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HOIL-1L and its binding partner, HOIL-1L interacting protein (HOIP), are essential components of linear ubiquitin (Ub) chain assembly complex (LUBAC), a 600-kDa enzyme complex catalyzing elongation of a tandemly connected Ub chain, which serve as a regulator of NF-κB activation. Specific interaction between the N-terminal Ub-like domain (UBL) of HOIL-1L and the Ub-associated domain (UBA) located at the central region of HOIP is shown to be involved in the formation of LUBAC. For better understanding of the mechanisms underlying the generation of the linear Ub chains by LUBAC, it is necessary to characterize the UBL-UBA interaction on the basis of structural data, which, however, is not available to date. Here we report backbone and side chain NMR assignments of the UBL of human HOIL-1L. By inspection of chemical shift index, it was predicted that HOIL-1L-UBL assumes a Ub fold followed by an α-helical segment, offering the basis for determination its 3D structure and interaction with HOIP-UBA in solution.

HOIL-1L and its binding partner HOIP are essential components of the E3-ligase complex that generates linear ubiquitin (Ub) chains, which are critical regulators of NF-κB activation. Using crystallographic and mutational approaches, we characterize the unexpected structural basis for the specific interaction between the Ub-like domain (UBL) of HOIL-1L and the Ub-associated domain (UBA) of HOIP. Our data indicate the functional significance of this non-canonical mode of UBA-UBL interaction in E3 complex formation and subsequent NF-κB activation. This study highlights the versatility and specificity of protein-protein interactions involving Ub/UBLs and their cognate proteins.

PUB domains are identified in several proteins functioning in the ubiquitin (Ub)-proteasome system and considered as p97-binding modules. To address the further functional roles of these domains, we herein characterized the interactions of the PUB domain of peptide:N-glycanase (PNGase) with Ub and Ub-like domain (UBL) of the proteasome shuttle factor HR23. NMR data indicated that PNGase-PUB exerts an acceptor preferentially for HR23-UBL, electrostatically interacting with the UBL surface employed for binding to other Ub/UBL motifs. Our findings imply that PNGase-PUB serves not only as p97-binding module but also as a possible activator of HR23 in endoplasmic reticulum-associated degradation mechanisms., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)