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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.


Comparison of the intra-molecular inter-subunit paramagnetic relaxation enhancement (PRE) data with an EDTA-Mn2+ (red circles) or MTS probe (blue circles) conjugated at N25C site.Lines simply connect the data points. The PREs obtained using EDTA-Mn2+ are larger than those using the MTS probe owing to the larger paramagnetic dipole of the former.DOI:http://dx.doi.org/10.7554/eLife.05767.009
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fig1s6: Comparison of the intra-molecular inter-subunit paramagnetic relaxation enhancement (PRE) data with an EDTA-Mn2+ (red circles) or MTS probe (blue circles) conjugated at N25C site.Lines simply connect the data points. The PREs obtained using EDTA-Mn2+ are larger than those using the MTS probe owing to the larger paramagnetic dipole of the former.DOI:http://dx.doi.org/10.7554/eLife.05767.009

Mentions: We previously reported that ubiquitin monomer dimerizes non-covalently with an apparent KD value of 4.9 ± 0.3 mM (Liu et al., 2012). Therefore, the PRE effect could arise both intramolecularly and inter-molecularly. The inter-molecular PREs were measured on an equimolar mixture of K63-Ub2 (each at 500 µM), with paramagnetic tagging and isotope labeling on different subunits in separate proteins. With a paramagnetic probe conjugated at either N25C or K48C site and using the inter-molecular data for reference, our measurements revealed large intra-molecular inter-subunit PREs for many residues in the proximal unit (Figure 1A,B). At relatively low protein concentration (50 µM), the inter-molecular contribution to the overall PRE is negligible. We found that the relative decreases in peak intensities between the paramagnetic and diamagnetic spectra recorded at 50 µM are highly correlated with the relative decreases between the paramagnetic and inter-molecular spectra recorded at 500 µM (Figure 1—figure supplement 5). This corroborates the PRE measurement at the higher concentration. In addition, when a different paramagnetic probe, EDTA-Mn2+, was conjugated at N25C, the PRE profile is similar to that using the MTS probe (Figure 1—figure supplement 6). Thus, the intra-molecular inter-subunit PREs are independent of the paramagnetic probe used, and reveal intrinsic structural features of ligand-free K63-Ub2.10.7554/eLife.05767.003Figure 1.Intra-molecular inter-subunit paramagnetic relaxation enhancements (PREs) measured for ligand-free K63-Ub2.


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)

Comparison of the intra-molecular inter-subunit paramagnetic relaxation enhancement (PRE) data with an EDTA-Mn2+ (red circles) or MTS probe (blue circles) conjugated at N25C site.Lines simply connect the data points. The PREs obtained using EDTA-Mn2+ are larger than those using the MTS probe owing to the larger paramagnetic dipole of the former.DOI:http://dx.doi.org/10.7554/eLife.05767.009
© Copyright Policy
Related In: Results  -  Collection

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

fig1s6: Comparison of the intra-molecular inter-subunit paramagnetic relaxation enhancement (PRE) data with an EDTA-Mn2+ (red circles) or MTS probe (blue circles) conjugated at N25C site.Lines simply connect the data points. The PREs obtained using EDTA-Mn2+ are larger than those using the MTS probe owing to the larger paramagnetic dipole of the former.DOI:http://dx.doi.org/10.7554/eLife.05767.009
Mentions: We previously reported that ubiquitin monomer dimerizes non-covalently with an apparent KD value of 4.9 ± 0.3 mM (Liu et al., 2012). Therefore, the PRE effect could arise both intramolecularly and inter-molecularly. The inter-molecular PREs were measured on an equimolar mixture of K63-Ub2 (each at 500 µM), with paramagnetic tagging and isotope labeling on different subunits in separate proteins. With a paramagnetic probe conjugated at either N25C or K48C site and using the inter-molecular data for reference, our measurements revealed large intra-molecular inter-subunit PREs for many residues in the proximal unit (Figure 1A,B). At relatively low protein concentration (50 µM), the inter-molecular contribution to the overall PRE is negligible. We found that the relative decreases in peak intensities between the paramagnetic and diamagnetic spectra recorded at 50 µM are highly correlated with the relative decreases between the paramagnetic and inter-molecular spectra recorded at 500 µM (Figure 1—figure supplement 5). This corroborates the PRE measurement at the higher concentration. In addition, when a different paramagnetic probe, EDTA-Mn2+, was conjugated at N25C, the PRE profile is similar to that using the MTS probe (Figure 1—figure supplement 6). Thus, the intra-molecular inter-subunit PREs are independent of the paramagnetic probe used, and reveal intrinsic structural features of ligand-free K63-Ub2.10.7554/eLife.05767.003Figure 1.Intra-molecular inter-subunit paramagnetic relaxation enhancements (PREs) measured for ligand-free K63-Ub2.

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.