Molecular Insights into the Ubiquitin Transfer Mechanism of RING-in-between-RING Ubiquitin ligases

Abstract

Ubiquitination is a posttranslational modification that regulates virtually every aspect of cellular function in eukaryotes including cell cycle progression, endocytosis, cell signaling, transcription, translation, DNA damage and even autophagy. Substrate modification with ubiquitin (Ub) requires the coordination of two types of enzymes: Ub-conjugating enzymes (E2s) and Ub ligases (E3s). While E3s are generally thought to bind substrates, substrate ubiquitination can be performed by either an E2 or an E3, depending on the type of E3. There are three classes of eukaryotic E3s: the RING (Really Interesting New Gene) E3s, do not contain an active site. They bind E2~Ub and activate Ub transfer directly onto a substrate. RBR (RING-in-between-RING) and HECT (Homologous to E6AP C-Terminus) E3s contain an active site Cys residue to which Ub is transferred from the E2~Ub to generate a covalent E3~Ub thioester that transfers Ub to a substrate. It was only six years ago that a landmark study discovered that RBR E3 are indeed not RING-type Es3 as had been assumed for many years. In 2011, when I began my studies, very little was known about how RBR E3s function. In fact, RBR E3s were originally termed RING-type E3s based on their primary sequence analysis of the two domains: RING1 (binds E2) and RING2 (contains active site). Based on structural work by other groups, we know today that RING2 does not contain a typical RING fold whereas RING1 domains are structurally similar to canonical RINGs. Nevertheless, in my graduate work I demonstrate that RING1 domains perform opposing functions to canonical RING E3s: instead of promoting closed E2~Ub conformations, RING1s actively favor open E2~Ub conformations. This strategy ensures that the transfer of Ub proceeds via the active site Cys on RING2 and therefore that the type of product generated is determined by the RBR E3 and not by the E2 (as done in the context of canonical RING domains). That RBR RING1 domains have opposing functions to canonical RING domains can be explained functionally, yet a structural explanation for this observation is not immediately apparent. I found that a two-residue extension of the second Zn2+-loop - unique to RING1 domains - is largely responsible for promoting open E2~Ubs. Three years ago, an initial study proposed that the RBR E3 HHARI is able to bind to neddylated cullins (N8-CUL) which are RING-type E3s; and that this interaction results in activation of the normally auto-inhibited HHARI. The biological significance of this complex formation was perplexing. In conjunction with others, my work shows that two types of E3s, the RBR E3 HHARI and the RING E3 N8-CUL-1, work together with their respective E2s to coordinately ubiquitinate a common substrate and that this plays an essential role in a developmental pathway in C. elegans. This work was based on the combination of structural, biochemical and organismal studies that led to a deeper understanding of how RBR E3s work. Ubiquitination is a posttranslational modification that regulates virtually every aspect of cellular function in eukaryotes including cell cycle progression, endocytosis, cell signaling, transcription, translation, DNA damage and even autophagy. Substrate modification with ubiquitin (Ub) requires the coordination of two types of enzymes: Ub-conjugating enzymes (E2s) and Ub ligases (E3s). While E3s are generally thought to bind substrates, substrate ubiquitination can be performed by either an E2 or an E3, depending on the type of E3. There are three classes of eukaryotic E3s: the RING (Really Interesting New Gene) E3s, do not contain an active site. They bind E2~Ub and activate Ub transfer directly onto a substrate. RBR (RING-in-between-RING) and HECT (Homologous to E6AP C-Terminus) E3s contain an active site Cys residue to which Ub is transferred from the E2~Ub to generate a covalent E3~Ub thioester that transfers Ub to a substrate. It was only six years ago that a landmark study discovered that RBR E3 are indeed not RING-type Es3 as had been assumed for many years. In 2011, when I began my studies, very little was known about how RBR E3s function. In fact, RBR E3s were originally termed RING-type E3s based on their primary sequence analysis of the two domains: RING1 (binds E2) and RING2 (contains active site). Based on structural work by other groups, we know today that RING2 does not contain a typical RING fold whereas RING1 domains are structurally similar to canonical RINGs. Nevertheless, in my graduate work I demonstrate that RING1 domains perform opposing functions to canonical RING E3s: instead of promoting closed E2~Ub conformations, RING1s actively favor open E2~Ub conformations. This strategy ensures that the transfer of Ub proceeds via the active site Cys on RING2 and therefore that the type of product generated is determined by the RBR E3 and not by the E2 (as done in the context of canonical RING domains). That RBR RING1 domains have opposing functions to canonical RING domains can be explained functionally, yet a structural explanation for this observation is not immediately apparent. I found that a two-residue extension of the second Zn2+-loop - unique to RING1 domains - is largely responsible for promoting open E2~Ubs. Three years ago, an initial study proposed that the RBR E3 HHARI is able to bind to neddylated cullins (N8-CUL) which are RING-type E3s; and that this interaction results in activation of the normally auto-inhibited HHARI. The biological significance of this complex formation was perplexing. In conjunction with others, my work shows that two types of E3s, the RBR E3 HHARI and the RING E3 N8-CUL-1, work together with their respective E2s to coordinately ubiquitinate a common substrate and that this plays an essential role in a developmental pathway in C. elegans. This work was based on the combination of structural, biochemical and organismal studies that led to a deeper understanding of how RBR E3s work.