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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.


Cross-validation of paramagnetic relaxation enhancement (PRE) data.The closed-state structure of K63-Ub2 was refined against N25C data only with a four-conformer representation. The PREs for the K48C site were back-calculated (blue line), which agree well with the experimental data (red circles) with a free Q-factor of 0.46. Residues that are completely broadened out are denoted with asterisks at the top.DOI:http://dx.doi.org/10.7554/eLife.05767.016
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fig3s1: Cross-validation of paramagnetic relaxation enhancement (PRE) data.The closed-state structure of K63-Ub2 was refined against N25C data only with a four-conformer representation. The PREs for the K48C site were back-calculated (blue line), which agree well with the experimental data (red circles) with a free Q-factor of 0.46. Residues that are completely broadened out are denoted with asterisks at the top.DOI:http://dx.doi.org/10.7554/eLife.05767.016

Mentions: To characterize the closed-state structure of ligand-free K63-Ub2, we performed rigid-body simulated annealing refinement against the inter-subunit PREs. The linker between the two subunits (Lys63 side chain in the proximal unit and C-terminal flexible residues 72–76 of the distal unit) was given full torsional freedom. A grid search was performed by varying the number of conformers representing the closed state (from a single conformer to a five-conformer ensemble), and by varying the overall population of the closed state (from 10% to 90%). A single-conformer representation for the closed state does not satisfy the inter-subunit PREs, as assessed by the PRE Q-factor (Iwahara et al., 2004). This means that the closed state of ligand-free K63-Ub2 should exist in multiple conformations. The PRE Q-factor rapidly decreases as the number of conformers representing the closed state increases, and levels off with four or more conformers (Figure 3A). On the other hand, a closed-state population of at least 30% is required to achieve a good fit to the PRE data (Figure 3A). For reasons that will be discussed below, the population of the K63-Ub2 closed state is about 70%. At a 70% population for the closed state with a four-conformer representation, the back-calculated PREs agree well with the experimental ones, affording a PRE Q-factor of 0.22 and correlation coefficient of 0.94 (Figures 1, 3B). Importantly, the two paramagnetic conjugation sites, N25C and K48C, provide cross-validating PRE measurements—when refining the ensemble structure of the K63-Ub2 closed state against the N25C data alone, the PRE values predicted for the K48C site largely agree with the experimental values, affording a free Q-factor of 0.46 (Figure 3—figure supplement 1). On the other hand, the SAXS profiles computed for the PRE-based closed-state structures differ from the experiment curve, with the calculated intensities larger at scattering angles between 0.5 and 1 nm−1 (Figure 2—figure supplement 2B).10.7554/eLife.05767.015Figure 3.Ensemble refinement of the closed-state structure of K63-Ub2 against intra-molecular inter-subunit paramagnetic relaxation enhancements (PREs).


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)

Cross-validation of paramagnetic relaxation enhancement (PRE) data.The closed-state structure of K63-Ub2 was refined against N25C data only with a four-conformer representation. The PREs for the K48C site were back-calculated (blue line), which agree well with the experimental data (red circles) with a free Q-factor of 0.46. Residues that are completely broadened out are denoted with asterisks at the top.DOI:http://dx.doi.org/10.7554/eLife.05767.016
© Copyright Policy
Related In: Results  -  Collection

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

fig3s1: Cross-validation of paramagnetic relaxation enhancement (PRE) data.The closed-state structure of K63-Ub2 was refined against N25C data only with a four-conformer representation. The PREs for the K48C site were back-calculated (blue line), which agree well with the experimental data (red circles) with a free Q-factor of 0.46. Residues that are completely broadened out are denoted with asterisks at the top.DOI:http://dx.doi.org/10.7554/eLife.05767.016
Mentions: To characterize the closed-state structure of ligand-free K63-Ub2, we performed rigid-body simulated annealing refinement against the inter-subunit PREs. The linker between the two subunits (Lys63 side chain in the proximal unit and C-terminal flexible residues 72–76 of the distal unit) was given full torsional freedom. A grid search was performed by varying the number of conformers representing the closed state (from a single conformer to a five-conformer ensemble), and by varying the overall population of the closed state (from 10% to 90%). A single-conformer representation for the closed state does not satisfy the inter-subunit PREs, as assessed by the PRE Q-factor (Iwahara et al., 2004). This means that the closed state of ligand-free K63-Ub2 should exist in multiple conformations. The PRE Q-factor rapidly decreases as the number of conformers representing the closed state increases, and levels off with four or more conformers (Figure 3A). On the other hand, a closed-state population of at least 30% is required to achieve a good fit to the PRE data (Figure 3A). For reasons that will be discussed below, the population of the K63-Ub2 closed state is about 70%. At a 70% population for the closed state with a four-conformer representation, the back-calculated PREs agree well with the experimental ones, affording a PRE Q-factor of 0.22 and correlation coefficient of 0.94 (Figures 1, 3B). Importantly, the two paramagnetic conjugation sites, N25C and K48C, provide cross-validating PRE measurements—when refining the ensemble structure of the K63-Ub2 closed state against the N25C data alone, the PRE values predicted for the K48C site largely agree with the experimental values, affording a free Q-factor of 0.46 (Figure 3—figure supplement 1). On the other hand, the SAXS profiles computed for the PRE-based closed-state structures differ from the experiment curve, with the calculated intensities larger at scattering angles between 0.5 and 1 nm−1 (Figure 2—figure supplement 2B).10.7554/eLife.05767.015Figure 3.Ensemble refinement of the closed-state structure of K63-Ub2 against intra-molecular inter-subunit paramagnetic relaxation enhancements (PREs).

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.