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SspH2: A Novel, Autoregulated E3 Ubiquitin Ligase

Protein ubiquitination is a multistep enzymatic process resulting in the formation of an isopeptide bond between the C-terminal glycine of ubiquitin and internal lysine residues of the substrate protein. The process involves a ubiquitin-activating enzyme (E1), which transfers ubiquitin to a family of ubiquitin-conjugating enzymes (E2s). Ubiquitin-loaded E2s are then recruited to their substrates by a family of ubiquitin ligases (E3s), which play a critical role in conferring specificity to the reaction. Eukaryotic cells possess 2 known classes of E3 ubiquitin ligases that target substrates for modification, the RING-finger and HECT families, which possess distinct structural and functional properties. Whereas some bacterial ubiquitin ligases display structural and functional homology to RING-finger and HECT domains, another class of recently discovered bacterial E3 ligases does not show any primary amino acid sequence similarity to any host proteins.

To understand how this class of bacterial mimics functions, we undertook a biochemical, structural, and cell biological study of one member of this family from Salmonella enterica serovar Typhimurium (S. typhimurium), the multidomain protein SspH2. Along with its close homologue SspH1, this protein is delivered into host cells by a Type III secretion system (T3SS) machine encoded within the pathogenicity island 2 of the S. typhimurium chromosome. Both SspH1 and SspH2 contribute to virulence in animal models of infection by mechanisms that are currently not well understood. Whereas SspH2 is widely distributed among Salmonella serotypes, SspH1 has a more restricted distribution. Recent studies have shown that SspH1 belongs to a new class of E3 ubiquitin ligase. Like its Shigella spp. homologue IpaH9.8, SspH1 showed E3 ligase activity in vitro and catalyzed the ubiquitination of its proposed host cell target PKN1. Although highly related at the primary amino acid sequence level, SspH1 and SspH2 are likely to play different roles during S. typhimurium infection. For example, whereas SspH1 can be delivered by both S. typhimurium T3SSs encoded in pathogenicity island 1 (SPI-1) and 2 (SPI-2), SspH2 is exclusively delivered by the SPI-2 T3SS, which is only expressed when S. typhimurium resides within cells. Furthermore, SspH1 and SspH2 exhibit different localization when transiently expressed in cultured cells. Consequently, the differential temporal and spatial regulation of these two highly related effector proteins suggests distinct roles during infection.

We determined the crystal structure of the Salmonella SPI-2 effector SspH2, an E3 ubiquitin ligase with a unique fold. We show that SspH2 localizes to the apical plasma membrane and propose a mechanism for the activation of its enzymatic activity involving a dramatic conformational change between two subunits of the protein.

The multidomain effector, SspH2, therefore belongs to an exquisitely evolved protein family with interacting subunits, an N-terminal domain that targets the enzymatic activity to the plasma membrane, thereby triggering a conformational change that releases the NEL domain to function in a localized and controlled fashion. The specific localization of SspH2 to the brush border of polarized epithelial cells is intriguing given the prominent role that intestinal epithelial cells play during the interaction of S. typhimurium, an enteropathogen, with its various hosts. Although the actual target(s) of the E3 ligase activity of SspH2 have yet to be identified, its restricted localization should aid this search and help to put its enzymatic activity in a firm physiologic context.

Comparison of the SspH2 NEL domain with 2 bacterial E3 ligases: SopA, a HECT family of cysteine-dependent E3 ubiquitin ligases from Salmonella (PDB ID 2QYU), and AvrPtoB, a RING finger/U-box protein (PDB ID 2FD4). The catalytic cysteine residues are shown in a space-filling format (blue).

Protein secretion mechanisms like the T3SS used by Salmonella have existed for nearly a billion years, bringing bacterial proteins into intimate contact with host cell biology and allowing the selective pressures of coevolutionary forces to shape the biochemical interactions between these very different forms of life. The highly sophisticated nature of bacterial virulence factors underscores this observation, and the targeting of the eukaryotic ubiquitination system represents a remarkable example of biochemical mimicry of host cell biology. In addition, the insights into the structure and function of SspH2 may illuminate the mechanisms of function and activity in other infectious agents sharing homology, opening new directions for the design of novel anti-infectives, as well as providing insights into the host ubiquitination system.

 

C.M. Quezada, S.W. Hicks, J.E. Galán, and C.E. Stebbins. (2009) "A family of Salmonella virulence factors functions as a distinct class of autoregulated E3 ubiquitin ligases." Proc Natl Acad Sci U S A., 6(12):4864-4869, PMID: 19273841. [Abstract] [pdf] [pdb]

 

Dr. Cindy Quezada was the lead scientist on the SspH2 project. Our collaborators were the group of Jorge Galan at Yale University.