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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.


Isothermal calorimetry (ITC) measurements for the binding affinities (A–C) between K63-Ub2 and tUIM, and (D–F) between K63-Ub2 and NZF.The titrations were performed for (A, D) wild type K63-Ub2, (B, E) N25C mutant conjugated with an MTS paramagnetic probe, and (C, F) K48C mutant conjugated with an MTS paramagnetic probe. The binding affinities KD values were averaged over four independent titrations with SD reported. The averaged enthalpy and entropy changes (±SD) are also labeled.DOI:http://dx.doi.org/10.7554/eLife.05767.006
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fig1s3: Isothermal calorimetry (ITC) measurements for the binding affinities (A–C) between K63-Ub2 and tUIM, and (D–F) between K63-Ub2 and NZF.The titrations were performed for (A, D) wild type K63-Ub2, (B, E) N25C mutant conjugated with an MTS paramagnetic probe, and (C, F) K48C mutant conjugated with an MTS paramagnetic probe. The binding affinities KD values were averaged over four independent titrations with SD reported. The averaged enthalpy and entropy changes (±SD) are also labeled.DOI:http://dx.doi.org/10.7554/eLife.05767.006

Mentions: To visualize the quaternary arrangement between the subunits of K63-Ub2, we resorted to PRE NMR. We prepared K63-Ub2 protein with the proximal unit 15N-labeled and the distal unit unlabeled. A cysteine point mutation was introduced to Asn25 or Lys48 in the distal unit of K63-Ub2. An MTS paramagnetic probe was conjugated at N25C or K48C site. Each of the conjugation sites was designed so that the paramagnetic probe was away from the binding partners of K63-Ub2, which include Rap80 tUIM domain (Sekiyama et al., 2012), TAB2 NZF domain (Kulathu et al., 2009; Sato et al., 2009b), and A20 ZnF4 domain (Bosanac et al., 2010), and therefore the conjugation does not interfere with ligand binding (Figure 1—figure supplement 2). Indeed, the binding affinities between K63-Ub2 and tUIM or NZF remain unchanged for the paramagnetically tagged K63-Ub2 proteins (Figure 1—figure supplement 3). In addition, the paramagnetic NMR spectrum can be overlaid onto the diamagnetic spectrum, except for residues that are completely broadened out due to the PRE effect (Figure 1—figure supplement 4). Together, the modifications do not perturb the conformational space of K63-Ub2 or have an effect on K63-Ub2 function.


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)

Isothermal calorimetry (ITC) measurements for the binding affinities (A–C) between K63-Ub2 and tUIM, and (D–F) between K63-Ub2 and NZF.The titrations were performed for (A, D) wild type K63-Ub2, (B, E) N25C mutant conjugated with an MTS paramagnetic probe, and (C, F) K48C mutant conjugated with an MTS paramagnetic probe. The binding affinities KD values were averaged over four independent titrations with SD reported. The averaged enthalpy and entropy changes (±SD) are also labeled.DOI:http://dx.doi.org/10.7554/eLife.05767.006
© Copyright Policy
Related In: Results  -  Collection

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

fig1s3: Isothermal calorimetry (ITC) measurements for the binding affinities (A–C) between K63-Ub2 and tUIM, and (D–F) between K63-Ub2 and NZF.The titrations were performed for (A, D) wild type K63-Ub2, (B, E) N25C mutant conjugated with an MTS paramagnetic probe, and (C, F) K48C mutant conjugated with an MTS paramagnetic probe. The binding affinities KD values were averaged over four independent titrations with SD reported. The averaged enthalpy and entropy changes (±SD) are also labeled.DOI:http://dx.doi.org/10.7554/eLife.05767.006
Mentions: To visualize the quaternary arrangement between the subunits of K63-Ub2, we resorted to PRE NMR. We prepared K63-Ub2 protein with the proximal unit 15N-labeled and the distal unit unlabeled. A cysteine point mutation was introduced to Asn25 or Lys48 in the distal unit of K63-Ub2. An MTS paramagnetic probe was conjugated at N25C or K48C site. Each of the conjugation sites was designed so that the paramagnetic probe was away from the binding partners of K63-Ub2, which include Rap80 tUIM domain (Sekiyama et al., 2012), TAB2 NZF domain (Kulathu et al., 2009; Sato et al., 2009b), and A20 ZnF4 domain (Bosanac et al., 2010), and therefore the conjugation does not interfere with ligand binding (Figure 1—figure supplement 2). Indeed, the binding affinities between K63-Ub2 and tUIM or NZF remain unchanged for the paramagnetically tagged K63-Ub2 proteins (Figure 1—figure supplement 3). In addition, the paramagnetic NMR spectrum can be overlaid onto the diamagnetic spectrum, except for residues that are completely broadened out due to the PRE effect (Figure 1—figure supplement 4). Together, the modifications do not perturb the conformational space of K63-Ub2 or have an effect on K63-Ub2 function.

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.