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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.


Structural basis for the perturbation of K63-Ub2 conformational space upon E64RP mutation.Representative (A) C1 and (B) C2 closed-state structures. Glu64 in the proximal unit opposes Arg72 and Arg74 in the distal unit, shown as sticks. Judging from the structures, the charge reversal mutation could have a larger impact on the stability of C1 closed state than on C2 closed state of K63-Ub2.DOI:http://dx.doi.org/10.7554/eLife.05767.021
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fig5s1: Structural basis for the perturbation of K63-Ub2 conformational space upon E64RP mutation.Representative (A) C1 and (B) C2 closed-state structures. Glu64 in the proximal unit opposes Arg72 and Arg74 in the distal unit, shown as sticks. Judging from the structures, the charge reversal mutation could have a larger impact on the stability of C1 closed state than on C2 closed state of K63-Ub2.DOI:http://dx.doi.org/10.7554/eLife.05767.021

Mentions: How does K63-Ub2 inter-convert among the preexisting conformations? To address this, we introduced a charge reversal mutation to residue Glu64 in the proximal unit, resulting an E64RP mutant of K63-Ub2. Glu64 is located at the interface between the two subunits in both C1 and C2 closed states, opposing the positively charged residues Arg72 and Arg74 in the distal unit (Figure 5—figure supplement 1). We reasoned that this mutation should affect the conformational space of K63-Ub2. Indeed, the E64RP mutation results in chemical shift perturbations (CSPs) in the K63-Ub2 distal unit (Figure 5—figure supplement 2). Although the perturbations are small, almost the same residues are perturbed upon E64RP mutation as upon the covalent linkage of ubiquitin monomers (Figure 5A and Figure 1—figure supplement 1D). However, the NMR peaks for the perturbed residues in the mutant do not simply move in the direction towards the chemical shift values of the ubiquitin monomer. This can be either due to altered non-covalent interactions around the mutation site, or to a change in the relative population of the conformational states. Therefore, it is difficult to quantitate the CSPs in terms of K63-Ub2 structural change.10.7554/eLife.05767.020Figure 5.Changes in NMR parameters for K63-Ub2 upon E64RP mutation.


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)

Structural basis for the perturbation of K63-Ub2 conformational space upon E64RP mutation.Representative (A) C1 and (B) C2 closed-state structures. Glu64 in the proximal unit opposes Arg72 and Arg74 in the distal unit, shown as sticks. Judging from the structures, the charge reversal mutation could have a larger impact on the stability of C1 closed state than on C2 closed state of K63-Ub2.DOI:http://dx.doi.org/10.7554/eLife.05767.021
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4507786&req=5

fig5s1: Structural basis for the perturbation of K63-Ub2 conformational space upon E64RP mutation.Representative (A) C1 and (B) C2 closed-state structures. Glu64 in the proximal unit opposes Arg72 and Arg74 in the distal unit, shown as sticks. Judging from the structures, the charge reversal mutation could have a larger impact on the stability of C1 closed state than on C2 closed state of K63-Ub2.DOI:http://dx.doi.org/10.7554/eLife.05767.021
Mentions: How does K63-Ub2 inter-convert among the preexisting conformations? To address this, we introduced a charge reversal mutation to residue Glu64 in the proximal unit, resulting an E64RP mutant of K63-Ub2. Glu64 is located at the interface between the two subunits in both C1 and C2 closed states, opposing the positively charged residues Arg72 and Arg74 in the distal unit (Figure 5—figure supplement 1). We reasoned that this mutation should affect the conformational space of K63-Ub2. Indeed, the E64RP mutation results in chemical shift perturbations (CSPs) in the K63-Ub2 distal unit (Figure 5—figure supplement 2). Although the perturbations are small, almost the same residues are perturbed upon E64RP mutation as upon the covalent linkage of ubiquitin monomers (Figure 5A and Figure 1—figure supplement 1D). However, the NMR peaks for the perturbed residues in the mutant do not simply move in the direction towards the chemical shift values of the ubiquitin monomer. This can be either due to altered non-covalent interactions around the mutation site, or to a change in the relative population of the conformational states. Therefore, it is difficult to quantitate the CSPs in terms of K63-Ub2 structural change.10.7554/eLife.05767.020Figure 5.Changes in NMR parameters for K63-Ub2 upon E64RP mutation.

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.