To date, structural insights into the RBR ligase supradomain are limited to structures solved for individual component domains: there are two structures of IBR domains, and one structure each of a RING1 and RING2 domain [26
] [PDB ID 1WIM
, PDB ID 2CT7
]. The dearth of structural information is probably due to the difficulty of expressing and purifying these proteins in large amounts. With such a small sampling, it is unclear whether these structures will be representative of the entire family of RBR ligases. However, the sequence similarity of these domains among human members of the RBR ligase family suggests we may expect to see similar structures.
Though limited, the available structures do provide some functional insights. The structure of the RNF144 RING1 is similar to that of other RING-type E3s such as BRCA1, with the E2-binding interface readily apparent (PDB ID 1WIM
, Figure ). E2-E3 disrupting mutations that are structurally analogous to those in bona fide
RING-type E3s abrogate the binding of the E2 UbcH7 to HHARI [5
]. Although the E2-E3 binding interactions of RBR E3 ligases seem to be analogous to those of the RING E3 ligases, the functional consequence of the interaction is not. Whereas binding of the BRCA1/BARD1 RING domain leads to a significant enhancement of the thioester reactivity of the E2~Ub conjugate (in this case UbcH5c~Ub) towards lysine, no enhancement of E2 reactivity was detected for a construct composed of the RING1 and IBR domains of HHARI [3
]. Thus, although the RING1 domain of HHARI serves as an E2-binding domain, it lacks the catalytic capacity of other canonical RING domains. Whether this is a general feature of the RING1 domains of RBRs remains to be tested experimentally.
Figure 2 RING1 of RBRs maintains features characteristic of canonical RINGs. (a) Structures of RING domains are displayed with Zn2+ coordinating residues as yellow sticks and Zn2+ ions displayed as grey spheres. A conserved hydrophobic residue important for E2 (more ...)
The function of the IBR domains of RBR ligases is even less clear. The IBR domains of Parkin and RNF31 (HOIP) are structurally sparse outside of the two Zn2+
binding-centers (Figure ) [26
]. Shaw and colleagues noted that the amino- and carboxy-termini of the IBR domain are close to one another and may therefore serve to bring the RING1 and RING2 domains together [26
]. Such a function would be analogous to that of the flexible linker found between the amino-lobe and the carboxy-lobe in HECT-type E3s, which allows conformational changes essential for E3 activity. If so, one would predict that mutations that either reduce the flexibility of the putative linker region or change the orientation of the two RING domains would disrupt ubiquitin transfer, as reported for the HECT ligase WWP1 [30
]. However, the structural conservation of the IBRs in terms of Zn2+
-binding residues, number of amino acids, and the fact that this domain is found almost exclusively in RBR E3s, suggest that the domain is unlikely to serve merely as an elaborate linker.
Figure 3 Conservation of the IBR domain. (a) Structures of IBR domains solved to date from RNF31 (left) (PDB ID 2CT7) and Parkin (right) (PDB ID 2JMO). (b) Multiple sequence alignment of the IBR domain from human RBR ligases. Residue numbers are shown at the beginning (more ...)
Our finding that Parkin RING2 residue C431 is an active site cysteine clarifies several observations about Parkin activity, while raising new questions about the structure and mechanisms of this family of ligases. The Parkin mutation C431F gives rise to a loss-of-function phenotype and is associated with juvenile onset Parkinson's disease, a phenotype that was earlier attributed to structural destabilization, under the presumption that C431 was involved in Zn2+
]. More recent experiments, however, in which the conserved cysteines of Parkin were systematically mutated to alanine, showed that most mutants were insoluble and mislocalized, except the C431A mutant, suggesting that this residue is not part of the Zn2+
coordination network of Parkin [32
]. The only RING2 structure to date is of HHARI RING2 [27
] (Figure ). Unlike the RING1 domain, whose structure led to the notion that the mechanism of the RBR ligases would be like that of the canonical RING ligases, the HHARI RING2 domain structure looks nothing like a canonical E2-binding RING: we discuss below the implications of this structural dissimilarity. Despite the presence of six well conserved cysteines, the structure binds only one Zn2+
ion, leaving two cysteine residues unliganded [27
]. We have identified one of the free cysteines, C357, as the active site cysteine of HHARI. The single Zn2+
-binding site in HHARI RING2 contrasts with electrospray mass spectrometry data that predict two Zn2+
ions for Parkin RING2 [33
]. The coordination of Zn2+
residues in Parkin may well differ from that of HHARI, and how this is accomplished remains to be determined structurally. Rankin and colleagues propose a Parkin RING2 model with two Zn2+
-binding sites, composed of residues C418, C421, C441, C436 and C446, C449, H461, C457 [34
]. This model leaves C431 free to form a thioester bond with ubiquitin (Figure ).
Figure 4 Comparison of HHARI RING2 with Parkin RING2. (a) Multiple sequence alignment of HHARI RING2 and Parkin RING2. Zn2+-liganding residues determined structurally for HHARI RING2 are denoted in yellow. Potential additional Zn2+ coordinating residues in Parkin (more ...)