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Lys63-linked ubiquitin chain adopts multiple conformational states for specific target recognition.

Liu Z, Gong Z, Jiang WX, Yang J, Zhu WK, Guo DC, Zhang WP, Liu ML, Tang C - Elife (2015)

Bottom Line: Free or bound to ligands, polyubiquitins are found in different arrangements of ubiquitin subunits.A point mutation that shifts the equilibrium between the different states modulates the binding affinities towards K63-Ub2 ligands.This conformational selection mechanism at the quaternary level may be used by polyubiquitins of different lengths and linkages for target recognition.

View Article: PubMed Central - PubMed

Affiliation: CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, China.

ABSTRACT
A polyubiquitin comprises multiple covalently linked ubiquitins and recognizes myriad targets. Free or bound to ligands, polyubiquitins are found in different arrangements of ubiquitin subunits. To understand the structural basis for polyubiquitin quaternary plasticity and to explore the target recognition mechanism, we characterize the conformational space of Lys63-linked diubiquitin (K63-Ub2). Refining against inter-subunit paramagnetic NMR data, we show that free K63-Ub2 exists as a dynamic ensemble comprising multiple closed and open quaternary states. The quaternary dynamics enables K63-Ub2 to be specifically recognized in a variety of signaling pathways. When binding to a target protein, one of the preexisting quaternary states is selected and stabilized. A point mutation that shifts the equilibrium between the different states modulates the binding affinities towards K63-Ub2 ligands. This conformational selection mechanism at the quaternary level may be used by polyubiquitins of different lengths and linkages for target recognition.

No MeSH data available.


Related in: MedlinePlus

The interactions between K63-Ub2 mutant with tUIM and with NZF.(A, B) Illustration of the E64RP mutation in the complex structures (Protein Data Bank [PDB] codes 2RR9 and 2WX0). The point mutation is distant from the K63-Ub2 interfaces with tUIM and NZF. (C, D) Isothermal calorimetry (ITC) measurements for the bindings with tUIM and NZF. The raw data (top panels) are converted to heat per injection, and the fitted curves using one-site binding model are shown as solid lines (bottom panels). The binding affinities KD, binding enthalpy changes ΔH, and entropy changes ΔS values are averaged over four independent experiments.DOI:http://dx.doi.org/10.7554/eLife.05767.023
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fig6: The interactions between K63-Ub2 mutant with tUIM and with NZF.(A, B) Illustration of the E64RP mutation in the complex structures (Protein Data Bank [PDB] codes 2RR9 and 2WX0). The point mutation is distant from the K63-Ub2 interfaces with tUIM and NZF. (C, D) Isothermal calorimetry (ITC) measurements for the bindings with tUIM and NZF. The raw data (top panels) are converted to heat per injection, and the fitted curves using one-site binding model are shown as solid lines (bottom panels). The binding affinities KD, binding enthalpy changes ΔH, and entropy changes ΔS values are averaged over four independent experiments.DOI:http://dx.doi.org/10.7554/eLife.05767.023

Mentions: Upon the E64RP mutation, the binding between K63-Ub2 and the open-state ligand tUIM becomes tighter, with the KD value decreasing by more than fourfold to 2.2 ± 0.1 µM (Figure 6C). On the other hand, the binding towards closed-state ligands weakens—the KD value of K63-Ub2 binding towards NZF increases by ∼50% to 17.8 ± 1.1 µM (Figure 6D). Importantly, the enthalpy change ΔH values are almost identical for the bindings involving the wild type and mutant proteins (Figure 6 and Figure 1—figure supplement 3). For the interaction between K63-Ub2 mutant and A20 ZnF4, more peaks at the interface (residues 50–62 of the distal unit) can be traced, which can be attributed to a faster exchange than that of the wild type. Significantly, the KD value increases by almost threefold to 1199.9 ± 104.9 µM (Figure 7). Together, the binding affinity towards the open-state ligand increases at the expense of the binding affinities towards the closed-state ligands, and the changes in binding affinities are caused entropically due to the perturbation of K63-Ub2 conformational space.


Lys63-linked ubiquitin chain adopts multiple conformational states for specific target recognition.

Liu Z, Gong Z, Jiang WX, Yang J, Zhu WK, Guo DC, Zhang WP, Liu ML, Tang C - Elife (2015)

The interactions between K63-Ub2 mutant with tUIM and with NZF.(A, B) Illustration of the E64RP mutation in the complex structures (Protein Data Bank [PDB] codes 2RR9 and 2WX0). The point mutation is distant from the K63-Ub2 interfaces with tUIM and NZF. (C, D) Isothermal calorimetry (ITC) measurements for the bindings with tUIM and NZF. The raw data (top panels) are converted to heat per injection, and the fitted curves using one-site binding model are shown as solid lines (bottom panels). The binding affinities KD, binding enthalpy changes ΔH, and entropy changes ΔS values are averaged over four independent experiments.DOI:http://dx.doi.org/10.7554/eLife.05767.023
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4507786&req=5

fig6: The interactions between K63-Ub2 mutant with tUIM and with NZF.(A, B) Illustration of the E64RP mutation in the complex structures (Protein Data Bank [PDB] codes 2RR9 and 2WX0). The point mutation is distant from the K63-Ub2 interfaces with tUIM and NZF. (C, D) Isothermal calorimetry (ITC) measurements for the bindings with tUIM and NZF. The raw data (top panels) are converted to heat per injection, and the fitted curves using one-site binding model are shown as solid lines (bottom panels). The binding affinities KD, binding enthalpy changes ΔH, and entropy changes ΔS values are averaged over four independent experiments.DOI:http://dx.doi.org/10.7554/eLife.05767.023
Mentions: Upon the E64RP mutation, the binding between K63-Ub2 and the open-state ligand tUIM becomes tighter, with the KD value decreasing by more than fourfold to 2.2 ± 0.1 µM (Figure 6C). On the other hand, the binding towards closed-state ligands weakens—the KD value of K63-Ub2 binding towards NZF increases by ∼50% to 17.8 ± 1.1 µM (Figure 6D). Importantly, the enthalpy change ΔH values are almost identical for the bindings involving the wild type and mutant proteins (Figure 6 and Figure 1—figure supplement 3). For the interaction between K63-Ub2 mutant and A20 ZnF4, more peaks at the interface (residues 50–62 of the distal unit) can be traced, which can be attributed to a faster exchange than that of the wild type. Significantly, the KD value increases by almost threefold to 1199.9 ± 104.9 µM (Figure 7). Together, the binding affinity towards the open-state ligand increases at the expense of the binding affinities towards the closed-state ligands, and the changes in binding affinities are caused entropically due to the perturbation of K63-Ub2 conformational space.

Bottom Line: Free or bound to ligands, polyubiquitins are found in different arrangements of ubiquitin subunits.A point mutation that shifts the equilibrium between the different states modulates the binding affinities towards K63-Ub2 ligands.This conformational selection mechanism at the quaternary level may be used by polyubiquitins of different lengths and linkages for target recognition.

View Article: PubMed Central - PubMed

Affiliation: CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, China.

ABSTRACT
A polyubiquitin comprises multiple covalently linked ubiquitins and recognizes myriad targets. Free or bound to ligands, polyubiquitins are found in different arrangements of ubiquitin subunits. To understand the structural basis for polyubiquitin quaternary plasticity and to explore the target recognition mechanism, we characterize the conformational space of Lys63-linked diubiquitin (K63-Ub2). Refining against inter-subunit paramagnetic NMR data, we show that free K63-Ub2 exists as a dynamic ensemble comprising multiple closed and open quaternary states. The quaternary dynamics enables K63-Ub2 to be specifically recognized in a variety of signaling pathways. When binding to a target protein, one of the preexisting quaternary states is selected and stabilized. A point mutation that shifts the equilibrium between the different states modulates the binding affinities towards K63-Ub2 ligands. This conformational selection mechanism at the quaternary level may be used by polyubiquitins of different lengths and linkages for target recognition.

No MeSH data available.


Related in: MedlinePlus