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. 2023 Feb 14;32(3):e4583. doi: 10.1002/pro.4583

Quantification of carboxylate‐bridged di‐zinc site stability in protein due ferri by single‐molecule force spectroscopy

Zhiyi Wang 1, Mengdie Wang 1, Zhongxin Zhao 1, Peng Zheng 1,✉
PMCID: PMC9926469  PMID: 36718829

Abstract

Carboxylate‐bridged diiron proteins belong to a protein family involved in different physiological processes. These proteins share the conservative EXXH motif, which provides the carboxylate bridge and is critical for metal binding. Here, we choose de novo‐designed single‐chain due ferri protein (DFsc), a four‐helical protein with two EXXH motifs as a model protein, to study the stability of the carboxylate‐bridged di‐metal binding site. The mechanical and kinetic properties of the di‐Zn site in DFsc were obtained by atomic force microscopy‐based single‐molecule force spectroscopy. Zn‐DFsc showed a considerable rupture force of ~200 pN, while the apo‐protein is mechanically labile. In addition, multiple rupture pathways were observed with different probabilities, indicating the importance of the EXXH‐based carboxylate‐bridged metal site. These results demonstrate carboxylate‐bridged di‐metal site is mechanically stable and improve our understanding of this important type of metalloprotein.

Keywords: de novo‐designed protein, DFsc, metalloprotein, single‐molecule force spectroscopy

1. INTRODUCTION

Metalloproteins are an indispensable type of bio‐macromolecule in nature, in which the incorporation of different types of metal ions into protein stabilizes the structure and adds functions (Liu et al., 2014; Nguyen & Kleingardner, 2021; Waldron et al., 2009) For example, the carboxylate‐bridged diiron‐containing protein family involves many critical biological processes, such as oxygen carrier protein hemerythrin, ribonucleotide reductase R2, and methane monooxygenase. And the carboxylate‐bridged diiron site is the key feature for them (Bailey et al., 2008; Fushinobu et al., 2003; Hogbom et al., 2004). To further understand these important metal sites, we used atomic force microscopy (AFM)‐based single‐molecule force spectroscopy (SMFS) to quantify the strength of a di‐zinc site in a de novo‐designed single‐chain due ferri protein (DFsc) and to obtain its rupture mechanism.

SMFS is a powerful biophysical methodology that can manipulate individual molecules by external mechanical force and quantify their intermolecular and intramolecular stability (Cao & Li, 2007; Seifi et al., 2021; Suay‐Corredera et al., 2021; Yu et al., 2020; Zhao & Woodside, 2021). Thus, it is widely applied to studying/quantifying protein (un)folding (Alonso‐Caballero et al., 2018; Dietz & Rief, 2004; Marszalek et al., 1999; Ramírez et al., 2017; Rief et al., 1997), protein–protein interactions (Hinterdorfer et al., 1996; Liu et al., 2022; Manibog et al., 2014; Rico et al., 2019; Tian et al., 2021), and chemical bonds (Beedle et al., 2018; Grandbois et al., 1999; Pill et al., 2019; Xiang et al., 2019; Xue et al., 2014; Zhang et al., 2019, 2020). In particular, the metal site strength in metal‐binding proteins is often quantified by AFM‐SMFS, in which the metal site can be ruptured along the protein unfolding. The strength of several different metal sites in proteins has been measured. Nevertheless, most sites quantified so far are mononuclear metal sites (Schmitt et al., 2000; Zheng & Li, 2011). The study of the more complex site with multiple metal ions, such as carboxylate‐bridged diiron protein, remains limited.

Here, we chose a de novo‐designed DFsc as a model protein to study the carboxylate‐bridged di‐zinc site. DFsc is 114 amino acids(aa)‐length, four‐helix protein (Calhoun et al., 2003; Chino et al., 2015; Lombardi et al., 2019.) Two EXXH motifs (Glu73–Glu74–Lys75–His76 and Glu103–Gln104–Arg105–His106) are present in the protein for di‐nuclear metal binding, which is characteristic of carboxylate‐bridged diiron proteins (Figure 1a,b). Glu73 and Glu103 coordinate simultaneously with two metal ions as the bridging ligand (Figure 1b,c). And DFsc can bind Fe(II) and Zn(II) with micromolar affinities. Therefore, to avoid the redox effect of ferrous ions, we chose zinc‐bound DFsc, the redox‐inert surrogate of Fe(II), as a starting point to study the carboxylate‐bridged di‐metal site (Capdevila et al., 2016).

FIGURE 1.

FIGURE 1

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Schematic of the structure and the metal binding sites of single‐chain due ferri protein (DFsc). (a) The de novo‐designed four helix‐bundle structure with a di‐ion metal binding site. (b) Enlarged structure of the metal binding site. Residues involved in chelation are shown in the stick model. Two bridging glutamic acids are colored in wheat, with oxygen atoms colored in red. (c) Simple line‐drawing schematic showing the planar structure of DFsc. The solid red lines between the zinc and α‐helix backbone represent the carboxylate‐bridged metal chelation bonds.

DFsc is a protein with a four‐helix bundle, whose stability generally relies on the charge attraction and the hydrophobic interactions of the amino acid side chains between the α‐helix. From a mechanical perspective, these weak interactions often lead to labile mechanical stability (Lee et al., 2012; Valle‐Orero et al., 2017). We first verified that the four‐helix bundle structure of DFsc is mechanically labile by AFM‐SMFS, similar to other helical proteins. Then, we measured a considerable force for the holo‐protein and demonstrated its unfolding mechanism.

2. RESULTS

2.1. Site‐specific protein immobilization for AFM‐SMFS system

Click chemistry and enzymatic ligation were applied for site specific, covalent immobilization of the target protein. In addition, an XDoc:Coh (XModule‐Dockerin: Cohesion) interaction pair was used as a reversible single‐molecule pulling handle (Stahl et al., 2012). First, we used the SPAAC (strain‐promoted azide‐alkyne cycloaddition) click reaction between azide and DBCO, as well as Michael addition reaction between thiol and maleimide to functionalize the peptide C‐ELP20‐NGL/GL‐ELP20‐C on the AFM tip and substrate, respectively (Figure 2a; Nie et al., 2022; Shi et al., 2022). Here, ELP is the abbreviation of elastin‐like polypeptide with a unit sequence of VPGXG as the single molecule spacer to avoid the short‐range nonspecfic interaction (Ott et al., 2017). Then, fusion protein GL‐CBM‐XDoc and Coh‐DFsc‐NGL were immobilized on the NGL‐functionalized AFM tip and GL‐functionalized substrate using ligase OaAEP1, respectively (Deng et al., 2019). The ligase can covalently link two proteins between their N‐terminal GL dipeptide sequence and C‐terminal NGL tripeptide sequence (Ding et al., 2022; Harmand et al., 2021; Yang et al., 2017).

FIGURE 2.

FIGURE 2

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Atomic force microscopy‐based single‐molecule force spectroscopy (AFM‐SMFS) measurements on apo‐single‐chain due ferri protein (DFsc). (a) Scheme of the AFM‐SMFS set‐up for polyprotein measurements. First, ELP20 with NGL and GL peptide tags was immobilized via strain‐promoted azide‐alkyne cycloaddition (SPAAC) click reaction. Then, fusion proteins GL‐CBM‐XDoc and Coh‐DFsc‐NGL were immobilized by ligase OaAEP1 via ligation between the NGL and GL peptide sequences. (b) Representative force‐extension curves (Liu et al., 2014; Waldron et al., 2009) showing the polyprotein unfolding. Besides the 57 nm peak from fingerprint CBM, no other peak was observed for the apo‐DFsc (Curve 1 and 2). A control experiment (Curve 3) without DFsc showed similar result, whereas another experiment without neither DFsc nor CBM (Curve 4) showed no signal but a last high‐force peak. Taking together, they confirmed the 57‐nm peak was from CBM, the last peak was from the stable XDoc:Coh complex, and apo‐DFsc is mechanically labile.

Next, AFM‐SMFS measurement of the strength of DFsc can be performed. First, the AFM probe approached the substrate, forming a stable protein complex between XDoc on the probe and Coh on the substrate (rupture force >500 pN). Then the probe retracted under a constant velocity, delivering tensile force to the target protein DFsc. Force is transmitted along the protein domains/backbone, inducing the protein unfolding, metal site rupture, and dissociation of the protein complex along the pathway.

2.2. AFM‐SMFS measurements on DFsc

To verify the four‐helix bundle structure of DFsc is mechanically labile, we first performed AFM experiment on apo‐DFsc without bound metal. The protein was obtained by preincubated with 1 mM EDTA after purification. In the force‐extension curves of apo‐DFsc, we only observed two peaks which can be fitted by worm‐like chain model (Figure 2b, dash line, Curves 1 and 2). The first of them shows an unfolding force of ~150 pN and a ΔLc (contour length increment) of ~57 nm, which belongs to fingerprint protein CBM unfolding. And the second peak of ~500 pN belongs to the rupture of XDoc:Coh complex. No other unfolding signal was observed.

We also performed a control experiment using Coh‐NGL without DFsc on the surface, and a similar result with a 57 nm peak was observed (Figure 2b, Curve 3). In addition, a further control experiment using GL‐XDoc with neither CBM nor DFsc showed only the last peak (Figure 2b, Curve 4). Taking together, they confirmed the 57‐nm peak was from CBM, the last peak was from the stable XDoc:Coh complex, and apo‐DFsc is most likely mechanically labile, with an unfolding force lower than the detection limit of our AFM (~20 pN).

Next, we studied the mechanical stability of the carboxylate‐bridged di‐zinc site in DFsc. After protein purification, we conducted AFM‐SMFS experiments using the same experimental set‐up as apo‐form experiments (Figure 3a). Indeed, additional signals besides CBM unfolding and XDoc:Coh complex rupture were observed, which should be derived from the unfolding of Zn‐DFsc. First, a new ~32 nm force peak was observed (Figure 3b), which agrees with the one‐step rupture of the complete di‐zinc site in DFsc. Since 97 residues are present in the zinc site between Glu10 and His106, the theoretical ΔLc is 34.1 nm upon protein unfolding (97aa × 0.36 nm/aa – 0.76 nm, the average backbone length of one amino acid is 0.36 nm, and the distance in the folded protein is 0.76 nm).

FIGURE 3.

FIGURE 3

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Full‐length unfolding on Zn‐single‐chain due ferri protein (DFsc). (a) Scheme of the atomic force microscopy (AFM)‐based single‐molecule force spectroscopy set‐up for Zn‐DFsc measurement. (b–d) Representative force‐extension curves from unfolding scenarios of complete Zn‐DFsc are shown at the bottom, and their corresponding di‐zinc site rupture pathway is shown at the top. (b) The one‐step unfolding scenario shows a single ~32 nm length peak, corresponding to the rupture of the entire di‐zinc site in one step. (c) The two‐step unfolding scenario shows a first ~12 nm peak from the extension of residues between Glu10 and Glu43 (dash line), followed by a ~22 nm peak from Glu43 to His106. (d) Another two‐step scenario shows a first ~22 nm peak from Glu10 to Glu73 (dash line), followed by a ~12 nm peak from Glu73 to His106.

In addition to the one‐step unfolding event, we also observed two‐step unfolding events whose added length of ΔLc is ~34 nm. The first stepwise unfolding event shows a ~12 nm peak, followed by a ~22 nm peak. The first peak is mostly like from the rupture of the single Zn—O(Glu10) bond in the di‐zinc site, leading to the extension of residues between Glu10 and Glu43 (34 × 0.36–0.82 = 11.4 nm; Figure 3c). Then, the second peak is from the rupture of the remaining metal site, from Glu43 to His106 (64 × 0.36–1.27 = 21.7 nm). Here, we believe that the di‐zinc site initially ruptures from the N‐terminus of protein due to the less and possibly weaker metal–ligand bond in this direction. A similar ΔLc result will be obtained as well if the site ruptures from the C‐terminus. However, there are three zinc‐ligand bonds present at the C‐terminus part. And the N‐terminus part of the di‐zinc site should be much less stable. Finally, for the rupture mechanism of the zinc‐coordination bonds from Glu103 to His106 in the same EHHX motif, we think their rupture is simultaneous as they are close to each other and hold the zinc ion together. Thus, we used His106 instead of Glu103 to calculate the theoretical ΔLc.

Indeed, another stepwise unfolding event shows a first ~22 nm peak, followed by a ~12 nm peak (Figure 3d). The first peak can be from the partial rupture of the di‐zinc site between Glu10 and Glu73 (64 × 0.36–0.94 = 22.1 nm), in which the two single Zn—O bonds are rupture. Then, the second peak is from the rupture of the remaining metal site, from Glu73 to His106 (34 × 0.36–0.99 = 11.3 nm). These stepwise unfolding events, which agree well with the stepwise rupture of the metal center, further prove that the signal is from the di‐zinc metal site in the protein.

In addition to full‐length unfolding events (32, 12 + 22, and 22 + 12 nm), we also observed a large population of partial unfolding scenarios. Force‐extension curve shows a single ~12 or ~22 nm unfolding event from DFsc (Figure 4a,b). In summary, Zn‐DFsc exhibits a trimodal distribution of ΔLc upon unfolding, peaking at 11.5 ± 2.0, 21.8 ± 2.2, and 31.9 ± 2.7 nm (Figure 4c). The one‐step rupture of the di‐zinc site (32 nm) accounted for 29%. The partial unfolding event with the 12 nm peak accounted for 20% and the 22 nm peak for 44% (Figure 4e). Thus, the carboxylate‐bridge zinc site is relatively stable yet dynamic with a partially unfolded state. This may arise from its helical structure, leading to several intermediate states. Indeed, the unfolding force of 32 nm events is 163 ± 84 pN, of 22 nm events is 199 ± 127 pN, and of 12 nm events is 201 ± 131 pN, respectively (Figure 4d).

FIGURE 4.

FIGURE 4

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Partial unfolding on Zn‐single‐chain due ferri protein (DFsc) and the summary of unfolding statistics. (a,b) Representative force‐extension curves from the single‐step unfolding event of Zn‐DFsc are shown at the bottom, and their corresponding di‐zinc site rupture process is shown at the top. (a) The unfolding scenario shows only a single ~12 nm peak corresponding to rupture from Glu73 to His106. (b) Another single‐step unfolding scenario shows only a ~22 nm peak corresponding to rupture from Glu43 to His106. (c) ΔLc histogram shows a trimodal distribution of Zn‐DFsc (11.5, 21.8, and 31.9 nm) and the value of CBM (56.2 nm). (d) Force histogram of Zn‐DFsc shows a similar average force of the single‐step protein unfolding evens, 163 pN for the 32 nm event, 199 pN for the 22 nm event, and 201 pN for the 12 nm event. (e) Pie chart of the proportion of different unfolding scenarios in which full‐length unfolding accounted for 36% (29% + 4% + 3%), 22 nm partial‐unfolding accounted for 44%, and 12 nm partial‐unfolding accounted for 20%. N = 781.

2.3. Dynamic force spectroscopy for Zn‐DFsc

We performed the dynamic force spectroscopy experiment to investigate the kinetics of the di‐zinc chelation center. Upon stretching the target protein under different velocities, the rupture force shows a linear relationship with the logarithm of the force loading rates (Figure 5). Fitted by Bell–Evans model, two key kinetic parameters, k 0 (the bond dissociation rate at zero force), and Δx (the distance between the bonded state and transition state) were obtained. The exact values are summarized in Table 1. For 22 and 12 nm partial unfolding events, they share almost the same Δx of 0.085 nm and a similar k0 value of ~2.0 s−1. However, 32 nm full‐length unfolding events have a larger Δx value of 0.094 nm and a higher k 0 value of 2.5 s−1, implying lower dynamic stability than 22 and 12 nm events.

FIGURE 5.

FIGURE 5

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Dynamic force spectrum results for Zn‐single‐chain due ferri protein (DFsc). The rupture force follows a linear relationship with the logarithm of the force loading rate. Bell–Evans model was used to fit the plots and the standard deviations were marked by error bars. For full‐length unfolding events (a) Δx is 0.094 nm and k 0 is 2.5 s−1. For partial unfolding events (b,c) Δx is 0.085 nm and k 0 is 2.0 s−1.

TABLE 1.

AFM‐SMFS results of the di‐zinc site in DFsc.

ΔLc (nm) F (pN) Δx (nm) k 0 (s−1)
32 163 0.094 2.50
22 199 0.085 2.00
12 201 0.083 1.91
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Abbreviations: AFM, atomic force microscopy; DFsc, single‐chain due ferri protein; SMFS, single‐molecule force spectroscopy.

3. DISCUSSION

Here, we employed AFM‐SMFS to study the stability of the carboxylate‐bridged di‐zinc site in de novo‐designed protein DFsc. First, we showed apo‐DFsc is mechanically liable with an unfolding force lower than 20 pN, which agrees well with the previous conclusion of helical‐structured proteins, such as spectrin, coiled‐coil, and SAH (Lopez‐Garcia et al., 2019; Serquera et al., 2010; Wolny et al., 2014). When compared with many mechanically stable proteins with β‐strand/sheet structure, such as titin, most helical structure proteins are labile. Protein secondary structure is the dominant factor for protein mechanical stability (Crampton & Brockwell, 2010). It becomes clear that this rule applies to naturally occurring and de novo‐designed proteins (Carrion‐Vazquez et al., 2000; Chakraborty et al., 2011; Wang et al., 2021; Wang, Zhao, et al., 2022).

Then, we focused on measuring the stability of the carboxylate‐bridged di‐zinc site in DFsc and found a considerable unfolding force of ~200 pN. So far, most metal sites in metalloprotein studied by AFM are mononuclear, such as iron–sulfur protein, zinc‐finger protein, and copper‐containing protein (Beedle et al., 2015; Perales‐Calvo et al., 2015). Metallothionein and ferredoxin with multiple metal ions and inorganic sulfur are very few exceptions (Lei et al., 2017; Li & Li, 2018; Yuan et al., 2019; Yuan et al., 2021). Here, the di‐zinc site has been investigated, and the role of the carboxylate bridge is highlighted. To avoid the redox issue, we studied the zinc substituted, which is also an essential metal ion and can be a proper starting point (Wang, Kuci, et al., 2022).

We found that the two EXXH motifs are essential in maintaining the metal site's overall stability. Besides the high unfolding force of 200 pN, the two single‐rupture events (22 and 12 nm) share similar kinetic properties. These two events respond to the partial rupture of the two EXXH motifs bound with metals. After the dissociation of the N‐terminal α‐helix upon the rupture of the Zn—O(Glu10) and Zn—O(Glu43) bond, the interactions between di‐zinc ions and the C‐terminal α‐helix (bridging Glu73 and Glu103, His76, and His106) can be stable enough to support the partially unfolded protein.

In addition, the charge changes of the metal site during the protein unfolding process are interesting. Under our experimental conditions (pH 7.0), NHis atom (pKa ~6.0) and OGlu (pKa ~4.2) in the metal‐binding residues involved in the coordination are both negatively charged. However, di‐Zn(II) has a strong positive charge. When DFsc is fully folded, these atoms (six OGlu atoms and two NHis atoms) share di‐Zn(II) charge. However, once the protein is partially unfolded, the positive charge of di‐Zn(II) is polarized, and more positive charges will interact with the OGlu atoms in the bridging glutamic acid and NHis atoms. The polarization of the positive charge might increase the coordination bond strength, as well as their mechanical and kinetic stability (Cheng et al., 2015; Koone et al., 2022). This may be a self‐protection mechanism developed by multimetal‐containing enzymes, where the charge in the metal center can be redistributed when the enzyme partially unfolds due to the interference caused by shearing force or reaction substrate moving.

As a single‐strand helical structure protein, DFsc provides an excellent model to study the metal sites in de novo‐designed protein with a mechanically labile protein structure and detectable ΔLc signal. Nevertheless, the symmetrical structure of the protein complicates the peak assignment. For the ~12 nm event, we think it is more likely from Glu73 to His106 because it is more stable than the Glu10–Glu43. The two residues belong to the bridging ligand in which two carboxyl groups of the glutamic acid side chain can coordinate with two different Zn(II) simultaneously, and multiple bonds coordinate the di‐zinc site. Thus, if the coordination bonds on the other side (Glu10, Glu43) are unstable and self‐dissociate, the di‐Zn(II) site still coordinates with the two EXXH motifs and remains relatively stable. Thus, the unfolding of such a structure leads to the 12 nm event. Similarly, for the ~22 nm event, we think it is more likely from Glu43 to His106 because it is more stable than Glu10–His76.

In conclusion, we used AFM‐SMFS to investigate the carboxylate‐bridged di‐Zn(II) site in de novo‐designed due ferri protein and measured the strength and kinetics of Zn‐DFsc. Our work revealed the stabilizing role of carboxylate‐bridge in the EXXH motif for the metal site, adding more knowledge on this important type of metalloproteins/metalloenzymes.

AUTHOR CONTRIBUTIONS

Zhiyi Wang: Data curation (lead); formal analysis (lead); investigation (lead); writing – original draft (equal). Mengdie Wang: Investigation (supporting). Zhongxin Zhao: Investigation (supporting). Peng Zheng: Project administration (lead); supervision (lead); writing – original draft (equal); writing – review and editing (lead).

FUNDING INFORMATION

This work was supported by the National Natural Science Foundation of China (Grant No. 21977047) and by Natural Science Foundation of Jiangsu Province (Nos. BK20200058 and BK20202004).

CONFLICT OF INTEREST STATEMENT

The authors declare no competing interests.

Supporting information

Appendix S1. Supporting information.

Click here for additional data file. (48.8KB, docx)

Wang Z, Wang M, Zhao Z, Zheng P. Quantification of carboxylate‐bridged di‐zinc site stability in protein due ferri by single‐molecule force spectroscopy. Protein Science. 2023;32(3):e4583. 10.1002/pro.4583

Review Editor: John Kuriyan

Funding information National Natural Science Foundation of China, Grant/Award Number: 21977047; Natural Science Foundation of Jiangsu Province, Grant/Award Numbers: BK20202004, BK20200058

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Appendix S1. Supporting information.

Click here for additional data file. (48.8KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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