidine residues of recombinant human granulocyte-colony stimulating factor (Cys17Ser) impacts on interaction with chelated metal ions and refolding in aqueous two-phase systems

Site directed mutagenesis of Cys17-->Ser17 form of recombinant human granulocyte colony stimulating factor (rhG-CSF C17S) for sequential replacing of surface His(43) and His(52) with alanine was used to identify residues critical for the protein interaction with metal ions, in particular Ni(2+) chelated by dye Light Resistant Yellow 2 KT (LR Yellow 2KT)-polyethyleneglycol (PEG), and refolding after partitioning of inclusion bodies in aqueous two-phase systems. Strong binding of rhG-CSF (C17S) to PEG-LR Yellow 2KT-Cu(II) complex allowed for the adoption of affinity chromatography on Sepharose-LR Yellow 2KT-Cu(II) that appeared to be essential for the rapid isolation of mutated forms of rhG-CSF. Efficiency of that purification stage is exemplified by isolation of rhG-CSF (C17S, H43A) and rhG-CSF (C17S, H43A, H52A) mutants in correctly folded and highly purified state. Affinity partitioning of rhG-CSF histidine mutants was studied in aqueous two-phase systems containing Cu(II), Ni(II) and Hg(II) chelated by LR Yellow 2KT-PEG at pH 7.0 and Cu(II)-at pH 5.0. It was determined, that affinity of rhG-CSF mutants for metal ions decreased in the order of C17S>C17S, H43A>C17S, H43A, H52A for Cu(II), and C17S=C17S, H43A>C17S, H43A, H52A for Ni(II) ions, while affinity of all rhG-CSF mutants for Hg(II) ions was of the same order of magnitude. Influence of His(43) and His(52) mutation on protein refolding was studied by partitioning of the respective inclusion body extract in aqueous two-phase systems containing Ni(II) and Hg(II) ions. Data on rhG-CSF histidine mutant partitioning and refolding indicated, that His(52) mutation is crucial for the strength of protein interaction with chelated Ni(II) ions and refolding efficiency.