The RING domain was previously considered sufficient to conf
The RING domain was previously considered sufficient to confer ubiquitination by RING-type E3 ligases. However, recent reports have shown that sequences closest to the RING domain are also important in E3 ligase function. Residues like Tyr193 in RNF4 , Phe296 and Arg294 in BIRC7 , and Lys65 and Asn71 in TRIM25 , , which lie immediately after the RING domain, make extensive contacts with the Ub moiety to maintain a “closed” conformation. Moreover, sequence alignment shows that these amino acids lie at a similar position and can be clustered together (Fig. 4b). However, in the case of LNX1, additional amino CP-673451 residues are needed for such contacts, and these residues are found in the C-terminus, away from the RING domain (Fig. 4b). Interestingly, the buried surface area between the LNX1A and UbB was more than the area shared between LNX1A and UbA. This is contrary to the other reported structures for E3–E2~Ub. This may be due to the longer (128 aa) sequence of the LNX1 construct required for ubiquitination activity in our study, and this may explain the additional contacts shared between LNX1A and UbB. Moreover, we identified the interaction of Lys33 on UbB with the C-terminus of LNX1A, which is critical for the function of LNX1.
From our mutational studies, we found that both the N- and C-terminal Zn-finger motifs are indispensable for the activity of LNX1, unlike LNX2, which only requires the N-terminal motif for function . To our knowledge, this is the first reported case where two Zn-finger motifs are necessary for RING domain ubiquitination function. The C-terminal Zn-finger motif of one monomer directly contacts the Ub molecule of other monomer and provides stability to the closed conformation of the E2~Ub complex. We propose that the C-terminal Zn-finger motif provides a structural support for the residues on LNX1 to interact with Ub. Our identification of a novel interface between the E3 and Ub that promotes LNX1-mediated ubiquitination activity could be the basis of study for proteins with similar domain architecture and other members of the LNX family.
Acknowledgment This work was supported by Ministry of Education, Singapore (MoE Tier-2) grant (R-154-000-625-112) and R154-000-A72-114 (AcRF Tier 1 grant) respectively. We acknowledge the Advanced Photon Source Beamline 24-ID-C, Argonne National Laboratory, USA. D.N. is a graduate scholar in receipt of a research scholarship from NUS. Author Contributions: J.S. conceived and designed the study. D.N. performed all the experiments. D.N. and J.S. analyzed the data and wrote the paper. Conflict of Interest Statement: The authors declare that they have no conflicts of interest.
Introduction Ubiquitin and ubiquitin-like pathways are important for the normal functioning of eukaryotic cells (Joazeiro and Weissman, 2000, Tanaka et al., 2001, Dohmen, 2004, Pan et al., 2004, Pickart and Eddins, 2004, Aragon, 2005, Mani and Gelmann, 2005). Ubiquitination promotes protein turnover, trafficking and regulation of protein function (Sawasdikosol et al., 2000, Marmor and Yarden, 2004). The lack of ubiquitination procedures has been described in the pathogenesis of several human diseases, including cancer. The addition of many ubiquitin moieties leads to degradation of the protein by the 26S proteasome. The E3 enzymes with RING finger domain, such as ring finger proteins BCA2, Mdm2 and BRCA1, have been shown to play an important biological role in breast cancer and other cancers (Joazeiro and Weissman, 2000, Burger and Seth, 2004, Burger et al., 2005). Breast cancer associated gene 2 (BCA2), also known as ring finger protein 115 (RNF115) or Rabring7, belongs to the E3 ubiquitin ligase family with a ring finger domain, which is overexpressed in more than 50% of infiltrating breast cancers compared with normal tissues and can be associated with proliferation of breast cancer cells in vitro (Burger et al., 1998, Burger et al., 2005). Overexpression of BCA2 increases the proliferation of NIH3T3 fibroblasts, while siRNA inhibits the growth of BCA2-expressing breast cancer cells (Burger et al., 2005). The BCA2 protein has autoubiquitination activity, which depends on its RING domain, and mutation at two lysines (K26 and K32) in the BCA2 zinc finger (BZF) domain also can eliminate autoubiquitination activity. Meanwhile, BCA2 E3 ligase has been shown to be an important factor in regulating the migration of breast cancer cells (Amemiya et al., 2008). Although it has been widely accepted that E3 enzyme can play an active role in the carcinogenesis process (Lipkowitz, 2003, Ohta and Fukuda, 2004, Burger et al., 2005), how deregulation of an E3 may occur is less well understood.