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Mechanisms of Ubiquitin and Ubiquitin-like Protein Conjugation and Regulation
Summary: Brenda Schulman works on the mechanisms underlying protein modification by the family of ubiquitin-related proteins.
Post-translational covalent attachment of ubiquitin-like proteins (Ubls) to protein targets is a predominant eukaryotic regulatory mechanism. In higher eukaryotes, more than a dozen Ubls—such as ubiquitin, NEDD8, ISG15, and SUMO—covalently modify myriad substrates. The best understood function of a Ubl modification is ubiquitin-mediated proteasomal degradation. However, different Ubls alter the functions of their targets in different ways, such as by changing the target's subcellular localization, enzymatic activity, or interactions with other proteins or DNA. Our goals are to understand (1) the basic enzymatic mechanisms underlying Ubl attachment to targets, (2) how Ubls are attached selectively, and (3) how Ubl modifications alter target functions.
Ubls are attached to protein targets by a series of molecular handoffs involving an E1-activating enzyme, an E2-conjugating enzyme (or Ubc), an E3 ligase, and the target. First, at the apex of each Ubl's cascade, a dedicated E1 enzyme selects its Ubl and catalyzes adenylation of the Ubl's C terminus. The E1 then forms a thioester intermediate between the E1's catalytic cysteine and the Ubl's C terminus and ultimately catalyzes Ubl transfer to an E2's catalytic cysteine to generate a thioester-linked E2~Ubl covalent product. The E2~Ubl complex typically associates with an E3, which facilitates transfer of the Ubl to the target. The ubiquitin pathway involves two E1s, tens of E2s, hundreds of E3s, and thousands of protein targets with diverse biological functions. By contrast, the enzymatic cascade for attaching the Ubl NEDD8 involves one E1, one E2, and a few E3s directing NEDD8 to relatively few targets. We have exploited the relatively minimalist nature of the NEDD8 pathway to identify protein-protein interactions specific for a particular Ubl's conjugation cascade.
Selective Initiation of Ubl Transfer by an E1
An E1 enzyme efficiently selects the correct Ubl for its pathway, chemically activates the Ubl's C terminus for the transfer reactions, and coordinates the Ubl with the correct downstream pathway, through four catalytic steps. To establish the molecular basis for these multiple E1 activities, we obtained the crystal structure of NEDD8's E1. This first E1 structure led to a model for the efficient initiation of Ubl conjugation by E1s: the many catalytic sites are organized into distinct domains so that Ubl activation occurs in an assembly-line manner (Figure 1).
Our subsequent crystal structure of NEDD8's E1 in complex with NEDD8 and ATP revealed how an E1 selects its particular Ubl, suggested how an E1 catalyzes adenylation of a Ubl's C terminus, and provided insights into conformational changes that underlie the E1 reaction cycle. A single arginine in the 110-kDa NEDD8 E1 was found to prevent misactivation of ubiquitin. Moreover, NEDD8 residues that interact with E1 correspond to residues in ubiquitin that are important for binding to downstream recognition/effector machineries such as the proteasome. Thus, a Ubl's conjugation machinery and downstream recognition machinery have evolved to selectively recognize a single Ubl surface. The E1-NEDD8-ATP structure also revealed flexibility in loops linking the E1 domains and showed plasticity of NEDD8's C-terminal tail, suggesting that conformational changes may accompany reactions in Ubl transfer cascades.
Selective Coordination of Enzymes in the NEDD8 Conjugation Cascade
We identified a basis for selective E1-E2 interactions in the NEDD8 pathway. Our structural studies showed that a unique insertion in the sequence of NEDD8's E2 docks in a groove formed by unique loop insertions in the sequence of NEDD8's E1. This first structure of any E1-E2 interaction led to a model for the tethering together of enzymes within a particular Ubl's conjugation cascade by unique protein-protein interactions distinct from their common structural scaffolds.
Coordination of E1-E2-E3 Cascades
A major focus of the lab has been to understand the central role of E2-conjugating enzymes in Ubl transfer cascades: the E2 accepts the Ubl from the E1 enzyme and then interacts with an E3, which promotes transfer of the Ubl to the target. We found that noncovalent E1-E2 interactions are required for an E2 to form a thioester intermediate with its particular Ubl. Moreover, an E2 was found to transfer different Ubls in a manner that depends on E1-E2 interactions: UbcH8, originally identified as an E2 for ubiquitin, was discovered, in a collaboration with Robert Krug (University of Texas at Austin), to also be an E2 for the Ubl ISG15. Interferon-α induces ISG15's E1, which causes a switch in UbcH8's Ubl selection from ubiquitin to ISG15. Thus, signaling pathways can impinge on the selection of a Ubl used by enzymes in a conjugation cascade. This finding challenged the concept that ubiquitin and Ubl conjugation pathways are strictly parallel and non-overlapping.
Our structural studies provided the first detailed insights into the noncovalent E1-E2 interactions involving domains common to all E1s and E2s. Unexpectedly, the E1's E2-binding domain adopts a structure resembling ubiquitin. The structure of the NEDD8 E1's ubiquitin-fold domain bound to the core domain from NEDD8's E2 showed that E1 recruits E2 in a manner mimicking ubiquitin's interactions with ubiquitin-binding domains (Figure 2). Structural comparison with E2-E3 complexes revealed overlap between E1- and E3-binding sites on E2s (Figure 3).
Subsequent biochemical studies with three distinct sets of E1-E2-E3 enzymes confirmed that E2s cannot bind their E1 and E3 partners simultaneously. This raises the question of how Ubl transfer cascades can proceed without the E2 becoming trapped in complex with either E1 or E3. One possible answer is that E1s bind free E2s but release their E2~Ubl products, which are then available to bind E3s and transfer their associated Ubls (Figure 4). It also follows that polyubiquitination may require E3s to exchange free E2s for charged E2~Ubl complexes after each round of Ubl transfer. We are attempting to elucidate the molecular switches between different enzyme forms that drive successive steps in conjugation.
Structural Basis for a Ubl Thioester Switch Toggling E1-E2 Interactions
A fundamental aspect of Ubl pathways is that the Ubl is passed—covalently—from enzyme active site to enzyme active site and ultimately to the target. How do the structural properties of conjugation enzymes drive successive steps in the cascade? We recently addressed this question for NEDD8 E1 and E2 enzymes. E1s activate Ubls via multiple steps. First, E1 binds ATP, Mg2+, and the Ubl and catalyzes adenylation of the Ubl's C terminus. Second, E1's catalytic cysteine attacks the Ubl~adenylate, producing a covalent thioester linkage between the E1's catalytic cysteine and the Ubl's C terminus. Third, the E1 binds another Ubl molecule at the high-affinity adenylation active site.
Thus, during the activation cycle, the E1 binds two Ubl molecules, each at a distinct site: Ubl(T) is linked to the E1's catalytic cysteine via a thioester, and Ubl(A) is bound noncovalently at the adenylation active site. Next, this doubly Ubl-loaded E1 associates with an E2. Finally, a transthiolation reaction ensues whereby Ubl(T) is transferred from the E1's catalytic cysteine to the E2's catalytic cysteine, the E2~Ubl thioester product is released from E1, and the activation cycle continues for the noncovalently associated Ubl(A) molecule. Consequently, the E1 cycles back and forth between doubly Ubl-loaded and singly Ubl(A)-loaded forms as it binds each free E2 substrate and releases each E2~Ubl product.
Recent structures of the NEDD8 and SUMO E1s, alone and in noncovalent singly loaded preadenylation intermediate complexes with NEDD8 and SUMO, respectively, revealed similar overall domain orientations. However, several previous studies suggested distinct structural properties for E1 and E2 forms involved in the latter steps of Ubl activation. First, E1s and E2s have been reported to display different relative affinities for each other in their free and covalent thioester-linked enzyme~Ubl states: free E1s display low affinity for E2s, doubly Ubl-loaded E1s bind their free E2 substrates with high affinity, and E2~Ubl thioester products are released from E1s. Second, these differential affinities between distinct enzyme forms are required for progression of E1-E2-E3 cascades, because we showed that there is structural overlap between the E1- and E3-binding sites on E2s and that E2s cannot bind their E1 and E3 partners simultaneously.
Finally, upon docking our structure of a complex between E1 and E2 domains onto our full-length structures of apo or singly Ubl-loaded (preadenylation) E1s, an E2 would bind the opposite side and face away from the E1's catalytic cysteine. Thus, significant conformational changes would be required for the E1 and E2 catalytic cysteines to face each other. This raises questions about what roles different E1 conformations might play during the activation cycle and what would drive E1 conformational changes.
To understand the molecular switches influencing E1, E2, and Ubl interactions, we determined the structure of a trapped activation complex, containing NEDD8's heterodimeric E1 (APPBP1-UBA3), two NEDD8s (one thioester linked to E1, one noncovalently associated for adenylation), a catalytically inactive E2 (Ubc12), and MgATP. The results suggest that a thioester switch toggles E1-E2 affinities. Two E2-binding sites depend on NEDD8 being thioester-linked to E1. One is unmasked by a striking E1 conformational change. The other involves noncovalent contacts to the thioester-bound NEDD8. After NEDD8 transfer to E2, reversion to an alternate E1 conformation would facilitate release of the E2~NEDD8 thioester product. Thus, transferring the Ubl's thioester linkage between successive conjugation enzymes can induce conformational changes and alter interaction networks to drive consecutive steps in Ubl cascades (Figure 5).
We are continuing our studies of Ubl cascades. Defects in these pathways have been widely associated with diseases such as cancer, neurodegenerative disorders, and viral infections. Therefore, determining the mechanisms by which enzymes transfer Ubls will be of broad importance.
These studies are partially supported by ALSAC/St. Jude Children's Research Hospital and the National Institutes of Health.
Last updated: April 29, 2008
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