Identifying the complement of molecules that interact with a given protein is an important step toward understanding the functional responsibilities of that protein in the cell. In addition, there are many protein domains which, although conserved and identifiable, are poorly understood or have unknown functions. Probing the functional properties of a protein by investigating possible interaction partners of its domains may illuminate functional properties utilized in the cell. We applied this principle to the ubiquitination system by looking for all possible interactions between a specific ubiquitin ligase (E3) and the family of ubiquitin-conjugating enzymes (E2s). Here, we review our strategy, which can be used for other E3s as well as for other weakly interacting systems.
Ubiquitination is a diverse signaling mechanism that involves multiple protein–protein interactions. Transfer of ubiquitin to a target substrate requires sequential protein–protein interactions among ubiquitin-activation (E1), E2 and E3 enzymes (). Although many of the basic principles of ubiquitination are well understood, the full functional extent of proteins involved in the ubiquitination pathway remains to be discovered. For example, there are over 30 different E2s and many hundreds of E3s encoded by the human genome, raising the question of what E2/E3 pairs are functional. Also, ubiquitin can be conjugated to a substrate in multiple forms, including monoubiquitination and polyubiquitin chains of up to seven different linkage types, raising the question of how the exact nature of the product is specified. These two properties are at the heart of the remarkable diversity of the ubiquitination system [1
Fig. 1 The ubiquitination pathway. Diagram illustrating protein–protein interactions that mediate the E3 dependent ubiquitination of substrates. (A) The pathway utilized by RING and U-box E3s and (B) the pathway utilized by HECT domain E3s. Proteins (more ...)
Although there are a growing number of examples of E3s that are active with more than one E2, the relationships have mostly been identified from activity assays that include only a limited number of E2s. Until our study involving the E3 ligase breast cancer susceptibility gene 1 (BRCA1), no exhaustive screen to identify all possible E2s for a given E3 had been performed [3
]. Unexpectedly we found that 10 of the ~ 30 human E2s can bind to BRCA1 and that different E2s have different ubiquitin-transfer properties in conjunction with BRCA1. E2/E3 interactions pose several challenges that must be overcome in a successful screen. First, the complexes are of modest affinity and often do not survive conventional approaches such as pull-down assays or co-immunoprecipitation. Second, a significant number of E3s function as dimers (homo-and heterodimeric E3s are known) or as multicomponent protein complexes and are only functional within those contexts. In this minireview, we outline a strategy that addresses these challenges and we illustrate its use using the heterodimeric ubiquitin ligase encoded by BRCA1 and its partner, BRCA1-associated RING domain (BARD1).
There are two distinct classes of E3s that function in mechanistically different manners. HECT domain E3s contain a cysteine residue that, similar to the E1 and E2 enzymes, forms a thiolester intermediate with the C-terminus of activated ubiquitin. In this case, ubiquitin is transferred from an E2 to an E3 and finally to a lysine side chain of a substrate protein. RING and U-box E3s do not possess an active-site cysteine residue; these E3s act by bringing the E2–ubiquitin (Ub) complex and substrate in close proximity to mediate the transfer of ubiquitin from the E2 directly to the substrate. Identification of substrates of specific E3s is a major goal in the ubiquitination field, but this has proven to be a difficult task. We believe that one limitation in previous efforts to discover and/or confirm substrates using biochemical assays may be the absence of the correct E2.
The RING-domain E3s are by far the largest family of ubiquitin ligases, with > 600 RING-containing proteins encoded in the human genome [4
]. RING-domain E3s come in four molecular architectures: single-chain, homodimeric, heterodimeric and multi-component. In all known cases, a RING domain interacts directly with an E2. However, in an increasing number of RING and U-box E3s, additional structural elements contribute significantly to the interaction. In some cases, elements proximal to the RING are responsible for either homo- or heterodimerization of the RING domains [5
], which modulates their activity and substrate specificity, and for BRCA1/BARD1 only the dimeric species binds an E2 [3
]. In gp78, a single-chain RING E3, a sequence outside the RING domain forms a second E2-binding site [9
]. The E3 Rad18 also contains a second E2 binding region distal to its RING [10
]. At present, it is not known how many or which E3s use and/or require additional elements for their E2 interactions, but given the number already known, it seems imprudent to assume that residues encompassed entirely within the zinc-chelating cysteine and histidine residues which define the RING domain, will be sufficient when investigating an unknown E3.
All E2s are recognizable by their conserved catalytic domains (referred to as Ubc) which contain the active cysteine residue, and 3D structures solved for dozens of E2 Ubcs reveal a conserved architecture. In addition, E2s are categorized into four classes, depending on whether they consist of only a Ubc (Class I), or have additional sequences N-terminal, C-terminal or both to the Ubc domain (Classes II, III and IV, respectively). Although several co-crystal structures and NMR mapping studies of E2/E3 complexes confirm that Ubcs interact directly with E3s, there are examples of non-Class I E2s that use elements outside the Ubc in their E3 interaction (UbcH10 [13
] and UbcM2 [14
]). Of the ~ 30 human E2s, 11 are Class I. Thus, although the functions of non-Ubc regions remain to be determined for most E2s, strategies that utilize only the Ubc may miss important interactions or features.
Below, we describe the method we devised to detect the human E2s that bind to BRCA1/BARD1. We took advantage of the 3D structure of the heterodimeric RING domain solved in 2001 [5
]. Subsequent structures of other heterodimeric RINGs, homodimeric RINGs and U-box dimers are strikingly similar [1
]. Therefore, approaches that are successful with BRCA1/BARD1 are likely to be generalizable to many other E3s. As illustrated in , the approach is comprised of: (a) a directed yeast two-hybrid assay to identify putative binding partners, (b) protein NMR to confirm direct binding and to map binding interfaces, (c) structure-directed sequence analyses to identify binding determinants, and (d) in vitro
ubiquiti-nation assays to assess the activity of each E2/E3 pair.
Fig. 2 Strategy to identify all E2s that interact with an E3. (A) Generalized flow chart for identification, confirmation, modeling and evaluation of interactions between members of two domain families. (B) Methods used to identify new E2 interactions with BRCA1/BARD1. (more ...)