Relations between Structure and Zn(II) Binding Affinity Shed Light on the Mechanisms of Rad50 Hook Domain Functioning and Its Phosphorylation
Abstract
:1. Introduction
2. Results
2.1. Folding of the Hook Domain Induced by Zn(II)
2.2. Stability of the Zinc Hook Domain
2.3. Small Angle X-ray Scattering (SAXS) Confirms Rod-Shaped Phosphomimetic Zinc Hook
2.4. X-ray Absorption Spectrum in the Near-Edge Structure and Extended X-ray Absorption Fine Structure Show Tetrathiolate Zn(II) in HsHk183
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Peptide Synthesis
4.3. Expression and Purification of Metal-Free HsHk183
4.4. XANES and EXAFS
4.5. SAXS
4.6. Spectroscopic Studies
4.6.1. Electron Absorption Spectroscopy in UV Range
4.6.2. Spectropolarimetric Titrations with Zn(II)
4.7. Competitive Titrations
4.8. Potentiometry
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
ATM | ataxia-telangiectasia mutated kinase |
CD | circular dichroism |
CI | competitivity index |
EXAFS | extended X-ray absorption fine structure |
HsHk | zinc hook from H. sapiens Rad50 |
MRN | Mre11, Rad50, Nbs1 complex |
NHEJ | non-homologous end joining |
PDB | Protein Data Bank |
PfHk | zinc hook from P. furiosus Rad50 |
SAXS | Small angle X-ray scattering |
XANES | X-ray absorption near-edge structure |
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Zinc Hook Peptide | logK12–Competition | logK12–Potentiometry |
---|---|---|
HsHk4 | n.d. a | 14.93 b |
HsHk6 | n.d. | 15.13 |
HsHk10 | 16.5 ± 0.4 | 16.15 |
HsHk14 | 15.8 ± 0.4 | n.d. |
HsHk42 | 17.26 ± 0.08 | n.d. |
HsHk42 pT690 | 16.4 ± 0.2 | n.d. |
HsHk42 T690E | 16.5 ± 0.1 | n.d. |
HsHk72 | 18.3 ± 0.4 | n.d. |
HsHk140 | 19.5 ± 0.2 | n.d. |
HsHk183 | 19.53 ± 0.06 | n.d. |
HsHk183 T690E | 19.27 ± 0.05 | n.d. |
Protein Complex | Residues in Coordination Sphere (Complex Stoichiometry) | Experimental Conditions | logK a | logK Estimated to pH 7.4 b | CI c Estimated to pH 7.4 b | Reference |
---|---|---|---|---|---|---|
CD4-Lck kinase complex (H. sapiens) | 2 × CC (ZnL′L″) | 150 mM NaCl, 10 mM Na2SO4, 0.08%, β-ME, 50 mM ZnSO4, 10 mM HEPES, pH 7.0 | 6.4 | 8 | 4.7 | [51] |
CD4-Lck kinase complex (H. sapiens) | 2 × CC (ZnL′L″) | 20 mM Tris, 10 mM NaClO4, 15 μM TCEP, pH 7.4 | 18.97 | 18.97 | 15.67 | [52] |
CD8α-Lck kinase complex (H. sapiens) | 2 × CC (ZnL′L″) | 150 mM NaCl, 10 mM Na2SO4, 0.08%, β-ME, 50 mM ZnSO4, 10 mM HEPES, pH 7.0 | 6.05 | 7.65 | 4.35 | [51] |
Rad50 (P. furiosus) | 2 × CC (ZnL2) | 20 mM Tris-HCl, 100 mM NaClO4, pH 7.4 | 20.82 | 20.82 | 17.52 | [27] |
Rad50 (H. sapiens) | 2 × CC (ZnL2) | 20 mM Tris-HCl, 100 mM NaClO4, pH 7.4 | 19.52 | 19.52 | 16.22 | this study |
CP1 | CCCC (ZnL) | 50 mM HEPES, 100 mM NaCl, pH 7.0 | 12.0 | 13.6 | 13.6 | [60] |
CP1 | CCHH (ZnL) | 50 mM HEPES, 100 mM NaCl, pH 7.0 | 11.2 | 12 | 12 | [60] |
MTF1-1 (H. sapiens) | CCHH (ZnL) | 50 mM HEPES, pH 7.4, 100 mM NaClO4 | 11.6 | 11.6 | 11.6 | [61] |
MTF1-1 (H. sapiens) | CCHH (ZnL) | 100 mM HEPES, 50 mM NaCl, pH 7.0 | 10.5 | 11.3 | 11.3 | [62] |
PDLIM1 (H. sapiens) | CCCC (ZnL) | 50 mM Tris, 100 mM NaCl, pH 7.4 | 14.5 | 14.5 | 14.5 | [49] |
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Tran, J.B.; Padjasek, M.; Krężel, A. Relations between Structure and Zn(II) Binding Affinity Shed Light on the Mechanisms of Rad50 Hook Domain Functioning and Its Phosphorylation. Int. J. Mol. Sci. 2022, 23, 11140. https://doi.org/10.3390/ijms231911140
Tran JB, Padjasek M, Krężel A. Relations between Structure and Zn(II) Binding Affinity Shed Light on the Mechanisms of Rad50 Hook Domain Functioning and Its Phosphorylation. International Journal of Molecular Sciences. 2022; 23(19):11140. https://doi.org/10.3390/ijms231911140
Chicago/Turabian StyleTran, Józef Ba, Michał Padjasek, and Artur Krężel. 2022. "Relations between Structure and Zn(II) Binding Affinity Shed Light on the Mechanisms of Rad50 Hook Domain Functioning and Its Phosphorylation" International Journal of Molecular Sciences 23, no. 19: 11140. https://doi.org/10.3390/ijms231911140