JBIC Journal of Biological Inorganic Chemistry

, Volume 10, Issue 2, pp 167–180

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

Authors

  • Wenpeng Kou
    • Department of ChemistryThe University of Texas at Dallas
  • Harsha S. Kolla
    • Department of ChemistryThe University of Texas at Dallas
  • Alfonso Ortiz-Acevedo
    • Department of ChemistryThe University of Texas at Dallas
  • Donovan C. Haines
    • Department of ChemistryThe University of Texas at Dallas
  • Matthew Junker
    • Department of Molecular and Cell BiologyThe University of Texas at Dallas
    • Department of ChemistryThe University of Texas at Dallas
Original Article

DOI: 10.1007/s00775-005-0625-9

Cite this article as:
Kou, W., Kolla, H.S., Ortiz-Acevedo, A. et al. J Biol Inorg Chem (2005) 10: 167. doi:10.1007/s00775-005-0625-9

Abstract

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)3His 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 KAZn/KACo of 2.8(±2.0)×103; (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 β-sheet hydrophobic cluster increases KAZn and KACo; (4) mutation in the β-hairpin turn, His20 to Asn generating an Asn-Gly turn, also increases KAZn and KACo; (5) the combination of His20 to Asn and Tyr24 to Trp mutations also increases KAZn and KACo, but the increments versus C3H are less than those of the single mutations. Furthermore, circular dichroism, size-exclusion chromatography, and 1D and 2D 1H 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.

Keywords

Zinc ribbonMetal binding constantsTotal correlation spectroscopyNuclear Overhauser enhancement spectroscopyThiolate-rich zinc site

Abbreviations

CD

Circular dichroism

DI

Deionized

DTNB

5,5′-Dithiobis(2-nitrobenzoic acid)

ESI-MS

Electrospray ionization mass spectrometry

Fmoc

9-Fluorenylmethoxycarbonyl

HEPES

N-(2-Hydroxyethyl)piperazine-N′-ethanesulfonic acid

HPLC

High performance liquid chromatography

ICP-MS

Inductively coupled plasma mass spectrometry

LFSE

Ligand field stabilization energy

LMCT

Ligand-to-metal charge transfer

NOESY

Nuclear Overhauser enhancement spectroscopy

SEC

Size-exclusion chromatography

TCEP·HCl

Tris(2-carboxyethyl)phosphine hydrochloride

TFA

Trifluoroacetic acid

TOCSY

Total correlation spectroscopy

Introduction

Zinc is an essential trace element that plays a critical role in the functioning of numerous macromolecules in biological systems. Zinc is favored because of several factors, including its accommodation of multiple coordination geometries owing to its d10 electron configuration, as well as its ability to utilize both hard (nitrogen and oxygen) and soft (sulfur) ligands. Sites that bind Zn(II) in proteins fall into two broad categories [1, 2]:
  1. 1.

    Catalytic Zn(II) sites contain carboxylate-rich and/or imidazole-rich ligation environments with an open coordination site for the solvent. The Zn(II) functions as a Lewis acid to activate a nucleophile (typically the coordinated solvent).

     
  2. 2.

    Structural Zn(II) sites stabilize a protein’s native fold or a protein–protein interface. These sites are thiolate-rich and are typically coordinately saturated.

     
Recently a third group of Zn(II) proteins have been identified which play a role in alkyl transfer reactions [3, 4]. These Zn(II)-binding sites do not fit into either the catalytic or the structural categories: they effect alkyl transfer and are therefore chemically reactive, yet they contain thiolate-rich coordination spheres. Examples of this group include the Escherichia coli DNA repair protein Ada [5, 6], cobalamin-dependent [7] and cobalamin-independent [8] methionine synthases, farnesyltransferase [9] and epoxide thiol-conjugating enzymes [10].

We are interested in how the protein environment ultimately controls the properties (i.e., reactivity, metal affinities, etc.) of thiolate-rich Zn(II)-binding sites, causing some to be only structural, while others with similar coordination environments perform chemistry. Previous computational [11, 12] and small molecule model studies [1316] suggest that both the first ligation shell and the surrounding environment can play important roles in determining these properties. Our complementary approach is to develop a simple model system through reengineering a small, naturally occurring Zn(II)-binding motif, modifying either the zinc ligands or distant sites in the protein, and then characterizing their metal-binding and reactivity properties. Previous Zn(II)-binding proteins studied this way include carbonic anhydrase II [17], TFIIIA-like zinc fingers [18, 19] and the phage T4 gene 32 protein [20].

This paper describes our initial studies which focus on the zinc ribbon protein L36, the smallest protein in the large prokaryotic ribosomal subunit from Thermus thermophilius. Although its function is unknown, it is believed to interact with ribosomal RNA [21, 22]. A number of features make L36 an attractive system to study and a potentially useful model for future structure/reactivity studies:
  1. 1.

    The protein is small (37 residues), simplifying synthesis and characterization.

     
  2. 2.

    The solution structure is known (PDB no. 1DFE) [23]. L36 adopts a zinc ribbon folding motif containing a three-stranded β-sheet with a (Cys)3His Zn(II)-binding site formed by two loops at one end of the protein (Fig. 1).

     
  3. 3.

    The metal ligands display different solvent accessibilities in the folded protein, providing a potential variance in reactivities.

     
  4. 4.

    Only one chromophore (Tyr24) is present, and is part of one of two small hydrophobic clusters that likely enable the protein to fold (Val7, Val16, Val23 and Val25 on one side of the β-sheet; Ile17, Tyr24 and Ile26 on the other side). Replacement of Tyr with Trp could provide a useful future reporter of local environmental changes via fluorescence/absorbance changes.

     
  5. 5.

    The Zn(II)-binding site contains several backbone-to-Cys NH–S hydrogen bonds [23], similar to those observed in other Zn(II)-binding proteins [2426]. Protein modifications that remove/moderate their strength would allow us to test their importance in determining structural and reactivity properties.

     
  6. 6.

    A previous study reported that L36 binds Co(II) preferentially over Zn(II) [27].

     
Since this latter metal-binding preference is unique when compared with other Zn(II)-binding sites in proteins, we are interested in understanding how the protein confers this interesting specificity.
Fig. 1

Average NMR structure of L36 [23]. β-strands are shown as red arrows, the Zn(II) ion is represented as a pink sphere, and the metal-binding ligands and key amino acids are represented as ball-and-stick illustrations. The labels indicate the various residue changes investigated in this study. Atom colors: C green; N blue; O red; S yellow

In this paper we describe our initial characterization of several L36 derivatives designed to probe the role of the protein environment in determining metal-binding site properties. We are interested in how distant locations in a protein can affect a metal site (i.e., can the metal-binding site act as a reporter for events, such as folding, that occur at remote locations in the protein?). The derivatives (Table 1) fall into three categories: (1) mutation of the Zn(II)-binding site, (2) mutation of the turn region of the β-hairpin in the zinc ribbon intended to stabilize the protein fold, and (3) mutation in one of the hydrophobic clusters on one face of the three-stranded β-sheet. Our results from metal binding, circular dichroism (CD) and 1D and 2D NMR show that the zinc ribbon motif is tolerant of the different types of sequence changes we have incorporated. In addition, the studies support the idea that nonlocal changes in the protein sequence designed to stabilize the native protein fold increase both the Zn(II) and Co(II) metal-binding affinities. Furthermore, we find that all derivatives of L36 display relative metal-binding affinities typically found in typical Zn(II)-binding sites (i.e., KAZn >KACo), contrary to results from a previous study of L36 [27].
Table 1

L36 derivatives and amino acid sequences

Materials and methods

Peptide synthesis and purification

Peptides were synthesized at a 0.1 mmol scale with an Applied Biosystems 433A solid-phase peptide synthesizer equipped with an Alltech model 450 UV detector using 9-fluorenylmethoxycarbonyl (Fmoc)-protected amino acids and standard solid-phase peptide synthetic methods [28]. Fmoc-amide resin was used, which produces an amide-protected C-terminus upon peptide cleavage from the resin. The N-terminus of each peptide was acetylated prior to cleavage. Cleavage of the peptide from the resin and side chain deprotection were effected using a 9:0.5:0.3:0.2 trifluoroacetic acid (TFA):thioanisole:ethanedithiol:anisole mixture. Crude peptides were precipitated with cold diethyl ether, lyophilized to dryness, and purified through reversed-phase high-performance liquid chromatography (HPLC) using a Waters Delta 600 preparative HPLC instrument and Vydac C4 columns with doubly distilled deionized (DI) water/acetonitrile/0.1% TFA mobile phase gradients. During purification, excess tris[2-carboxyethyl]phosphine hydrochloride (TCEP·HCl; Pierce) was added to the crude peptide solution, and the fractions containing pure peptide were frozen immediately to limit Cys side chain oxidation. The identities of the peptides were verified using electrospray ionization mass spectrometry (HT Laboratories, San Diego, CA, USA).

Preparation of peptide, Co(II) and Zn(II) solutions

All peptide stock solutions were prepared by resuspending lyophilized peptide solids in doubly distilled DI water under anaerobic conditions. Peptide concentration was determined spectrophotometrically with a Cary 50 UV–vis spectrophotometer using the absorbance of Tyr (ε275=1,420 M−1 cm−1) or Trp (ε280=5,600 M−1 cm−1). Co(II) and Zn(II) stock solutions were prepared by dissolving CoCl2·6H2O (Mallinckrodt) or ZnCl2 (EM Science), respectively, in degassed, doubly distilled DI water. Co(II) and Zn(II) concentrations in the metal stock solutions were measured by flame atomic absorption spectroscopy using a Varian SpectrAA-5 atomic absorption spectrophotometer. For Co(II), a cobalt hollow cathode lamp (Fisher Scientific) was used with a detection wavelength of 240.7 nm and a slit width of 0.2 nm. For Zn(II), a zinc hollow cathode lamp (Fisher Scientific) was used with a detection wavelength of 213.9 nm and a slit width of 1.0 nm. The background concentrations of Co(II), Zn(II) and other metals in the DI water were measured by inductively coupled plasma mass spectrometry (ICP-MS) and were low enough to not influence the results significantly. The concentrations were as follows: Zn, 5.58 ppb (8.5×10−2 μM); Co, below detection limits, or less than 0.10 ppb (less than 1.7×10−3 μM); Cd, 0.23 ppb (2.1×10−3 μM); Fe and Ni, below detection limits.

Free thiol quantitation

The number of free thiols in apo-peptide solutions was determined using a 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB; Acros Organics) colorimetric assay [29, 30]. All solutions were prepared and reactions performed under anaerobic conditions. A homocysteine calibration curve was generated using standards between 20 and 100 μM prepared from a 100 μM stock in 0.1 M NaH2PO4·H2O buffer (pH 8.0). To each standard, 50 μl of a 0.01 M DTNB solution was added and the resulting mixture was incubated for 30 min. The absorbance was measured between 240 and 600 nm and corrected for the buffer absorbance. The free thiol concentration was determined using ε410=13,650 M−1 cm−1 for the aromatic thiolate ion produced from the reaction of DTNB with reduced thiols. Peptide samples were prepared in 0.1 M NaH2PO4·H2O buffer (pH 8.0) with peptide concentrations between 20 and 80 μM, and were then treated with DTNB for 30 min. The resulting absorbance at 410 nm was compared with the calibration curve to determine the free thiol content.

Co(II)-binding titrations

Co(II) titrations were carried out at 25°C in 20 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) buffer (pH 6.0) under anaerobic conditions. Three CoCl2 stock solutions with varying metal concentrations were used to reduce dilution effects. For each titration point, an appropriate volume of stock CoCl2 solution was added to an approximately 100 μM peptide solution and allowed to stand for 2 min (control experiments demonstrated no change in absorbance of a mixture after 2 min—data not shown). Absorbance spectra were collected from 250 to 900 nm at a scanning rate of 1 nm s−1 for each sample using a 1.0-cm quartz cuvette. Three titrations were conducted for each peptide. Spectra were corrected for buffer and peptide absorbance and for dilution from the addition of Co(II) stock. For all titrations reported in this paper, the shape of the dd transition region (500–750 nm) did not change during the course of the experiment, indicative of a single type of tetrahedral environment present.

For each titration, the Aλ versus [Co(II)]tot data were best fit using a model where two species (SP1 and SP2) are present, each of which can bind Co(II) in a 1:1 stoichiometry with a different binding affinity (tight binder SP1 and weak binder SP2; see “Results” section for details). Fits were performed using the program DynaFit (BioKin), which simulates multiple complex equilibria to perform nonlinear least-squares regression. Experimental parameters included the percentage of SP1 and SP2 in the titration (based on DTNB titration results; percent SP1 equated with the percent reduced, percent SP2 equated with percent oxidized). Fitted parameters included KDCo [dissociation constant for Co(II)] for species SP1 and SP2, and the extinction coefficient (ελ) for the weaker binder SP2 (ελ for SP1 could principally be defined from the titration data). KACo was calculated by taking the inverse of KDCo.

Zn(II)-binding titrations

Zn(II) binding was monitored using a Zn(II) versus Co(II) competition experiment [20, 31]. First, a saturated Co(II)-bound peptide sample was generated by the addition of 100 equiv or more of Co(II) from a Co(II) stock solution to a peptide solution (between 50 and 100 μM) in 20 mM HEPES buffer (pH 6.0) at 25°C under anaerobic conditions. For each titration point, an appropriate volume of stock Zn(II) solution was added to the Co(II)-peptide solution and allowed to stand for at least 1 min. Bleaching of the Co(II) signal for each sample was monitored by collecting absorbance spectra from 250 to 900 nm at a scanning rate of 1 nm s−1 using a 1.0-cm quartz cuvette. Spectra were corrected for buffer and peptide absorbance and for dilution from the addition of the Zn(II) stock.

For each spectrum, the absorbance was converted to the fraction of protein bound by Co(II) (νCo) using Eq. 1:
$$ \nu _{{\text{Co}}} = \frac{{A_i - A_0 }} {{A_{{\text{max}}} - A_0 }} $$
(1)
where Ai is the absorbance at λmax in the dd transition region for the ith point of the titration, A0 is the initial absorbance at λmax, and Amax is the maximum absorbance at λmax. The νCo versus [Zn(II)]tot data were then fit using a competitive inhibitor model which assumes that all peptide is in 1:1 complexes bound by either Co(II) or Zn(II) [32]. 1
$$ \nu _{{\text{Co}}} = \frac{{ - b + \left( {b^2 - 4ac} \right)^{{1 \mathord{\left/ {\vphantom {1 2}} \right. \kern-\nulldelimiterspace} 2}} }} {{2a}} $$
(2)
where
$$ \begin{array}{*{20}l} {a \hfill} & { = \hfill} & {{{\left[ {{\text{peptide}}} \right]}^{{\text{2}}}_{{{\text{tot}}}} \cdot {\left( {K^{{{\text{Co}}}}_{{\text{D}}} \cdot K^{{{\text{Zn}}}}_{{\text{A}}} - 1} \right)}} \hfill} \\ {b \hfill} & { = \hfill} & {{{\left[ {{\text{peptide}}} \right]}_{{{\text{tot}}}} \cdot \left( {{\left[ {{\text{Zn}}{\left( {{\text{II}}} \right)}} \right]}_{{{\text{tot}}}} \cdot K^{{{\text{Co}}}}_{{\text{D}}} \cdot K^{{{\text{Zn}}}}_{{\text{A}}} + {\left[ {{\text{peptide}}} \right]}_{{{\text{tot}}}} } \right.} \hfill} \\ {{} \hfill} & {{} \hfill} & {{\left. { + {\left[ {{\text{Co}}{\left( {{\text{II}}} \right)}} \right]}_{{{\text{tot}}}} - {\left[ {{\text{peptide}}} \right]}_{{{\text{tot}}}} \cdot K^{{{\text{Co}}}}_{{\text{D}}} \cdot K^{{{\text{Zn}}}}_{{\text{A}}} } \right)} \hfill} \\ {c \hfill} & { = \hfill} & {{ - {\left[ {{\text{peptide}}} \right]}_{{{\text{tot}}}} \cdot {\left[ {{\text{Co}}{\left( {{\text{II}}} \right)}} \right]}_{{{\text{tot}}}} } \hfill} \\ {{K^{{{\text{Zn}}}}_{{\text{A}}} } \hfill} & { = \hfill} & {{{\text{association}}\;{\text{constant}}\;{\text{for}}\;{\text{Zn}}{\left( {{\text{II}}} \right)}} \hfill} \\ \end{array} $$
and KAZn is the association constant for Zn(II). Zn(II) titration fits were performed using the program KaleidaGraph (Synergy Software).

CD spectroscopy

CD spectral measurements were made using a sealed 0.1-cm rectangular quartz cuvette and an Aviv Instruments model 202 CD spectrometer equipped with a thermoelectric sample changer to control the temperature. Samples were prepared under anaerobic conditions and contained 100 μM peptide in 20 mM HEPES buffer (pH 6.0). When appropriate, aliquots from a Zn(II) stock were added to yield a final metal concentration between 150 and 200 μM. All CD data were corrected for buffer contributions by subtracting a buffer blank. Wavelength scans were collected from 190 to 320 nm at 25°C with a bandwidth of 1 nm and averaging for 2 s nm−1.

The mean residue ellipticity, [θ], was calculated using Eq. 3:
$$ \left[ \theta \right] = \frac{{\theta _{{\text{obs}}} }} {{10lcn}} $$
(3)
where θobs is the ellipticity in millidegrees, l is the path length of the cuvette in centimeters, c is the peptide concentration in moles per liter, and n is the number of residues per peptide. This gives units of deg cm2 dmol−1 res−1 for [θ].

Size-exclusion chromatography

Size-exclusion chromatography (SEC) was used to determine the association state of Zn(II)-bound derivatives of L36. Zinc-bound protein samples (100 μM) were prepared in 200 mM HEPES buffer (pH 6.0) under anaerobic conditions. For each sample, 4 μl was loaded onto a Tosoh Super SW2000 SEC column (4.6 mm×300 mm; 4-μm particle size) attached to an HP1100 analytical HPLC. Chromatography runs were performed under isocratic conditions at a flow rate of 0.35 ml min−1 and monitored at 280 nm (350 nm reference) using a diode-array detector. The column was calibrated using horse heart cytochrome c (12.4 kDa), bovine lung aprotinin (6.5 kDa) and vitamin B12 (1.4 kDa).

NMR spectroscopy

NMR spectra were collected on a 500 MHz Varian Unity-INOVA spectrometer equipped with a z-axis gradient. Samples were prepared under anaerobic conditions by dissolving dry peptide in 90% H2O/10% D2O containing 2.0 mM Zn(II) to give a final peptide concentration of 1.0 mM. The sample pH was adjusted to 6.0 with NaOH, with the peptide acting as the sole buffer. NMR experiments were performed at 25°C. The 1D experiments and 2D nuclear Overhauser enhancement spectroscopy (NOESY) experiments employed WATERGATE for suppression of the water signal [33]. Total correlation spectroscopy (TOCSY) experiments used presaturation for water suppression and an MLEV-17 sequence for the spin lock [34]. The 2D NOESY and TOCSY experiments used sweep widths of 6,300.2 Hz in the direct and indirect dimensions, and collected 2,048 complex points (real plus imaginary) in the direct dimension and typically 150 (TOCSY) or 200 (NOESY) complex points in the indirect dimension. The data were processed using FELIX (Accelyrys) and analyzed with NMRView [35]. For the quantitation of NOESY cross peak volumes, NOESY data were processed identically and apodized with sinebell squared window functions that were shifted by 90° in each dimension.

Results

Selection of derivatives

We utilized the zinc ribbon protein L36 as a model system to study how changes in the protein matrix can affect metal binding. The derivatives studied fall into three categories (Table 1):
  1. 1.

    Mutation of the binding site. The C3H group of derivatives contain the native (Cys)3His Zn(II)-binding site, while the members of the C4 group each contain a His32-to-Cys mutation, yielding a (Cys)4 coordination site; these two groups allow us to compare S3N and S4 metal ligation.

     
  2. 2.

    Mutation of the β-hairpin turn. L36 contains a single type I′ β-hairpin turn between β-strands 1 (residues 15–19) and 2 (residues 22–26), with His and Gly at positions i+1 and i+2 of the turn (Fig. 1). The derivatives C3H_N and C4_N contain a His20-to-Asn mutation, which is expected to stabilize a type I′ β-turn [3638]. We expect mutations that stabilize the protein fold to lead to higher metal-binding affinities, as observed for β-sheet mutations in traditional zinc finger domains [39, 40]. A stabler fold should also lead to a decrease in the reactivity of a subset of the Cys ligands that become more effectively buried as the protein folds [12].

     
  3. 3.

    Mutation in the hydrophobic cluster. Tyr24 is part of a hydrophobic cluster on one face of the three-stranded β-sheet in L36. C3H_W and C4_W contain a Tyr24-to-Trp mutation, which introduces a better fluorescent probe for future folding studies. We are interested in the effect this larger aromatic residue will have on the hydrophobic cluster.

     
Other derivatives include C3H_NW and C4_NW, which contain the double mutation His20 to Asn/Tyr24 to Trp, and finally C3H_26, the same 26-mer used in a previous mass spectrometry study on L36 [27], which contains the core 26 residues (9 through 34) that make up the three-stranded β-sheet and metal-binding site. Interestingly, the C3H_26 derivative was shown to have unique metal-binding specificity, binding Co(II) tighter than Zn(II) [27].

Co(II) binding

Co(II) substitution monitored by UV–vis spectroscopy is a useful technique for characterizing the identity and geometric arrangement of ligands in Zn(II)-binding sites in proteins. In the presence of an excess of Co(II) (greater than 100 equiv), the C3H derivatives of L36 exhibit intense absorption bands around 323 nm (average ε=1,750 M−1 cm−1) and 360 nm (average ε=1,150 M−1 cm−1) (Fig. 2a). These transitions are in the wavelength and intensity range observed for S→Co(II) ligand-to-metal charge transfer (LMCT) transitions [4143]. Three bands are also observed between 500 and 700 nm: at 609 nm (average ε=180 M−1 cm−1), 653 nm (average ε=300 M −1 cm−1), and a maximum at 689 nm (average ε=400 M−1 cm−1) (Fig. 2b). These correspond to 4A24T1(P) dd transitions observed previously in high-spin tetrahedral Co(II) systems with S3N coordination environments [44, 45].
Fig. 2

Absorption spectra for Co(II)-bound L36 derivatives. a Full spectrum for C3H (red) and C4 (blue). b dd transition region for all peptide derivatives. Spectral contributions from Co(H2O)62+ have been removed.

For the C4 derivatives, the replacement of His32 with Cys leads to redshifted and broadened dd transition regions (Fig. 2). The formation of a shoulder at 732 nm is consistent with spectral changes observed in tetrahedral Co(II) compounds when a non-thiolate ligand is replaced with an additional thiolate ligand (Fig. 2b) [20, 46]. The dd transitions are observed at 614 nm (average ε=230 M−1 cm−1), a maximum at 688 nm (average ε=370 M−1 cm−1), and 732 nm (average ε=300 M−1 cm−1. The more broadened dd transition envelope is consistent with either a greater distortion from tetrahedral symmetry than for the C3H coordination site, or with an averaging of conformations [44]. A change is also observed in the LMCT band intensities at 305 nm (average ε=4,060 M−1 cm−1) and 350 nm (average ε=1,800 M−1 cm−1) and in their broadness, attributable to an increase in the number of coordinating thiolate ligands from 3 to 4 (Fig. 2a) [46]. The changes in the UV–vis spectra are therefore consistent with the designed change from a (Cys)3His Co(II)-binding site in the C3H derivatives to a (Cys)4 site in the C4 derivatives.

Co(II) versus Zn(II) specificity

The Co(II)- and Zn(II)-binding affinities of the various L36 derivatives were determined at pH 6.0 using anaerobic UV–vis titrations. All peptides in this study displayed similar behavior during these titration experiments—only C3H will be discussed in detail (see supplementary material for details for the other L36 derivatives). For each Co(II) titration, small aliquots of Co(II) were added to a solution of the apo-peptide. With each addition, an increase was observed in the LMCT and dd transition regions corresponding to formation of a tetrahedral Co(II) complex (Fig. 3a). No changes in the overall shape of the dd transition or LMCT regions were observed during the course of the titration for any peptide derivative, suggesting that one type of tetrahedral Co(II) coordination environment was generated during each titration. Once saturation of the peptide had been reached, the LMCT and dd transition regions stopped changing in intensity and a new set of peaks at 470 nm (average ε=28 M−1 cm−1) and 514 (average ε=37 M−1 cm−1) began growing into the spectrum. These bands are attributable to the formation of Co(H2O)62+ from excess Co(II) (Fig. 3a).
Fig. 3

Metal titrations of C3H. a Co(II) titration. b Zn(II) titration. Labeling indicates the number of equivalents of metal added per equivalent of peptide.

To observe Zn(II) binding and measure Zn(II)-binding constants, we used a metal competition assay in which Zn(II) is added to a Co(II)-saturated peptide sample. The Zn(II) is expected to displace the Co(II) and bind to the same coordination site. The addition of Zn(II) led to a bleaching of the Co(II)–peptide absorbance (LMCT and dd transition regions), with a corresponding increase in the octahedral Co(II) spectrum, consistent with Co(II) being replaced by Zn(II) in the tetrahedral binding site (Fig. 3b).

For each Co(II) titration, the shape of the plot of Aλ versus [Co(II)]tot could not be sufficiently described by a simple 1:1 protein:Co(II) binding model (yielded an underestimation of the initial slope, and reached the Amax value too early). Results from DTNB titrations also suggested that there was between 10 and 25% oxidized peptide species present in each Co(II)-binding titration that could participate in metal binding. Therefore, the Aλ versus [Co(II)]tot data were best fit using a model where two species (SP1 and SP2) are present, each of which can bind Co(II) in a 1:1 stoichiometry with a different binding affinity. On the basis of the DTNB results, the tight binder SP1 was determined to be the totally reduced peptide, and the weaker binder SP2 was one or more possible oxidized species (it was deemed unnecessary to determine how many or the exact identities of the oxidized species, since suitable fits were obtained assuming a single oxidized species). Fits were performed using the program DynaFit. The plots of Aλ versus [Co(II)]tot for all L36 derivatives and the corresponding fits are shown in Figs. 4a and S1, with the resulting Co(II) binding constants listed in Tables 2 and S1.
Fig. 4

Plots of fraction of C3H bound by a Co(II) or b Zn(II) versus total metal concentrations. Data are shown as circles, with curve fits shown as lines. Details of the plots and curve fits are given in the text.

Table 2

Co(II) and Zn(II) metal binding constants for L36 derivatives

Peptide

KAZn (M−1)

KACo (M−1)

KAZn/KACo

C3H

5.79(±0.57)×107

1.77(±0.11)×104

3.27(±0.39)×103

C3H_Na

9.1(±6.9)×108

1.19(±0.89)×105

<1.6×104

C3H_Wa

1.4(±1.0)×109

3.5(±2.6)×105

<8.0×103

C3H_NWa

7.9(±6.6)×108

1.00(±0.81)×105

<1.7×104

C4b

ND

ND

ND

C4_N

5.63(±0.86)×107

1.39(±0.05)×104

4.1(±0.6)×103

C4_W

4.9(±1.6)×107

6.2(±1.8)×104

7.9(±3.5)×102

C4_NW

1.65(±0.32)×107

6.38(±0.57)×103

2.6(±0.5)×103

C3H_26

1.50(±0.56)×108

4.3(±1.6)×104

3.5(±1.8)×103

Values for SP2 species listed in Table S1.

ND not determined

aThe large errors in the KACo values are due to two major contributing factors: limitations in our methodology for measuring larger Co(II) binding constants [very low free Co(II) concentrations], and an ill-defined break in the Aλ versus [Co(II)]tot plot from a relatively small amount of oxidation. These large errors lead to large errors in the KAZn values, and propagate to the KAZn/KACo ratio. In light of this, we only report upper limits for these derivatives

bA significant propensity to oxidize made C4 difficult to characterize quantitatively. However, as with all other L36 derivatives, Zn(II) easily displaced Co(II) in competition assays.

For Zn(II)-binding titrations, νCo was plotted versus [Zn(II)]tot and the data were fit using a competitive inhibitor model which assumes that all peptide is in 1:1 metal:peptide complexes bound by either Co(II) or Zn(II), with effectively no free peptide in solution (Figs. 4b and S1)1. The presence of two isobestic points in the Zn(II) titrations at approximately 420 and 560 nm (Fig. 3) demonstrates the conversion of Co(II)-peptide to Co(H2O)62+ with no Co(II) intermediates. The Zn(II)-binding constants for all L36 derivatives are shown in Table 2.

A series of trends is observed in the measured Co(II) and Zn(II) metal-binding constants:
  1. 1.

    Zn(II) binds tighter than Co(II). All L36 derivatives display a significant preference for Zn(II) over Co(II), with an average KAZn/KACo of 3(±2)×103 (Table 2). This selectivity for Zn(II) over Co(II) has been observed for other tetrahedral CxH(4−x) sites in proteins, with literature values for KAZn/KACo ranging from 3.1×102 at pH 7.5 for gp32 (with a HC3 binding site) to 3.2×105 at pH 7.0 for CP-1 (with a C4 binding site) [19, 20, 40, 47, 48].

     
  2. 2.

    C3H coordination sites bind both Co(II) and Zn(II) tighter than C4 sites. For each peptide derivative, it can be seen that the C3H metal-binding site has a higher affinity for Co(II) and Zn(II) than the corresponding C4 site. For example, the (KACo)C3H deriv/(KACo)C4 deriv values are 8.6±6.5 for C3H_N versus C4_N, 5.6±4.5 for C3H_W versus C4_W, and 16±13 for C3H_NW versus C4_NW. This trend has been reported previously for carbonic anhydrase-II [49], the zinc finger domain gp32 [20], and a consensus zinc finger sequence [19].

     
  3. 3.

    Tyr24 to Trp mutation leads to an increase in metal-binding constants. The replacement of Tyr with the larger and more hydrophobic side chain Trp yields an increase in the metal-binding constants. (KACo)C3H_W/(KACo)C3H is 20±15, and (KAZn)C3H_W/(KAZn)C3H is 24±17. Aromatic residues are also found in analogous positions in the β-sheet of other small β-sheet proteins, including the C-terminal zinc ribbon domain of human transcription elongation factor TFIIS (PDB no. 1TFI) [50] and the WW domain [51], suggesting a potential role as aromatic “anchors” or cores to help stabilize hydrophobic clusters.

     
  4. 4.

    His20 to Asn mutation in the β-hairpin loop increases the Co(II)- and Zn(II)-binding constants The His-Gly turn in the three-stranded β-sheet was mutated to Asn-Gly to stabilize the β-hairpin turn and potentially increase the metal-binding affinity. This is reflected in the binding constants, with (KACo)C3H_N/(KACo)C3H equal to 6.7±5.0 and (KAZn)C3H_N/(KAZn)C3H equal to 16±12.

     
The Tyr24 to Trp and His20 to Asn mutations each increase the affinity of the various L36 derivatives for Zn(II) and Co(II). We undertook structural studies of the various derivatives to determine if the changes in binding constants arise from modifications to the folded structure, or if the changes are likely attributable to a simple increase in the stability of the folded state.

CD studies

The secondary structure of all peptides in solution in the presence and absence of Zn(II) was studied using CD. Far-UV CD spectra were collected between 190 and 320 nm under anaerobic conditions to prevent oxidation of the Cys residues. In the absence of Zn(II), the peptides display spectral characteristics of unfolded, random coil structures with no distinct CD bands above 200 nm (Fig. 5). The addition of Zn(II) leads to the formation of a significant positive band at 228 nm, with the Trp derivatives displaying larger positive bands than the Tyr derivatives (Fig. 5). The spectra in the presence of Zn(II), presumably generated by the folded state of the peptides formed when binding the Zn(II), lack the negative band at 215–218 nm which is characteristic of a β-sheet secondary structure. However, the CD spectra of the Zn(II)-bound peptides resemble those observed previously for the WW domain, a similar small three-standed β-sheet motif which binds proline-rich sequences [5254]. The WW domain, which contains two Trp residues, also displays a CD spectrum in its folded state that contains a positive band near 230 nm attributed to the aromatic residues, yet no distinct band near 215 nm. Considering the similarity of the CD spectra and the similarity between the known structures of L36 and the WW domain, we interpret our CD results as evidence of the folding of all L36 derivatives into the expected three-stranded β-sheet structure in the presence of Zn(II).
Fig. 5

Circular dichroism spectra of L36 derivatives. For each derivative, the solid curve represents the peptide minus Zn(II) and the dotted curve represents the peptide in the presence of 1.5–2.0 equiv Zn(II).

Size-exclusion chromatography

The oligomeric state of L36 derivatives was assessed using SEC. We looked specifically at the C3H derivatives, since these peptides bind Zn(II) tighter than the C4 peptides and are therefore expected to be the most resistant towards oxidation [55]. Each derivative was eluted as a single symmetrical peak (data not shown). The apparent molecular masses based on a globular protein calibration curve, with the calculated monomer molecular masses in parentheses, are as follows (all in grams per mole): C3H, 5,270 (4,540); C3H_N, 5,310 (4,504); C3H_W, 4,800 (4,550); C3H_NW, 5,440 (4,493). All Zn(II)-bound derivatives are approximately monomeric under the same pH and concentration conditions used in the metal-binding studies.

NMR

Detailed structural information was obtained using both 1D and 2D 1H NMR. In the absence of Zn(II), all peptide derivatives displayed almost identical 1D spectra consistent with unfolded protein structures in solution, with poor peak dispersion in all spectral regions (shown for C3H in Fig. 6). The addition of 2.0 equiv of Zn(II) caused substantial changes in the spectra, including an increase in the dispersion of the amide proton resonances over a large parts per million range, as well as increased dispersion in the aliphatic region with shifts of methyl proton resonances upfield of the main methyl proton cluster around 0.9 ppm (Fig. 6). The spectral changes are similar to those seen for zinc finger domains when binding Zn(II) [56], and are consistent with the formation of a folded state. One notable exception is C3H_NW with Zn(II), which displays a 1D 1H NMR spectrum distinct from the spectra for other Zn(II)-bound L36 derivatives. Specifically, both the amide and aliphatic regions show a decrease in dispersion in the signals, characteristic of protein aggregation or unfolding. This behavior is not apparent for C3H_NW at the lower concentrations used for the SEC studies, suggesting that the high concentrations required for NMR samples gives rise to this problem. Importantly, the narrow line widths and high degree of dispersion seen in the 1D spectra for the other L36 derivatives leads us to believe that aggregation is not occurring in those NMR samples.
Fig. 6

1D 1H NMR spectra of L36 derivatives in the presence (+) or absence () of Zn(II). The spectra of all derivatives in the absence of Zn(II) were similar to that of C3H minus Zn(II).

We utilized 2D NMR techniques (TOCSY and NOESY) to examine in detail the folding of the various L36 derivatives in the presence of Zn(II). The goal was to determine what changes (if any) occur in the solution structure of L36 as a result of introduced modifications in the peptide sequence. We first compared our data for C3H with the data originally used by Härd et al. to determine the solution structure of L36 (kindly furnished to us by Härd). Cross peaks were assigned using the previously published chemical shift data [23] and verified by tracing sequential connectivities in overlays of 2D NOESY and TOCSY spectra (Fig. 7). Our chemical shift data are essentially identical, indicating that the folded structure of L36 is not noticeably perturbed when changing the pH from 7.0 (Härd data) to 6.0 (our conditions).
Fig. 7

Overlays of the Hα–HN region of 2D nuclear Overhauser enhancement spectroscopy (black) and total correlation spectroscopy (red) spectra of Zn(II)-bound C3H (top), C4 (middle) and C3H_N (bottom). Blue labels indicate sequence number

We next compared the 2D spectra of C3H with those obtained for C4 and C3H_N. Figure 7 shows a superposition of the Hα–HN region of the TOCSY spectrum onto the NOESY spectrum for C3H, C3H_N and C4. Strong Hα–HN nuclear Overhauser enhancements, characteristic of a β-structure, are observed between the Cα proton of residue i and the amide proton of residue i+1 for residues 15–19 and 22–26 of both derivatives; these two segments form the β-strands of a β-hairpin in the solution structure of native L36. Figure 8a shows a comparison of the integrated values for the Hαi–HNi+1 nuclear Overhauser enhancement cross peaks. A similar intensity pattern is observed for the three derivatives. A comparison of ΔHα values is shown in Fig. 8b, where ΔHα is calculated using Eq. 4 [57, 58]:
$$ \Delta {\text{H}}\alpha = \Delta \delta _{{\text{H}}\alpha } \left( {{\text{ppm}}} \right) = \delta _{{\text{H}}\alpha } \left( {{\text{protein}}} \right) - \delta _{{\text{H}}\alpha } \left( {{\text{random}}\,{\text{coil}}} \right) $$
(4)
Downfield shifts of Δδ (+0.1 ppm or greater) for a stretch of consecutive residues are indicative of β-sheet secondary structure, while upfield shifts of Δδ (−0.1 ppm or lower) indicate an α-helical structure. As can be seen in Fig. 8b, the three derivatives show the same secondary structure profile, with two β-strand segments separated by non-β structures (turns). Finally, we compared the amide proton chemical shifts for C3H_N and C4 with those observed for C3H. ΔHN was calculated using Eq. 5 and is plotted in Fig. 8c.
$$ \Delta {\text{HN plot}} = {\left| {\delta _{{{\text{HN}}}} {\left( {{\text{C}}_{3} {\text{H}}} \right)} = \delta _{{{\text{HN}}}} {\left( {{\text{C}}_{4} \,{\text{or}}\,{\text{C}}_{3} {\text{H}}\_{\text{N}}} \right)}} \right|} $$
(5)
Only minor changes are observed for the derivatives. C3H_N and C3H amide proton chemical shifts are essentially identical. For C4, the largest differences versus C3H are seen around the mutated site (metal-binding site). The smaller side chain of Cys (as compared with His) requires a slight adjustment of the loop containing Cys32 in order to allow coordination to Zn(II) (which occurs based on the UV–vis results); this localized structural movement to facilitate metal-binding highlights the flexibility of the metal-binding site. Taken in total, the NMR results indicate that (1) all L36 derivatives fold in the presence of Zn(II), and (2) replacement of His32 with Cys in the coordination shell of Zn(II) or the change from a His-Gly to an Asn-Gly β-hairpin turn do not change the fold of the peptide in solution. Furthermore, the NMR data support the interpretation of the CD spectra described earlier, even though the CD spectra lack the shape characteristic of a β-sheet structure.
Fig. 8

Analysis of 2D 1H NMR data for various L36 derivatives. a Comparison of Hα–HN nuclear Overhauser enhancement intensities. Nuclear Overhauser enhancements not shown in the figure were ambiguous and so were not assigned. b Comparison of Hα chemical shifts. c Comparison of Hα chemical shifts. Details for each graph are given in the text.

Discussion

In this study we investigated the consequences of changing ligands to the Zn(II), as well as other amino acids expected to perturb the stability of the protein fold, on the metal-associated properties of the zinc ribbon protein L36. The structural studies indicate that the zinc ribbon motif is tolerant of changes in the Zn(II) coordination shell, the β-turn located at the opposite end of the protein, and in one of the hydrophobic clusters of the β-sheet. All derivatives are monomeric at 100 μM concentrations, with only C3H_NW showing signs of aggregation at higher concentrations (1 mM). The Zn(II)-bound L36 derivatives fold into a similar overall structure on the basis of CD and NMR studies, with small changes created in the metal-binding site when the coordinating ligand His32 is replaced by Cys with a shorter side chain. Optical spectra of the Co(II)-bound derivatives indicate that all utilize a tetrahedral coordination geometry to bind the Co(II). Comparison of the KACo and KAZn values obtained from the Co(II)- and Zn(II)-binding titrations clearly shows that remote changes in the protein (including the β-turn at the opposite end of the domain) can significantly perturb the properties of the metal-binding site. A number of important trends are apparent in the KACo and KAZn values:
  1. 1.

    Zn(II) binds tighter than Co(II) The lower affinity of the L36 derivatives for Co(II) versus Zn(II), which is demonstrated by the ability of Zn(II) to displace Co(II) in competition titrations, has been assigned in large part to a loss of ligand field stabilization energy (LFSE) for Co(II) upon going from the octahedral Co(H2O)62+ to the tetrahedral Co(II)–peptide complex [32, 59]. In contrast, Zn(II) has no LFSE for any coordination geometry, so there is no change when Zn(II) goes from an octahedral to a tetrahedral complex. The KACo and KAZn values determined for the L36 peptides are slightly smaller than many reported in the literature, but compare favorably to those measured at similar pHs [48, 60]. Interestingly, our measured preference of Zn(II) over Co(II) for L36 is opposite to that observed by Boysen et al. [27] for a 26-residue derivative of the same protein (residues 9–34). We have tested the same 26-mer used by Boysen et al. and observe no difference in metal specificity between full-length L36 (C3H) and this 26-residue derivative (see C3H_26 in Table 2). Although it is not clear why this discrepancy exists, it should be noted that in their study, they observed Zn(II)-dependent changes in the CD of the 26-mer consistent with our results, yet they saw no significant population of Zn(II)-bound 26-mer under their electrospray ionization mass spectrometry (ESI-MS) conditions used to measure relative metal-binding affinities, even in the absence of competing metals. This calls into question the ability of their ESI-MS conditions to obtain accurate relative metal-binding information.

     
  2. 2.

    The C3H coordination site binds both Co(II) and Zn(II) tighter than the C4 site. We observed an increase in the specificity for binding of Zn(II) over Co(II) as the number of thiolate ligands was increased from 3 to 4 (Table 2). This is consistent with differences observed in several other naturally occurring Zn(II)-binding proteins [17, 20, 60] and designed sites [61], and is attributed to a decrease in the energy split between the e and t2 sets of d orbitals in a tetrahedral field [19].

     
  3. 3.

    Co(II)- and Zn(II)-binding is increased when His20 is replaced with Asn in the β-hairpin turn. It is becoming increasingly evident that turns in proteins can play important roles as nucleation sites for protein folding [62], and can also affect folding and unfolding rates [63, 64]. It would be interesting to observe how different turn sequences affect the interactions of metal cofactors with metal-binding domains. Blasie and Berg [39] and Kim and Berg [65] have demonstrated stabilization of the native protein fold in zinc finger domains through incorporation of stabilizing interstrand side chain/side chain contacts, which also leads to increases in the measured metal-binding constants. The metal-binding and protein-folding processes are thermodynamically coupled, since the zinc finger is only folded in the presence of Zn(II) or Co(II) [65]. In other words, a change in the equilibrium metal-binding constant that results from a change in an amino acid not directly involved in metal chelation is correlated with a change in ΔGfolding (conformational stability of the protein) [39, 40].

     
We also predicted that stabilization of the β-hairpin at one end of the zinc ribbon motif of L36 should increase metal-binding constants by increasing the population of peptide molecules with ligands in an appropriate position to form a tetrahedral metal-binding site, thus lowering the entropic penalty of protein folding upon binding a metal. In a survey of β-turns in the Protein Data Bank by Hutchinson and Thornton [66], a statistical preference for certain amino acids at the i to i+3 positions of different turn types was observed. Furthermore, β-hairpins have been shown to be stabilized by certain residues in positions i+1 and i+2, with DPro-Gly and Asn-Gly being the best for type I′ turns [3638]. We looked at the family of 38 structures generated by NMR for L36 [PDB no.1DGZ] [23]. The structures fall into three classes based on the ϕ/ψ backbone dihedral angles for i+1 and i+2 residues of a β-hairpin [66]: type II (27 structures), type II′ (nine structures) and type I′ (two structures). A superposition of each structure onto the minimized average structure of L36 (PDB no. 1DFE) [using the backbone atoms of residues in β-strands 1 (residues 15–19) and 2 (residues 22–26)] gave an average root-mean-square deviation of 0.329 (standard deviation 0.091) Å2. This small root-mean-square deviation suggests that the same positioning of β-strands 1 and 2 can be accommodated by all three turn types. Consistent with this, the NMR, SEC and CD results indicate that this mutation does not significantly alter the secondary, tertiary or quaternary structures of L36. Therefore, just as in the case with the zinc finger domain, it is probable that the changes observed in metal-binding affinities are due to changes in the ΔGfolding of the protein caused by the turn mutation.

In summary, we have studied the Co(II) and Zn(II) binding of a series of derivatives of the zinc ribbon protein L36. CD, SEC, and 1D and 2D 1H NMR experiments have shown that the L36 motif is stable towards mutations introduced into the β-hairpin turn, a hydrophobic cluster on the β-sheet, and the tetrahedral coordination environment of the metal—no changes in the overall fold or association state of the various proteins are observed. The derivatives, however, do display changes in the Co(II)- and Zn(II)-binding constants, with an increase observed when the mutation is expected to stabilize the protein fold. These properties should make L36 a useful model system for future structure/reactivity studies.

Footnotes
1

Although one can envision a more thorough treatment with two metals [Co(II) and Zn(II)] competitively binding to two different protein forms (SP1 and SP2), the number of variables involved makes extracting useful information impossible. This model can be greatly simplified by the assumption that the SP1 and SP2 protein forms both have a similar ratio of KAZn to KACo, making them indistinguishable in the competitive titration. The validity of this assumption is supported by the quality of fits obtained with Eq. 2, which assumes both SP1 and SP2 have the same ratio of KAZn to KACo. It is further supported by the consistency of the trends obtained both internally and with literature on Zn-binding proteins, and that no change in the shape of the spectrum occurs during the course of the titration, suggesting that both protein forms have the same set of ligands and ligand geometry (possible if SP2 binds metals as an SP2–SP2 dimer).

 

Acknowledgements

We wish to thank Torleif Härd for providing us with the original NMR assignments for L36, Piu Zhao for assistance with the NMR studies, and Adam Lyons for assistance with peptide purification. Financial support for this work was provided by the American Chemical Society Petroleum Research Fund (PRF no. 40835-AC3; G.R.D.) and The Robert A. Welch Foundation (AT-1448; G.R.D.).

Supplementary material

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