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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.


Overlay of NMR spectra for K63-Ub2 and K63-Ub2 mixed with equimolar A20 ZnF4 at (A) 303 K and (B) 313 K.The protein concentrations are 50 µM. The timescale of the exchange between ZnF4-free and bound species is slow for residues 50–62 of K63-Ub2 distal unit, whose peaks disappear upon ZnF4 titration. At 313 K, the exchange is slightly faster, which allows the fitting of the KD value.DOI:http://dx.doi.org/10.7554/eLife.05767.026
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fig7s2: Overlay of NMR spectra for K63-Ub2 and K63-Ub2 mixed with equimolar A20 ZnF4 at (A) 303 K and (B) 313 K.The protein concentrations are 50 µM. The timescale of the exchange between ZnF4-free and bound species is slow for residues 50–62 of K63-Ub2 distal unit, whose peaks disappear upon ZnF4 titration. At 313 K, the exchange is slightly faster, which allows the fitting of the KD value.DOI:http://dx.doi.org/10.7554/eLife.05767.026

Mentions: Using isothermal calorimetry (ITC), we evaluated the binding affinities between wild type K63-Ub2 and tUIM, and between wild type K63-Ub2 and NZF. The respective KD values are 9.7 ± 0.3 µM and 12.2 ± 0.6 µM (Figure 1—figure supplement 3), which agree with the literature values (Kulathu et al., 2009; Sekiyama et al., 2012). We attempted to measure the binding affinity between A20 ZnF4 domain and K63-Ub2. However, the heat was too small to be fitted (Figure 7—figure supplement 1). Thus we resorted to NMR titration for the KD measurement. Upon titrating A20 ZnF4, a large set of residues in the distal unit of K63-Ub2 was perturbed (Figure 7A). Among the perturbed residues, residues 50–62 are located at the interface with ZnF4 (Bosanac et al., 2010), and their peaks disappear upon A20 ZnF4 titration, indicating a slow exchange between ZnF4-free and bound species (Figure 7—figure supplement 2). We were only able to fit the CSPs at an elevated temperature (313 K instead of 303 K), and determined the KD value at 384.1 ± 39.4 µM. A number of other residues in the K63-Ub2 distal unit also experience CSPs upon A20 ZnF4 titration. However, their peaks shift progressively at increasing ZnF4 concentrations, which indicates a fast exchange and should belong to a separate binding event.


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)

Overlay of NMR spectra for K63-Ub2 and K63-Ub2 mixed with equimolar A20 ZnF4 at (A) 303 K and (B) 313 K.The protein concentrations are 50 µM. The timescale of the exchange between ZnF4-free and bound species is slow for residues 50–62 of K63-Ub2 distal unit, whose peaks disappear upon ZnF4 titration. At 313 K, the exchange is slightly faster, which allows the fitting of the KD value.DOI:http://dx.doi.org/10.7554/eLife.05767.026
© Copyright Policy
Related In: Results  -  Collection

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

fig7s2: Overlay of NMR spectra for K63-Ub2 and K63-Ub2 mixed with equimolar A20 ZnF4 at (A) 303 K and (B) 313 K.The protein concentrations are 50 µM. The timescale of the exchange between ZnF4-free and bound species is slow for residues 50–62 of K63-Ub2 distal unit, whose peaks disappear upon ZnF4 titration. At 313 K, the exchange is slightly faster, which allows the fitting of the KD value.DOI:http://dx.doi.org/10.7554/eLife.05767.026
Mentions: Using isothermal calorimetry (ITC), we evaluated the binding affinities between wild type K63-Ub2 and tUIM, and between wild type K63-Ub2 and NZF. The respective KD values are 9.7 ± 0.3 µM and 12.2 ± 0.6 µM (Figure 1—figure supplement 3), which agree with the literature values (Kulathu et al., 2009; Sekiyama et al., 2012). We attempted to measure the binding affinity between A20 ZnF4 domain and K63-Ub2. However, the heat was too small to be fitted (Figure 7—figure supplement 1). Thus we resorted to NMR titration for the KD measurement. Upon titrating A20 ZnF4, a large set of residues in the distal unit of K63-Ub2 was perturbed (Figure 7A). Among the perturbed residues, residues 50–62 are located at the interface with ZnF4 (Bosanac et al., 2010), and their peaks disappear upon A20 ZnF4 titration, indicating a slow exchange between ZnF4-free and bound species (Figure 7—figure supplement 2). We were only able to fit the CSPs at an elevated temperature (313 K instead of 303 K), and determined the KD value at 384.1 ± 39.4 µM. A number of other residues in the K63-Ub2 distal unit also experience CSPs upon A20 ZnF4 titration. However, their peaks shift progressively at increasing ZnF4 concentrations, which indicates a fast exchange and should belong to a separate binding event.

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.