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.
Highlights
Sophisticated protein functions are, in many cases, mediated through the cooperative interplay between two or more domains [1]
In the cyclic forms, the C-terminus of one Ub unit is native and cyclic forms by in vitro enzymatic reactions conjugated to the Lys48 of its and mostn-tetraUb distal Ubinunit
As we reported previously [20], native form diUbinterchange (n-diUb) has a single set of chemical shifts, except for the peaks originating from the Lys48-Gly76 linkage site, indicating that the two Ub units in n-diUb are structurally equivalent
Summary
Sophisticated protein functions are, in many cases, mediated through the cooperative interplay between two or more domains [1]. Proteins with a modular architecture of multiple domains connected by linkers often exhibit diversity in relative positions of the individual domains organized through weak and even transient inter-domain interactions [1,2]. Polymeric ubiquitin (Ub) chains, in which several Ub proteins are connected through specific isopeptide bonds, are known to play regulatory roles in various cellular processes, including cell cycle progression, DNA repair, transcriptional regulation, and apoptosis [3,4,5]. The Ub chains conjugated by different linkages carry distinct biological information in the form of a “Ub code” that is read out by.
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