Structure of the Ubiquitin-interacting Motif of S5a Bound to the Ubiquitin-like Domain of HR23B

Abstract

Ubiquitination, a modification in which single or multiple ubiquitin molecules are attached to a protein, serves signaling functions that control several cellular processes. The ubiquitination signal is recognized by downstream effectors, many of which carry a ubiquitininteracting motif (UIM). Such interactions can be modulated by regulators carrying a ubiquitin-like (UbL) domain, which binds UIM by mimicking ubiquitination. Of them, HR23B regulates the proteasomal targeting of ubiquitinated substrates, DNA repair factors, and other proteins. Here we report the structure of the UIM of the proteasome subunit S5a bound to the UbL domain of HR23B. The UbL domain presents one hydrophobic and two polar contact sites for interaction with UIM. The residues in these contact sites are well conserved in ubiquitin, but ubiquitin also presents a histidine at the interface. The pH-dependent protonation of this residue interferes with the access of ubiquitin to the UIM and the ubiquitin-associated domain (UBA), and its mutation to a smaller residue increases the affinity of ubiquitin for UIM.

Conformational Dynamics of Wild-type Lys-48-linked Diubiquitin in Solution

“…S6B). The pK a value of His-68 in monomeric Ub has been reported to be 5.5 (37); this value coincides with the those of wild-type Lys-48-linked diUb (data not shown). The observations can be explained by the removal of the positive repulsive charges and enhancement of hydrophobicity at the Ub-Ub interface, resulting from valine substitution at His-68.…”

Multidimensional Protein Identification Technology (MudPIT) Analysis of Ubiquitinated Proteins in Plants

“…The specificities of the two domains were compared using pure forms of mono-Ub, Lys-48-conjugated chains, or Lys-63-conjugated chains. Our observation that mono-Ub could only be pulled down using the ISO fusion protein contrast with a previous report showing binding of mono-Ub to the UIM domain of the human proteasome subunit S5a (39). However, others have reported differential affinities between UIM-containing proteins toward various lengths of Ub chains (40).…”

The Ubiquitin-Interacting Motif–Containing Protein RAP80 Interacts with BRCA1 and Functions in DNA Damage Repair Response

“…UIMs consist of a short sequence motif of f20 amino acids that can bind ubiquitin and/or specific ubiquitinated proteins (17). UIM-containing proteins have been reported to direct (multi)monoubiquitination thereby regulating a broad range of cellular functions including membrane protein trafficking, histone function, transcriptional regulation, DNA repair, replication, and (de)ubiquitination control (17,18,35,36). Our results show that deletion of the UIMs (RAP80DUIM) abolished the ability of RAP80 to translocate to IRIF.…”