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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.


NMR chemical shift differences between ubiquitin monomer and K63-Ub2.Overlay of 2D 1H-15N correlation spectra for (A) ubiquitin monomer and K63-Ub2 proximal unit, and (B) ubiquitin monomer and K63-Ub2 distal unit. The ubiquitin monomers (also the reactants for preparing K63-Ub2) were modified (either by appending an Asp at the C-terminus or by mutating Lys63 to an Arg) so that only a single product (diubiquitin) was obtained. Residues with relatively large chemical shift differences (>0.01 ppm) were mapped to the surface of (C) the proximal unit and (D) the distal unit, and are colored red. The chemical shift differences in ppm is calculated as (ΔδH2/2 + ΔδN2/5)^0.5.DOI:http://dx.doi.org/10.7554/eLife.05767.004
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fig1s1: NMR chemical shift differences between ubiquitin monomer and K63-Ub2.Overlay of 2D 1H-15N correlation spectra for (A) ubiquitin monomer and K63-Ub2 proximal unit, and (B) ubiquitin monomer and K63-Ub2 distal unit. The ubiquitin monomers (also the reactants for preparing K63-Ub2) were modified (either by appending an Asp at the C-terminus or by mutating Lys63 to an Arg) so that only a single product (diubiquitin) was obtained. Residues with relatively large chemical shift differences (>0.01 ppm) were mapped to the surface of (C) the proximal unit and (D) the distal unit, and are colored red. The chemical shift differences in ppm is calculated as (ΔδH2/2 + ΔδN2/5)^0.5.DOI:http://dx.doi.org/10.7554/eLife.05767.004

Mentions: We first compared the chemical shift differences between the subunits in K63-Ub2 and ubiquitin monomer. Except for residues near the covalent ubiquitin linkage, the differences in chemical shifts are small (<0.04 ppm; Figure 1—figure supplement 1). Residues with relatively large chemical shift differences (>0.01 ppm) can be tentatively mapped, and form rather contiguous surfaces on each subunit (Figure 1—figure supplement 1, insets). However, it is unclear whether the perturbations are due to the covalent linkage, or due to non-covalent interactions between the two subunits. We also performed a half-filtered NMR experiment, which failed to reveal any inter-subunit nuclear Overhauser effects (NOEs) between the proximal and distal units of K63-Ub2. Our data are consistent with the previous NMR studies of K63-Ub2 (Tenno et al., 2004; Varadan et al., 2004); in the latter work, the authors failed to detect cross-saturations between the two subunits. Together, the diamagnetic NMR studies indicated that the closed-state structure of K63-Ub2, if existing, is loosely packed and possibly adopts multiple conformations.


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)

NMR chemical shift differences between ubiquitin monomer and K63-Ub2.Overlay of 2D 1H-15N correlation spectra for (A) ubiquitin monomer and K63-Ub2 proximal unit, and (B) ubiquitin monomer and K63-Ub2 distal unit. The ubiquitin monomers (also the reactants for preparing K63-Ub2) were modified (either by appending an Asp at the C-terminus or by mutating Lys63 to an Arg) so that only a single product (diubiquitin) was obtained. Residues with relatively large chemical shift differences (>0.01 ppm) were mapped to the surface of (C) the proximal unit and (D) the distal unit, and are colored red. The chemical shift differences in ppm is calculated as (ΔδH2/2 + ΔδN2/5)^0.5.DOI:http://dx.doi.org/10.7554/eLife.05767.004
© Copyright Policy
Related In: Results  -  Collection

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

fig1s1: NMR chemical shift differences between ubiquitin monomer and K63-Ub2.Overlay of 2D 1H-15N correlation spectra for (A) ubiquitin monomer and K63-Ub2 proximal unit, and (B) ubiquitin monomer and K63-Ub2 distal unit. The ubiquitin monomers (also the reactants for preparing K63-Ub2) were modified (either by appending an Asp at the C-terminus or by mutating Lys63 to an Arg) so that only a single product (diubiquitin) was obtained. Residues with relatively large chemical shift differences (>0.01 ppm) were mapped to the surface of (C) the proximal unit and (D) the distal unit, and are colored red. The chemical shift differences in ppm is calculated as (ΔδH2/2 + ΔδN2/5)^0.5.DOI:http://dx.doi.org/10.7554/eLife.05767.004
Mentions: We first compared the chemical shift differences between the subunits in K63-Ub2 and ubiquitin monomer. Except for residues near the covalent ubiquitin linkage, the differences in chemical shifts are small (<0.04 ppm; Figure 1—figure supplement 1). Residues with relatively large chemical shift differences (>0.01 ppm) can be tentatively mapped, and form rather contiguous surfaces on each subunit (Figure 1—figure supplement 1, insets). However, it is unclear whether the perturbations are due to the covalent linkage, or due to non-covalent interactions between the two subunits. We also performed a half-filtered NMR experiment, which failed to reveal any inter-subunit nuclear Overhauser effects (NOEs) between the proximal and distal units of K63-Ub2. Our data are consistent with the previous NMR studies of K63-Ub2 (Tenno et al., 2004; Varadan et al., 2004); in the latter work, the authors failed to detect cross-saturations between the two subunits. Together, the diamagnetic NMR studies indicated that the closed-state structure of K63-Ub2, if existing, is loosely packed and possibly adopts multiple conformations.

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.