Abstract
Ubiquitylation of proteins is a versatile posttranslational modification that can serve to promote protein degradation, or it can have nondegradative roles, such as mediating protein protein interactions. The Inhibitor of APoptosis (IAP) proteins are important regulators of pathways that control cell death, proliferation, and differentiation. A number of IAP family members are RING E3 ubiquitin protein ligases, which promote direct transfer of ubiquitin from charged E2 enzymes, or E2 similar to ubiquitin (E2 similar to Ub) conjugates, to substrate proteins. This results in the attachment of nondegradative ubiquitin signals to other proteins, or the autoubiquitylation and degradation of IAPs. Modulating ubiquitin transfer by IAPs is the focus of a number of drug development initiatives and these studies require a detailed understanding of ubiquitylation. Here, we describe preparation of stable E2 similar to Ub conjugates that can be used in biochemical and biophysical experiments to examine RING domain function. In the last 2 years, the availability of these conjugates has helped unveil a molecular understanding of the process of ubiquitin transfer by IAPs. The approaches described here will be suitable for studying other RING E3 ligases.