NMR Characterization of Conformational Interconversions of Lys48-Linked Ubiquitin Chains

Abstract

Ubiquitin (Ub) molecules can be enzymatically connected through a specific isopeptide linkage, thereby mediating various cellular processes by binding to Ub-interacting proteins through their hydrophobic surfaces. The Lys48-linked Ub chains, which serve as tags for proteasomal degradation, undergo conformational interconversions between open and closed states, in which the hydrophobic surfaces are exposed and shielded, respectively. Here, we provide a quantitative view of such dynamic processes of Lys48-linked triUb and tetraUb in solution. The native and cyclic forms of Ub chains are prepared with isotope labeling by in vitro enzymatic reactions. Our comparative NMR analyses using monomeric Ub and cyclic diUb as reference molecules enabled the quantification of populations of the open and closed states for each Ub unit of the native Ub chains. The data indicate that the most distal Ub unit in the Ub chains is the most apt to expose its hydrophobic surface, suggesting its preferential involvement in interactions with the Ub-recognizing proteins. We also demonstrate that a mutational modification of the distal end of the Ub chain can remotely affect the solvent exposure of the hydrophobic surfaces of the other Ub units, suggesting that Ub chains could be unique design frameworks for the creation of allosterically controllable multidomain proteins.

Mutational and Environmental Effects on the Dynamic Conformational Distributions of Lys48-Linked Ubiquitin Chains

“…We have developed an NMR technique to quantitatively determine the conformational distributions of Ub chains [ 12 , 13 ]. In this approach, we use monomeric Ub (monoUb) and a cyclic form of Lys48 di-Ub (c-diUb), in which the two Ub units are connected through two isopeptide linkages, as imitations of open and closed states regarding exposure of the hydrophobic surface.…”

“…In triUb, the hydrophobic surface of the central Ub unit (Ub2) is competitively shared by the most distal and most proximal Ub units (Ub1 and Ub3, respectively). Our prior NMR analysis indicated that, among the three Ub units of triUb, Ub1 is the one most inclined to expose its hydrophobic surface [ 13 ]. Therefore, if one could improve the intramolecular interaction between Ub1 and Ub2 in triUb, this would facilitate the exposure of the hydrophobic surface of the excluded Ub3 unit.…”

“…Our prior NMR analysis demonstrated that, in wild-type triUb, 76% of all conformers have the Ub1 hydrophobic surface exposed to solvent (States A and B): Ub1 interacted with Ub2 in only 19% of the conformers (State C) [ 13 ]. In contrast, in K48C-triUb, Ub1 interacting with Ub2 accounted for 62% of the conformers ( Figure 6 b,d).…”

“…Human Ub, C-terminally hexahistidine-tagged Ub (Ub-His 6 ), and K48S-mutated Ub were expressed and purified as described previously [ 12 , 13 ]. K48C-mutated Ub was produced using site-directed mutagenesis techniques and was purified by the same protocol employed for the wild-type Ub.…”

Rice SIAH E3 Ligases Interact with RMD Formin and Affect Plant Morphology

“…E3 ubiquitin ligases, the most abundant eukaryotic proteins (Mazzucotelli et al 2006 ; Liyuan et al 2013; Michael et al 2015 ), catalyze the covalent attachment of ubiquitin to substrates with the help of E1 (ubiquitin-activating enzyme) and E2 (ubiquitin-conjugating enzyme) proteins (Buetow and Huang 2016 ). A wide variety of ubiquitination processes participate in numerous biological pathways (Jmii and Cappadocia 2021 ; Wu et al 2021 ; Liu et al 2021 ; Uchida and Kitagawa 2016 ), and the predominant function, mediated by Lys48-linked ubiquitination, is to target proteins to the proteasome for degradation (Hiranyakorn et al 2020 ). Other types of linkages, e.g., Lys63-linkages, signal substrates for non-degradative functions, such as protein assembly or modification (Chen and Sun 2009 ) (Swatek and Komander 2016 ); or chromatin modelling or repair (Nakazawa et al 2020 ).…”