Ligand variation and metal ion binding specificity in zinc finger peptides

Krizek, Beth A.; Merkle, Denise L.; Berg, Jeremy M.

doi:10.1021/ic00058a030

Cited by 234 publications

(332 citation statements)

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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%

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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%

“…The optical absorption spectra of Co (I1 as the number of thiolate ligands increases (Moura et al, 1991;Krizek et al, 1993). This is consistent with the relative positions of histidine and cysteine in the spectrochemical series.…”

Section: Characterizationsupporting

confidence: 79%

The de novo design of a rubredoxin‐like fe site

Farinas

Regan

1998

Protein Science

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“…Cys-to-His mutations in MT-2 have been shown to enhance Zn 2ϩ binding (38), consistent with predictions based on the hard-soft acids and bases concept. A 100-fold preference for Cd 2ϩ over Zn 2ϩ for a ZnCys 4 zinc finger site has been reported, whereas a CCHC site has comparable affinity for Cd 2ϩ and Zn 2ϩ (39). 111 Cd NMR experiments showed that the Zn 2ϩ ion that is inert to Cd 2ϩ substitution is, curiously, in a Cys 4 site.…”

Section: Methodsmentioning

confidence: 99%

A metallothionein containing a zinc finger within a four-metal cluster protects a bacterium from zinc toxicity

Blindauer

Harrison

Parkinson

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

172

183

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Zinc is essential for many cellular processes, including DNA synthesis, transcription, and translation, but excess can be toxic. A zinc-induced gene, smtA, is required for normal zinc-tolerance in the cyanobacterium Synechococcus PCC 7942. Here we report that the protein SmtA contains a cleft lined with Cys-sulfur and His-imidazole ligands that binds four zinc ions in a Zn4Cys9His2 cluster. The thiolate sulfurs of five Cys ligands provide bridges between the two ZnCys 4 and two ZnCys3His sites, giving two fused six-membered rings with distorted boat conformations. The inorganic core strongly resembles the Zn4Cys11 cluster of mammalian metallothionein, despite different amino acid sequences, a different linear order of the ligands, and presence of histidine ligands. Also, SmtA contains elements of secondary structure not found in metallothioneins. One of the two Cys4-coordinated zinc ions in SmtA readily exchanges with exogenous metal ( 111 Cd), whereas the other is inert. The thiolate sulfur ligands bound to zinc in this site are buried within the protein.Regions of ␤-strand and ␣-helix surround the inert site to form a zinc finger resembling the zinc fingers in GATA and LIM-domain proteins. Eukaryotic zinc fingers interact specifically with other proteins or DNA and an analogous interaction can therefore be anticipated for prokaryotic zinc fingers. SmtA now provides structural proof for the existence of zinc fingers in prokaryotes, and sequences related to the zinc finger motif can be identified in several bacterial genomes.

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“…A peptide characterized in our laboratory, called CP-l(CCCC), has the sequence Pro Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Gln Lys Ser Asp Leu Val Lys Cys Gln-Arg Thr Cys Thr Gly, in which the two histidinxgands have beenreplaced with cysteines (Krizek et al, 1993). This peptide binds Co2+ tetrahedrally through the four cysteines.…”

Section: Fig 2 Amino Acid Sequences (In Singlementioning

confidence: 99%

Independence of metal binding between tandem Cys₂His₂ zinc finger domains

1993

Self Cite

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Most Cys2His2 zinc finger proteins contain tandem arrays of metal binding domains. The tandem nature of these arrays suggests that metal binding by these domains may not be independent but rather that metal binding may occur in a cooperative manner. This is especially true in light of the crystal structure of a three zinc finger array bound to DNA that revealed several types of interactions between domains. To address this question, peptides containing two tandem domains have been prepared. While metal binding studies do show that the two finger peptide has a metal ion affinity about threefold higher than that for a single domain peptide with the same sequence, additional studies reveal that this behavior is due to increased single site affinities in the context of the two domain peptide rather than to cooperativity. These studies indicate that domains of this type are independent of one another with regard to metal binding, at least in the absence of DNA. This observation has implications with regard to the question of whether the activities of proteins of this class might be modulated by available zinc concentrations.

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Ligand variation and metal ion binding specificity in zinc finger peptides

Cited by 234 publications

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The de novo design of a rubredoxin‐like fe site

The de novo design of a rubredoxin‐like fe site

A metallothionein containing a zinc finger within a four-metal cluster protects a bacterium from zinc toxicity

Independence of metal binding between tandem Cys₂His₂ zinc finger domains

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Ligand variation and metal ion binding specificity in zinc finger peptides

Cited by 234 publications

References 0 publications

The de novo design of a rubredoxin‐like fe site

The de novo design of a rubredoxin‐like fe site

A metallothionein containing a zinc finger within a four-metal cluster protects a bacterium from zinc toxicity

Independence of metal binding between tandem Cys2His2 zinc finger domains

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Independence of metal binding between tandem Cys₂His₂ zinc finger domains