Identification of cysteine ligands in metalloproteins using optical and NMR spectroscopy: Cadmium‐substituted rubredoxin as a model [Cd(CysS)<sub>4</sub>]<sup>2‐</sup> center

Henehan, Colin J.; Pountney, Dean L.; Vašák, Milan; Zerbe, Oliver

doi:10.1002/pro.5560021019

Cited by 77 publications

(87 citation statements)

References 33 publications

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“…In a titration experiment monitoring the formation of this band, saturation occurs at 1 equivalent of Cd(II) to . The extinction coefficient observed is comparable to reported Cd(II) complexes of S-Cys4 containing peptides (Krizek et al, 1993) and Cd(I1)-substituted rubredoxin (Henehan et al, 1993). Figure 4 shows a CD wavelength scan comparing the structure of the apo and Cd(I1)-bound forms of the [ pl-Cys41.…”

Section: Effect Of Metal-binding On Protein Structure and Stabilitysupporting

confidence: 79%

“…Cd(II) binds tightly to cysteine-rich systems due to the principles of hard and soft acids and bases (Pearson, 1997). Cd(II) is also known to retain tetrahedral geometry when substituted into metalloproteins that have tetrahedral metal chelating sites, for example, the S-Cys4 site in rubredoxin (Henehan et al, 1993). Furthermore, cadmium exists almost exclusively in the Cd(II) oxidation state and, therefore, does not have the potential for complications associated with varying oxidation states as may be encountered with Fe.…”

Section: Effect Of Metal-binding On Protein Structure and Stabilitymentioning

confidence: 99%

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The de novo design of a rubredoxin‐like fe site

Farinas

Regan

1998

Protein Science

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A redox center similar to that of rubredoxin was designed into the 56 amino acid immunoglobulin binding B 1 domain of Streptococcals protein G. The redox center in rubredoxin contains an iron ion tetrahedrally coordinated by four cysteine residues, [ F e ( S -c~s )~] ",-'. The design criteria for the target site included taking backbone movements into account, tetrahedral metal-binding, and maintaining the structure and stability of the wild-type protein. The optical absorption spectrum of the Co(I1) complex of the metal-binding variant is characteristic of tetrahedral chelation by four cysteine residues. Circular dichroism and nuclear magnetic resonance measurements reveal that the metal-free and Cd(I1)-bound forms of the variant are folded correctly and are stable. The Fe(II1) complex of the metal-binding mutant reproduces the optical and the electron paramagnetic resonance spectra of oxidized rubredoxin. This demonstrates that the engineered protein chelates Fe(II1) in a tetrahedral array, and the resulting center is similar to that of oxidized rubredoxin.Keywords: de novo design; metalloprotein; protein engineering; rubredoxin Protein engineering and de novo design approaches have begun to afford a more detailed understanding of the balance of the forces that determine protein structure and function (Regan & DeGrado, 1988;Regan, 1993;Bryson et al., 1995;Regan, 1995;Dalal et al., 1997;Smith & Regan, 1997;Hellinga, 1998). To date, there are just a few examples of designed proteins that adopt a specified fold, and number which display novel activities is still smaller (Dill, 1990; Schafmeister et al., 1993;Jackson et al., 1994;Quinn et al., 1994;Yan & Erickson, 1994;Baltzer et al., 1996;Dahiyat & Mayo, 1997;Hill & DeGrado, 1998;QuCnCneur et al., 1998). A particular focus of functional design efforts has been the engineering of novel metal-binding sites. Metal-binding sites represent an attractive target because they play a variety of functions from stabilization of protein structure to catalytic roles such as the activation of water for nucleophilic attack, electron transfer, and redox activity. More recently, several groups have focused strongly on the design of metal-binding sites with particular activities in mind. For example, two metal-binding histidine residues have been engineered into rat trypsin to "switch" the catalytic activity on or off by disrupting the catalytic triad. The protein can be turned off by Cu(I1) chelation in the designed site and reactivated upon removal of Cu(I1) with EDTA (Halfon & Craik, 1996). In another design, a metal-binding site, based on the metal center of carbonic

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Section: Effect Of Metal-binding On Protein Structure and Stabilitysupporting

confidence: 79%

Section: Effect Of Metal-binding On Protein Structure and Stabilitymentioning

confidence: 99%

The de novo design of a rubredoxin‐like fe site

Farinas

Regan

1998

Protein Science

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“…6). The binding of Cd 2ϩ to Cys-thiolate ligands in peptides and proteins can be followed by monitoring the absorption intensity of the Cys-S-Cd(II) ligand-metal charge-transfer transitions between 240 and 250 nm (37,38). Titration of TTK (Fig.…”

Section: Electronic Absorption and CD Spectroscopy Of Ttk Titrated Wimentioning

confidence: 99%

“…The calculated molar extinction coefficient (⑀) was about 12,000 M Ϫ1 cm Ϫ1 /Cd 2ϩ . The intensity of the Cys-S-Cd(II) charge transfer band is reported to be proportional to the number of Cys-S-Cd(II) coordinative bonds with ⑀ between 5500 and 6500 M Ϫ1 cm Ϫ1 /Cys-S-Cd bond (38). The value of about 12,000 M Ϫ1 cm Ϫ1 was consistent with the presence of two Cys ligands/Cd 2ϩ , the expected number of thiolate ligands present in the CCHH finger motif of TTK.…”

Section: Fig 3 Band Shift Gel Showing Effect Of Various Concentratimentioning

confidence: 99%

Modulation of DNA Binding of a Tramtrack Zinc Finger Peptide by the Metallothionein-Thionein Conjugate Pair

Roesijadi¹,

Bogumil²,

Vašák³

et al. 1998

Journal of Biological Chemistry

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The ability of metallothionein (MT) to modulate DNA binding by a two-finger peptide of Tramtrack (TTK), a CCHH zinc transcription factor, was investigated using metal-bound and metal-deficient forms of rabbit MT-2 and the TTK peptide. Thionein inhibited DNA binding by zinc-bound TTK, and Zn-MT restored DNA-binding by zinc-deficient apo-TTK. "Free" zinc at low concentrations was as effective as Zn-MT in restoring DNA binding by apopeptide but was inhibitory at concentrations equal to zinc bound to 2 mol eq and higher of Zn-MT. Substitution of cadmium for zinc reduced the affinity of the peptide for its DNA binding site. This effect was reversed by incubation with Zn-MT. The circular dichroic spectra of the TTK peptide indicated that zinc removal resulted in loss of ␣-helical structures, which are sites of DNA contact points. Reconstitution with cadmium resulted in stoichiometric substitution of 2 mol of Cd/mol of peptide but not recovery of ␣-helical structures. Incubation of Cd-TTK with Zn-MT restored the secondary structure expected for zinc-bound TTK. The ability of Zn-MT and thionein to restore or inhibit DNAbinding by TTK was associated with effects on the metallation status of the peptide and related alterations in its secondary structure.The low molecular weight metal-binding protein metallothionein (MT) 1 is proposed to have functions in metal ion regulation and detoxification (1) and as a scavenger of free radicals (2). Roles for MT in dynamic intermolecular metal exchange reactions are consistent with the high thermodynamic stability (3) and high kinetic lability of its metal binding sites (4, 5). Metal exchange can also be facilitated by cellular redox couples such as ). An early focus on enzyme activation by Zn-MT implicated MT as a zinc donor to apometallic forms of metalloenzymes such as aldolase, thermolysin, and carbonic anhydrase (10) and pyridoxal kinase (11). Recent studies on the effect of MTs on interactions between zinc transcription factors and their cognate DNAs implicate MTs in gene regulation. Incubation of metal-free thionein with CCHH zinc finger transcription factors Sp1 (12) and TFIIIA (13) inhibits DNA binding, suggesting zinc abstraction by thionein as a mechanism for regulating gene expression. Reciprocal zinc exchange is suggested as a mechanism for regulating gene expression in a study in which Zn-MT and thionein could activate or inhibit DNA binding by the estrogen receptor (14), a CCCC zinc finger protein.Zinc transcription factors appear uniquely qualified as potential partner molecules in metal exchange reactions with MT. Reported stability constants for zinc binding by zinc finger peptides and proteins are in a similar range, i.e. within 1 or 2 orders of magnitude, as that of . Additionally, it appears that the kinetics for zinc exchange in these structures are also relatively rapid (18). Direct examination of the metal transfer process has shown that zinc can be transferred from an ␣-domain peptide of human MT-2 (␣-hMT-2) to a peptide derived from the third finger of Sp1...

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“…The "'Cd NMR spectrum gives a single peak at 745 pprn , indicating the Cd is bound to four sulfurs (Armitage & Otvos, 1982;Summers, 1988;Coleman, 1993;Henehan et al, 1993). A single peak at 723 ppm was observed for the "'Cd form of Rd, the difference in chemical shift suggesting a slightly different geometry at the metal center for the two proteins.…”

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confidence: 99%

NMR determination of the global structure of the ¹¹³Cd derivative of desulforedoxin: Investigation of the hydrogen bonding pattern at the metal center

Goodfellow¹,

Rusnak²,

Moura³

et al. 1998

Protein Science

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Desulforedoxin (Dx) is a simple homodimeric protein isolated from Desulfovibrio gigas (Dg) containing a distorted rubredoxin‐like center with one iron coordinated by four cysteinyl residues (7.9 kDa with 36 amino acids per monomer). In order to probe the geometry and the H‐bonding at the active site of Dx, the protein was reconstituted with 113Cd and the solution structure determined using 2D NMR methods. The structure of this derivative was initially compared with the NMR solution structure of the Zn form (Goodfellow BJ et al., 1996, J Biol Inorg Chem 1:341‐353). Backbone amide protons for G4, D5, G13, L11 NH, and the Q14 NH side‐chain protons, H‐bonded in the X‐ray structure, were readily exchanged with solvent. Chemical shift differences observed for amide protons near the metal center confirm the H‐bonding pattern seen in the X‐ray model (Archer M et al., 1995, J Mol Biol 251:690‐702) and also suggest that H‐bond lengths may vary between the Fe, Zn, and 113Cd forms. The H‐bonding pattern was further probed using a heteronuclear spin echo difference (HSED) experiment; the results confirm the presence of NH‐S H‐bonds inferred from D2O exchange data and observed in the NMR family of structures. The presence of “H‐bond mediated” coupling in Dx indicates that the NH‐S H‐bonds at the metal center have significant covalent character. The HSED experiment also identified an intermonomer “through space” coupling for one of the L26 methyl groups, indicating its proximity to the 113Cd center in the opposing monomer. This is the first example of an intermonomer “through space” coupling. Initial structure calculations produced subsets of NMR families with the S of C28 pointing away from or toward the L26 methyl: only the subset with the C28 sulfur pointing toward the L26 methyl could result in a “through space” coupling. The HSED result was therefore included in the structure calculations. Comparison of the Fe, Zn, and 113Cd forms of Dx suggests that the geometry of the metal center and the global fold of the protein does not vary to any great extent, although the H‐bond network varies slightly when Cd is introduced. The similarity between the H‐bonding pattern seen at the metal center in Dx, Rd (including H‐bonded and through space‐mediated coupling), and many zinc‐finger proteins suggests that these H‐bonds are structurally vital for stabilization of the metal centers in these proteins.

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Identification of cysteine ligands in metalloproteins using optical and NMR spectroscopy: Cadmium‐substituted rubredoxin as a model [Cd(CysS)₄]^2‐ center

Cited by 77 publications

References 33 publications

The de novo design of a rubredoxin‐like fe site

The de novo design of a rubredoxin‐like fe site

Modulation of DNA Binding of a Tramtrack Zinc Finger Peptide by the Metallothionein-Thionein Conjugate Pair

NMR determination of the global structure of the ¹¹³Cd derivative of desulforedoxin: Investigation of the hydrogen bonding pattern at the metal center

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Identification of cysteine ligands in metalloproteins using optical and NMR spectroscopy: Cadmium‐substituted rubredoxin as a model [Cd(CysS)4]2‐ center

Cited by 77 publications

References 33 publications

The de novo design of a rubredoxin‐like fe site

The de novo design of a rubredoxin‐like fe site

Modulation of DNA Binding of a Tramtrack Zinc Finger Peptide by the Metallothionein-Thionein Conjugate Pair

NMR determination of the global structure of the 113Cd derivative of desulforedoxin: Investigation of the hydrogen bonding pattern at the metal center

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Identification of cysteine ligands in metalloproteins using optical and NMR spectroscopy: Cadmium‐substituted rubredoxin as a model [Cd(CysS)₄]^2‐ center

NMR determination of the global structure of the ¹¹³Cd derivative of desulforedoxin: Investigation of the hydrogen bonding pattern at the metal center