Conformational dynamics of wild-type Lys-48-linked diubiquitin in solution - PubMed (original) (raw)

Conformational dynamics of wild-type Lys-48-linked diubiquitin in solution

Takashi Hirano et al. J Biol Chem. 2011.

Abstract

Proteasomal degradation is mediated through modification of target proteins by Lys-48-linked polyubiquitin (polyUb) chain, which interacts with several binding partners in this pathway through hydrophobic surfaces on individual Ub units. However, the previously reported crystal structures of Lys-48-linked diUb exhibit a closed conformation with sequestered hydrophobic surfaces. NMR studies on mutated Lys-48-linked diUb indicated a pH-dependent conformational equilibrium between closed and open states with the predominance of the former under neutral conditions (90% at pH 6.8). To address the question of how Ub-binding proteins can efficiently access the sequestered hydrophobic surfaces of Ub chains, we revisited the conformational dynamics of Lys-48-linked diUb in solution using wild-type diUb and cyclic forms of diUb in which the Ub units are connected through two Lys-48-mediated isopeptide bonds. Our newly determined crystal structure of wild-type diUb showed an open conformation, whereas NMR analyses of cyclic Lys-48-linked diUb in solution revealed that its structure resembled the closed conformation observed in previous crystal structures. Comparison of a chemical shift of wild-type diUb with that of monomeric Ub and cyclic diUb, which mimic the open and closed states, respectively, with regard to the exposure of hydrophobic surfaces to the solvent indicates that wild-type Lys-48-linked diUb in solution predominantly exhibits the open conformation (75% at pH 7.0), which becomes more populated upon lowering pH. The intrinsic properties of Lys-48-linked Ub chains to adopt the open conformation may be advantageous for interacting with Ub-binding proteins.

PubMed Disclaimer

Figures

FIGURE 1.

FIGURE 1.

The crystal structure of wild-type Lys-48-linked diUb. The proximal domain is located on the left, and the distal domain is located on the right. His-68 (blue) and Val-70 (orange) are shown in a space-filling representation. By superimposing the Cα carbons of the distal and proximal Ub units, an r.m.s.d. value of 0.2 Å was obtained for 73 residues, excluding the three C-terminal residues.

FIGURE 2.

FIGURE 2.

1H-15N HSQC spectra of monomeric Ub (cyan), wild-type Lys-48-linked diUb (red), and cyclic Lys-48-linked diUb (black) at pH 7.0. The close views of spectral regions (boxed), including the peaks from Ile-13, Leu-43, Leu-69, and Val-70, are displayed at the bottom.

FIGURE 3.

FIGURE 3.

Three-dimensional structural model of cyclic Lys-48-linked diUb derived from the NMR data. His-68 (blue) and Val-70 (orange) are shown in a space-filling representation.

FIGURE 4.

FIGURE 4.

NMR characterization of conformational dynamics of Lys-48-linked diUb in solution. A, cartoon model of the conformational equilibrium of wild-type Lys-48-linked diUb. Cyclic Lys-48-linked diUb was used as a mimic of the closed state, whereas monomeric Ub was used as a mimic of the open state. The hydrophobic surface is colored red. The crystallographic data (

supplemental Fig. S2

) indicate that the distances between the two Ile-44 Cα atoms are 18.8 and 12.3 Å in the open and closed forms, respectively. B, 1H-15N HSQC peaks originating from Val-70 of monomeric Ub (cyan), wild-type Lys-48-linked diUb (red), and cyclic Lys-48-linked diUb (black) at the following different pH conditions: pH 7.0, 6.0, 5.0, and 4.6, starting from the left. C, pH dependence of the open conformation population of wild-type Lys-48-linked diUb (black) and its His-68→Val mutant (red) estimated on the basis of the dividing ratio of chemical shift differences of Val-70.

Similar articles

Cited by

References

    1. Hershko A., Ciechanover A. (1998) Annu. Rev. Biochem. 67, 425–479 - PubMed
    1. Pickart C. M., Eddins M. J. (2004) Biochim. Biophys. Acta 1695, 55–72 - PubMed
    1. Baboshina O. V., Haas A. L. (1996) J. Biol. Chem. 271, 2823–2831 - PubMed
    1. Peng J., Schwartz D., Elias J. E., Thoreen C. C., Cheng D., Marsischky G., Roelofs J., Finley D., Gygi S. P. (2003) Nat. Biotechnol. 21, 921–926 - PubMed
    1. Kirisako T., Kamei K., Murata S., Kato M., Fukumoto H., Kanie M., Sano S., Tokunaga F., Tanaka K., Iwai K. (2006) EMBO J. 25, 4877–4887 - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources