Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 May 4;14(21):5756-5767.
doi: 10.1039/d3sc00400g. eCollection 2023 May 31.

Arsenic binding to human metallothionein-3

Amelia T Yuan  1 , Martin J Stillman  1
Affiliations

Arsenic binding to human metallothionein-3

Amelia T Yuan et al. Chem Sci. .

Abstract

Arsenic poisoning is of great concern with respect to its neurological toxicity, which is especially significant for young children. Human exposure to arsenic occurs worldwide from contaminated drinking water. In human physiology, one response to toxic metals is through coordination with the metallochaperone metallothionein (MT). Central nervous system expression of MT isoform 3 (MT3) is thought to be neuroprotective. We report for the first time on the metalation pathways of As3+ binding to apo-MT3 under physiological conditions, yielding the absolute binding constants (log Kn, n = 1-6) for each sequential As3+ binding event: 10.20, 10.02, 9.79, 9.48, 9.06, and 8.31 M-1. We report on the rate of the reaction of As3+ with apo-MT3 at pH 3.5 with rate constants (kn, n = 1-6) determined for each sequential As3+ binding event: 116.9, 101.2, 85.6, 64.0, 43.9, and 21.0 M-1 s-1. We further characterize the As3+ binding pathway to fully metalated Zn7MT3 and partially metalated Zn-MT3. As3+ binds rapidly with high binding constants under physiological conditions in a noncooperative manner, but is unable to replace the Zn2+ in fully-metalated Zn-MT3. As3+ binding to partially metalated Zn-MT3 takes place with a rearrangement of the Zn-binding profile. Our work shows that As 3+ rapidly and efficiently binds to both apo-MT3 and partially metalated Zn-MT3 at physiological pH.

PubMed Disclaimer

Conflict of interest statement

There are no conflicts to declare.

Figures

Fig. 1
Fig. 1. Amino acid sequence of MT3 used in this study. Amino acids (AAs) 1 to 31 form the β domain, 32 to 34 form the linker region, and 35 to 73 form the α domain. The domains are labelled for the clusters formed when 7 equivalents of Zn2+ or Cd2+ bind to the cysteines (yellow). The AAs in pink are included in the recombinant protein for increased stability during the expression step. The AAs in blue are the S-tag for increased stability, removed in the protein purification process.
Fig. 2
Fig. 2. As3+ metalation of apo-MT3 at pH 7.4, monitored using ESI-MS. The concentration of apo-MT3 used was 50 μM and 8 molar equivalents of As3+ from As2O3 were added. (A) Charge state spectrum at 1 minute post metalation. (B) Deconvoluted spectrum 1 minute post metalation. (C) UV-visible absorption spectrum of apo-MT3 and As6MT3. The As6MT3 was confirmed to be the sole species using ESI-MS.
Fig. 3
Fig. 3. As3+ stepwise titration into apo-MT3 with 3 molar equivalents of reduced GSH at pH 7.4. A sample of charge state spectra (left panels) and deconvoluted spectra (right panels) for As3+ added at 1.15 (A), 2.30 (B), 4.60 (C), and 5.75 (D) molar equivalents. All spectra were recorded for 2 minutes at ambient temperature. (E) Speciation of AsnMT3 species as a function of mol. eq. As3+ added to apo-MT3 in solution with GSH at pH 7.4. Symbols represent experimental data and solid lines represent model fitted to data. The model was calculated using HySS software. (F) Corresponding log K inputted to HySS. The log K values determined for each As3+ binding event are as follows: 10.20, 10.02, 9.79, 9.48, 9.06, and 8.31 M−1.
Scheme 1
Scheme 1. The sequential binding pathway for As3+ binding to apo-MT3. The equilibrium constants for each step are indicated by K1–6 for each As3+ binding event.
Fig. 4
Fig. 4. As3+ binding to apo-MT3 as a function of time. 8 molar equivalents of As3+ were added to 20 μM apo-MT3 at pH 3.5. A total of 6 replicates were analyzed to ensure accuracy. (A–F) Charge state (left) and deconvoluted (right) mass spectra of As3+ metalation kinetics taken at 2 minutes (A), 4 minutes (B), 10 minutes (C), 16 minutes (D), 24 minutes (E), and 45 minutes (F) as a sample of data collected. Charge states are labelled on the first spectra and apply to the spectra below. Apo-MT3 and AsnMT3 species are labelled and applies to all spectra. (G) Speciation of AsnMT3 species as a function of time, with experimentally determined data represented by points and modelled speciation curves fitted by COPASI as solid lines. (H) Fitted rate constants for each As3+ binding event to MT3, with standard error represented by error bars.
Scheme 2
Scheme 2. The sequential binding pathway for As3+ binding to apo-MT3. The biomolecular rate constants for each step are indicated by k1–6 for each As3+ binding event.
Fig. 5
Fig. 5. As3+ binding to apo-MT3 at pH 3.5 monitored at 290 nm as a function of time. (A) Sample of absorption spectra taken at various time point intervals during the reaction. The arrow shows the 290 nm absorption band increasing in intensity as As3+ binds to apo-MT3. (B) Kinetic trace of As3+ binding determined by the 290 nm band (black line) and fitted trace determined by COPASI (red line). The fitted k = 16.6 M−1 s−1. (C) Kinetic trace of As3+ binding experimentally determined by UV-visible spectroscopy (blue line) compared with the time-dependence of As3+ binding calculated from mass spectral data (black line).
Fig. 6
Fig. 6. Ribbon and ball-and-stick molecular models of representative As-MT3 species. The energy-minimized structures were calculated for 300 K conditions for 1000 ps with 0.02 ps equilibration time. The protein backbone is represented by the grey ribbon, the S atoms in yellow, the As3+ ions in purple, and the rest of the protein in brown. (A) The As3+ was bound to three cysteinyl thiolates sequentially starting at the N-terminal β domain (Cys1–3, Cys4–6, Cys7–9, Cys10–12, Cys13–15, Cys18–20), with the last As3+ bound by the last three thiolates due to the sequential thiolates occurring on either side of the acidic loop of the α domain. (B) The As3+ was bound to three cysteinyl thiolates sequentially starting at the C-terminal α domain, in reverse order to (A).
Fig. 7
Fig. 7. As3+ binding to Zn7MT3 at pH 7.4. (A and B) Charge state (left) and deconvoluted (right) spectra of Zn7MT3 (A) and Zn7MT3 with 10 molar equivalents of As3+ added (B). The charge states are labelled in the first panel and apply to the charge state spectrum below. The mass of Zn7MT3 is labelled in the deconvoluted spectra. (C) Absorption spectra of Zn7MT3 (black line) and Zn7MT3 with 10 molar equivalents of As3+ added after 12 hours of reaction time.
Fig. 8
Fig. 8. As3+ titration into partially metalated Zn-MT3 at pH 7.4. (A–H) Charge state (left) and deconvoluted (right) spectra of As3+ added to the Zn-MT3 solution formed by adding 4 molar equivalents of Zn2+ to apo-MT3 with As3+ molar ratios of 0 molar equivalents (A), 0.5 molar equivalents (B), 1.0 molar equivalents (C), 1.5 molar equivalents (D) 2.0 molar equivalents (E), 2.5 molar equivalents (F), 3.0 molar equivalents (G), and 6.0 molar equivalents (H). (I) Speciation diagram of AsnMT3 species as a function of molar equivalents of As3+ added to the Zn-MT3 solution formed by adding 4 molar equivalents of Zn2+ to apo-MT3.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. IARC, Arsenic, Metals, Fibres, and Dusts, International Agency for Research on Cancer, Lyon, France, 2012
    1. Rahman S. Kim K.-H. Saha S. K. Swaraz A. M. Paul D. K. J. Environ. Manage. 2014;134:175–185. doi: 10.1016/j.jenvman.2013.12.027. - DOI - PubMed
    1. Chen Q. Y. Costa M. Annu. Rev. Pharmacol. Toxicol. 2021;61:47–63. doi: 10.1146/annurev-pharmtox-030220-013418. - DOI - PubMed
    1. Chung J. Y. Yu S. D. Hong Y. S. J. Prev. Med. Public Health. 2014;47:253–257. doi: 10.3961/jpmph.14.036. - DOI - PMC - PubMed
    1. Shen S. Li X.-F. Cullen W. R. Weinfeld M. Le X. C. Chem. Rev. 2013;113:7769–7792. doi: 10.1021/cr300015c. - DOI - PMC - PubMed