NMR structure note: UBA domain of CIP75

Abstract

Ubiquitination is a post-translational process in eukaryotes that covalently modifies substrate proteins with ubiquitin. Ubiquitin can act as a tag that signals the protein-transport machinery to shuttle the protein to the proteasome for degradation. Within the ubiquitin receptor family of proteins is a group that can directly connect ubiquitinated proteins to the proteasome for degradation. Of these receptors, the best-studied are the ubiquitin-like (UBL)ubiquitin-associated (UBA) proteins, Rad23A, PLIC2, and Ddi1. These proteins contain an UBL domain that interacts with the proteasome and the UBA domain that recognizes mono and polyubiquitinated proteins [see review (Su and Lau 2009)]. Recently, a potentially new member of this UBL-UBA domain-containing protein family, named connexin43interacting protein of approximately 75 kDa (CIP75), was identified in a yeast-two hybrid screen (mouse embryonic c-DNA library) to interact with the gap junction protein connexin43 (Cx43) (Li et al. 2008). CIP75 contains an UBL domain at its N-terminus and an UBA domain at its C-terminus; as well as a heat shock chaperonin-binding domain and a PEST sequence. PEST sequences have been shown to direct the ubiquitination and subsequent degradation of proteins undergoing rapid turnover (Roth et al. 1998). CIP75 has 596 amino acids and exhibits high sequence homology (75%) with the human A1Up protein (Davidson et al. 2000). The general domain organization of CIP75 also shows high similarity with the UBL-UBA domain-containing protein family members Ubiquilin-1, PLIC2, Rad23A, Rad23B, and the yeast protein Dsk2 (Li et al. 2008). CIP75 functions in a similar manner as these family members in that the UBL domain of CIP75 is essential for the interaction with the S2/RPN1 and S5a/ RPN10 subunits of the 19S subunit from the 26S proteasome complex. However, the UBA domain may associate with non-ubiquitinated Cx43, and this interaction appears to still regulate the turnover of Cx43 through the proteasomal pathway (Li et al. 2008). UBA domains typically consist of approximately 45 amino acids and are characterized by relatively poor sequence conservation (Hofmann and Bucher 1996). UBA domains adopt a common, compact fold comprising a bundle of three helices and the hydrophobic surface formed by the C-terminus of a-helix 1, loop 1, and a-helix 3, is the principal interface with ubiquitin [see review (Hurley et al. 2006)]. Studies using the two UBA domains from HHR23A established that there are functional differences (i.e. differential protein partners) between UBA domains (Withers-Ward et al. 2000), suggesting the hydrophobic surface may be a more general protein–protein interaction module. Support for this is provided from a survey of the ubiquitin interaction properties of 30 UBA domains, which Fabien Kieken and Gaelle Spagnol contributed equally to this work.