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

Abstract

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...

The ubiquitin system: a critical regulator of innate immunity and pathogen–host interactions

“…typhimurium, SopA, is structurally similar to the eukaryotic homologous to E6-AP C terminus (HECT) E3 ligase and interacts with the host E2 enzyme UbcH7. 116 Although the substrate in the host cells has not yet been demonstrated, it is believed that SopA-mediated ubiquitination is involved in the regulation of host inflammation. 117 Intriguingly, Sa.…”

Impact ofSalmonella entericaType III Secretion System Effectors on the Eukaryotic Host Cell

“…In addition to being a substrate for ubiquitination, SopA is itself an E3 ubiquitin ligase. Although it has little sequence identity to eukaryotic E3s, it is considered as a HECT-like E3 ligase because of its structure and proposed mechanism of action [101]. The substrates for the E3 activity of SopA are unknown, but this activity appears to be involved in Salmonella-induced PMN transepithelial migration [102].…”

“…It preferentially uses E2s UbcH5a, UbcH5c, and UbcH7 for its ubiquitination reaction. Although the E2-interacting surface of SopA has little similarity to those of eukaryotic E3s, structural analysis reveals that SopA binds to human UbcH7 on the same region as eukaryotic HECT E3s [101,257]. Thus, Salmonella SopA illustrates a functional mimicry of the mammalian HECT E3 ubiquitin ligase by a Gram-negative bacterial pathogen.…”

Exploitation of the host cell ubiquitin machinery by microbial effector proteins

“…The position of the E2-binding site in the N-lobe of the HECT domain of SopA and NleL differs from that found in eukaryotic HECT domains. However, the large movement of the C-lobe between the E2-binding site and the substrate protein binding site is reminiscent of that in eukaryotic HECT domains (Lin et al, 2012), indicating that the domain mobility in HECT E3s is universally important for the successful transfer of ubiquitin.…”

“…Even though there is only little sequence similarity between SopA or NleL and their eukaryotic counterparts, they possess a similar bilobal architecture to that of eukaryotic HECT domains (Diao et al, 2008;Lin et al, 2012). The HECT domain of SopA and NleL is composed of two lobes (the N-and C-lobe), which are connected by a long flexible helix.…”