Structures of the cIAP2 RING Domain Reveal Conformational Changes Associated with Ubiquitin-conjugating Enzyme (E2) Recruitment

Mace, Peter D.; Linke, Katrin; Feltham, Rebecca; Schumacher, Frances-Rose; Smith, Clyde A.; Vaux, David L.; Silke, John; Day, Catherine L.

doi:10.1074/jbc.m804753200

Cited by 162 publications

(182 citation statements)

References 32 publications

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“…1C). This surface, comprising residues on the N-terminal helix (H1), loop 4, and loop 7 (L4 and L7), is the canonical E3 interaction surface through which UbcH7 interacts with both HECT and RING E3 ligases (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). A highly conserved phenylalanine residue in L4 (F63 in UbcH7) seems to be the determinant for HECT E3 recognition.…”

Section: Resultsmentioning

confidence: 99%

Crystal structures of two bacterial HECT-like E3 ligases in complex with a human E2 reveal atomic details of pathogen-host interactions

Lin¹,

Diao

Chen

2012

Proc. Natl. Acad. Sci. U.S.A.

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In eukaryotes, ubiquitination is an important posttranslational process achieved through a cascade of ubiquitin-activating (E1), conjugating (E2), and ligase (E3) enzymes. Many pathogenic bacteria deliver virulence factors into the host cell that function as E3 ligases. How these bacterial "Trojan horses" integrate into the eukaryotic ubiquitin system has remained a mystery. Here we report crystal structures of two bacterial E3s, Salmonella SopA and Escherichia coli NleL, both in complex with human E2 UbcH7. These structures represent two distinct conformational states of the bacterial E3s, supporting the necessary structural rearrangements associated with ubiquitin transfer. The E2-interacting surface of SopA and NleL has little similarity to those of eukaryotic E3s. However, both bacterial E3s bind to the canonical surface of E2 that normally interacts with eukaryotic E3s. Furthermore, we show that a glutamate residue on E3 is involved in catalyzing ubiquitin transfer from E3 to the substrate, but not from E2 to E3. Together, these results provide mechanistic insights into the ubiquitin pathway and a framework for understanding molecular mimicry in bacterial pathogenesis.crystallography | microbe-host interaction | NleL | SopA | ubiquitination I n order to coexist with eukaryotic hosts, bacterial pathogens have evolved mechanisms to alter the physiological and immune response of the host. For example, many gram-negative bacteria use the type III or type IV secretion system to deliver a large number of virulence factors into the host cell cytosol. These virulence factors, also known as effector proteins, play vital roles in bacterial attachment, entry, and survival. They modulate numerous host cellular functions such as cytoskeleton dynamics, gene expression, and posttranslational modification. Understanding how bacterial virulence factors exert their effects on host cell processes will offer new insights into eukaryotic host cell physiology and possibly lead to new approaches to antimicrobial therapy.The ubiquitin (Ub) pathway is one of the host systems that is hijacked by bacterial pathogens (1, 2). In eukaryotes ubiquitination is central to many processes such as cell cycle, immune response, and DNA damage tolerance (3-6). The covalent attachment of Ub to a target protein requires the sequential actions of Ub-activating enzymes (E1), conjugating enzymes (E2), and ligases (E3). An Ub molecule is first attached to E1 through a thioester bond upon ATP hydrolysis and is subsequently transferred to the active site cysteine residue in E2. The E3 ligase is often needed to transfer Ub from E2 to a protein substrate. There are two major types of E3 ligases: the RING E3s that function as scaffold to bring E2 and substrate into proximity and HECT E3s that form a thioester intermediate with the Ub before transferring it to the substrate. Ubiquitination is absent in prokaryotes. However, some pathogenic bacteria deliver E3 ligases that interfere with the eukaryotic Ub pathway. These bacterial-encoded E3 ligases have litt...

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Section: Resultsmentioning

confidence: 99%

Crystal structures of two bacterial HECT-like E3 ligases in complex with a human E2 reveal atomic details of pathogen-host interactions

Lin¹,

Diao

Chen

2012

Proc. Natl. Acad. Sci. U.S.A.

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“…A highly conserved Phe is represented in green stick format. Coordinates are from Dueber and colleagues (Dueber et al 2011), PDB: 3T6P, and Mace and colleagues (Mace et al 2008) (Komander and Rape 2012). Whether ubiquitylation targets proteins for degradation or mediates nondegradative signaling depends on protein interactions between the ubiquitylated protein and Ub-binding proteins, which can therefore be considered as Ub "receptors" (Hoeller et al 2006).…”

Section: Ring Fingermentioning

confidence: 99%

“…cIAP1's E3 ligase activity can be activated following binding to a substrate. Substrate-binding to the BIR3 liberates the RING from BIR3-mediated inhibition, exposing two interaction surfaces required for RING dimerization and E2 binding, respectively (Mace et al 2008). RING dimerization is of particular importance as it is indispensable for the transfer of Ub from the E2 to a lysine residue of the target substrate.…”

Section: Iaps As Modulators Of Cell Death and Inflammationmentioning

confidence: 99%

Inhibitor of Apoptosis (IAP) Proteins-Modulators of Cell Death and Inflammation

Silke

Meier

2013

Cold Spring Harbor Perspectives in Biology

280

214

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“…Our previous structural studies showed that the RING domain of cIAP2 exists as a dimer and that the dimer bound directly to the E2 UbcH5b (20). To understand how IAP antagonists promote autoubiquitylation of cIAPs, we focused on identifying the essential features of the RING domain.…”

mentioning

confidence: 99%

“…Some IAPs, such as X-linked IAP, can directly inhibit caspases and prevent proteolytic cleavage, whereas cIAP1 and cIAP2 are recruited to tumor necrosis factor (TNF) receptor complexes (16) and modulate the receptor-mediated apoptotic pathways that lead to NF-B activation (17,18). The C-terminal RING domains of cIAP1 and cIAP2 are critical (19,20) with the RING domain required for dimerization, substrate ubiquitylation, and autoubiquitylation (21)(22)(23). In addition to the RING domain, all IAPs have N-terminal baculoviral IAP repeat (BIR) domains that can bind to other proteins, such as the IAP antagonist Smac/DIABLO (second mitochondrion-derived activator of caspase or direct IAP-binding protein with low pl) (24,25).…”

mentioning

confidence: 99%

Smac Mimetics Activate the E3 Ligase Activity of cIAP1 Protein by Promoting RING Domain Dimerization

Feltham

Bettjeman

Budhidarmo

et al. 2011

Journal of Biological Chemistry

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146

152

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The inhibitor of apoptosis (IAP) proteins are important ubiquitin E3 ligases that regulate cell survival and oncogenesis. The cIAP1 and cIAP2 paralogs bear three N-terminal baculoviral IAP repeat (BIR) domains and a C-terminal E3 ligase RING domain. IAP antagonist compounds, also known as Smac mimetics, bind the BIR domains of IAPs and trigger rapid RING-dependent autoubiquitylation, but the mechanism is unknown. We show that RING dimerization is essential for the E3 ligase activity of cIAP1 and cIAP2 because monomeric RING mutants could not interact with the ubiquitin-charged E2 enzyme and were resistant to Smac mimetic-induced autoubiquitylation. Unexpectedly, the BIR domains inhibited cIAP1 RING dimerization, and cIAP1 existed predominantly as an inactive monomer. However, addition of either mono-or bivalent Smac mimetics relieved this inhibition, thereby allowing dimer formation and promoting E3 ligase activation. In contrast, the cIAP2 dimer was more stable, had higher intrinsic E3 ligase activity, and was not highly activated by Smac mimetics. These results explain how Smac mimetics promote rapid destruction of cIAP1 and suggest mechanisms for activating cIAP1 in other pathways.The attachment of ubiquitin and ubiquitin-like molecules to intracellular proteins is a key process that regulates many cellular events (1). A hierarchical multienzyme cascade brings about the attachment of ubiquitin to substrate proteins. Ubiquitin is first activated by the ubiquitin-activating enzyme (E1) and then is transferred to the active site cysteine of the ubiquitin-conjugating enzyme (E2). Subsequently, ubiquitin-protein ligases (E3s) promote the transfer of ubiquitin from the E2 to lysine residues in target proteins. The RING 4 domain-containing E3 ligases, which are prevalent in mammals (Ͼ300), do not directly interact with ubiquitin; instead, the RING domain binds to the E2 and promotes the transfer of ubiquitin from the E2ϳubiquitin (E2ϳUb) thioester conjugate to the target protein (2). The mechanism by which the RING domain promotes ubiquitin transfer is not obvious because the RING-binding site on the E2 is distant from the active site. It has been proposed that the RING domain simply brings the E2ϳUb conjugate into close proximity with the substrate and that the increased availability of E2s promotes transfer of ubiquitin to the substrate. However, interactions between RING domains and E2s are generally transient and have modest affinity, and tighter binding does not always correlate with increased activity. In addition, not all RING-E2 complexes promote transfer, demonstrating that proximity alone is insufficient (3). Notably, the RING domains of Brca1 and c-Cbl interact with UbcH7 and UbcH5b, but only interaction with UbcH5b results in ubiquitin transfer (3, 4). Others have suggested that an allosteric mechanism is important whereby interaction of the RING domain with the E2 leads to changes at its active site that promote release of ubiquitin (5, 6). Consistent with this, binding of the G2BR domain of the E3 gp78...

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Structures of the cIAP2 RING Domain Reveal Conformational Changes Associated with Ubiquitin-conjugating Enzyme (E2) Recruitment

Cited by 162 publications

References 32 publications

Crystal structures of two bacterial HECT-like E3 ligases in complex with a human E2 reveal atomic details of pathogen-host interactions

Crystal structures of two bacterial HECT-like E3 ligases in complex with a human E2 reveal atomic details of pathogen-host interactions

Inhibitor of Apoptosis (IAP) Proteins-Modulators of Cell Death and Inflammation

Smac Mimetics Activate the E3 Ligase Activity of cIAP1 Protein by Promoting RING Domain Dimerization

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