Insights into Ubiquitin Transfer Cascades from a Structure of a UbcH5B∼Ubiquitin-HECTNEDD4L Complex

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...

Section: Resultsmentioning

confidence: 99%

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

“…During review of this manuscript a structure of a UbcH5B~Ub/ HECTNEDD4L complex was published revealing similar E2/E3 interactions to those previously observed 3081in a E6AP/UbcH7 complex. Ub is also involved in binding the C-terminal lobe of NEDD4L, resulting in the assembly of an overall compact structure (36).…”

Section: Methodsmentioning

confidence: 99%

Identification of an unconventional E3 binding surface on the UbcH5 ∼ Ub conjugate recognized by a pathogenic bacterial E3 ligase.

Levin

Eakin

Blanc

et al. 2010

Gram-negative bacteria deliver a cadre of virulence factors directly into the cytoplasm of eukaryotic host cells to promote pathogenesis and/or commensalism. Recently, families of virulence proteins have been recognized that function as E3 Ubiquitin-ligases. How these bacterial ligases integrate into the ubiquitin (Ub) signaling pathways of the host and how they differ functionally from endogenous eukaryotic E3s is not known. Here we show that the bacterial E3 SspH2 from S. typhimurium selectively binds the human UbcH5 ∼ Ub conjugate recognizing regions of both UbcH5 and Ub subunits. The surface of the E2 UbcH5 involved in this interaction differs substantially from that defined for other E2/E3 complexes involving eukaryotic E3-ligases. In vitro, SspH2 directs the synthesis of K48-linked poly-Ub chains, suggesting that cellular protein targets of SspH2-catalyzed Ub transfer are destined for proteasomal destruction. Unexpectedly, we found that intermediates in SspH2-directed reactions are activated poly-Ub chains directly tethered to the UbcH5 active site (UbcH5 ∼ Ub n ). Rapid generation of UbcH5 ∼ Ub n may allow for bacterially directed modification of eukaryotic target proteins with a completed poly-Ub chain, efficiently tagging host targets for destruction.

“…Crystal structures of the HECT domains of E6AP and Nedd4L bound to E2 enzyme show that the E2 binding site is a concavity on a specific subdomain whose surface is largely defined by an alpha helix, a beta strand, and a loop between them (19,20). Point mutations in the corresponding alpha helix of Nedd4 (EY680/1AH, based on ref.…”

Section: Significancementioning

confidence: 99%

“…Two regions, each covered by multiple peptides, showed significant reduction in exchange. Both of these are located in the E2 binding region, as defined by reference to the crystal structures of Nedd4L and E6AP HECT/E2 complexes (19,20) (Fig. 5).…”

Section: Significancementioning

confidence: 99%

Peptide and small molecule inhibitors of HECT-type ubiquitin ligases

Mund

Lewis

Maslen

2014

120

130

The human genome encodes several hundred E3 ubiquitin ligases containing RING domains, and around 28 containing HECT domains. These enzymes catalyze the transfer of ubiquitin from E2 enzyme thioesters to a huge range of substrates and play crucial roles in many cellular functions. This makes them attractive potential therapeutic targets. However, they have proven difficult to inhibit: very few good inhibitors exist for RING domain ligases, and none have been described for HECT ligases. Here we show that bicyclic peptides isolated by phage display [Heinis C, Rutherford T, Freund S, Winter G (2009) Nat Chem Biol. 5(7):502-507] can target the E2 binding sites on the HECT domains of Smurf2, Nedd4, Mule/ Huwe1, and WWP1, and thus act as specific inhibitors of these enzymes in vitro. By screening for displacement of one of these peptides from Smurf2, we were able to identify a small molecule, heclin (HECT ligase inhibitor), which inhibits several HECT ligases in tissue culture cells. In vitro, heclin does not block E2 binding but causes a conformational change that results in oxidation of the active site Cys. This demonstrates that HECT domains are potentially druggable and provides molecules that may be of experimental use. Heclin kills HEK293 cells growing in culture, consistent with an essential role for HECT ligase activity in mammalian cells.inhibitor | ubiquitin | HECT ligase | phage display U biquitin ligases are involved in almost every important function of cells, including the removal of misfolded proteins, the regulation of signaling pathways, DNA repair, the cell cycle, and apoptosis (1-4). This broad range of functions is mediated by a large number of E3 ubiquitin ligases, which recognize a similarly large range of substrates. Ubiquitination begins with the ATP-dependent formation of a ubiquitin (Ub)-E2 enzyme thioester intermediate by the E1 enzyme. The E3 enzymes then transfer ubiquitin from this intermediate to a lysine residue on the substrate (3). There are two main classes of E3: the RING domain ligases and their relatives, of which there are several hundred in humans, and the HECT domain ligases, which number around 28 (5-7). Because of their diversity and numerous functions and the fact that some ligases are frequently overexpressed in cancer cells, ubiquitin ligases have attracted much attention as possible targets for therapeutic intervention (2,5,6,8).Despite this interest, it has proven challenging to make small molecule inhibitors of ubiquitin ligases. The action of these enzymes involves primarily protein-protein interactions among them, the Ub-E2 conjugate, and the substrate. For the RING enzymes, this is simultaneous, and the Ub moiety is transferred directly from E2 to substrate; for the HECT ligases, the interaction is sequential, with a thioester-linked Ub-E3 intermediate forming transiently. Such protein-protein interactions are considered difficult to block with small molecules. Added to this difficulty has been the complexity of the ubiquitin system, with ligases having many substrat...