Substrate-binding destabilizes the hydrophobic cluster to relieve the autoinhibition of bacterial ubiquitin ligase IpaH9.8

Abstract

IpaH enzymes are bacterial E3 ligases targeting host proteins for ubiquitylation. Two autoinhibition modes of IpaH enzymes have been proposed based on the relative positioning of the Leucine-rich repeat domain (LRR) with respect to the NEL domain. In mode 1, substrate-binding competitively displaces the interactions between theLRR and NEL to relieve autoinhibition. However, the molecular basis for mode 2 is unclear. Here, we present the crystal structures of Shigella IpaH9.8 and the LRR of IpaH9.8 in complex with the substrate of human guanylate-binding protein 1 (hGBP1). A hydrophobic cluster in the C-terminus of IpaH9.8LRR forms a hydrophobic pocket involved in binding the NEL domain, and the binding is important for IpaH9.8 autoinhibition. Substrate-binding destabilizes the hydrophobic cluster by inducing conformational changes of IpaH9.8LRR. Arg166 and Phe187 in IpaH9.8LRR function as sensors for substrate-binding. Collectively, our findings provide insights into the molecular mechanisms for the actication of IpaH9.8 in autoinhibition mode 2.

Genomic and phenotypic analysis of SspH1 identifies a new Salmonella effector, SspH3

“…Amino acid sequences sourced from NCBI for SspH1 (WP_000481981), SspH2 (WP_001115840), SlrP (WP_000481997), and IpaH9.8 (WP_031942481) were compared to the consensus sequence for SspH3 from cluster 3 (same as WP_052938253) using BLASTp to determine protein sequence identify. The N‐terminal domain (NTD), leucine rich‐repeat(LRR) region, and novel E3 ubiquitin ligase (NEL) domain for SspH1, SspH2, SlrP, and IpaH9.8 were taken from previously published work (Keszei et al., 2014; Miao & Miller, 2000; Quezada et al., 2009; Ye et al., 2020; Zouhir et al., 2014). Domains in SspH3 were identified using the web‐based application InterPro by EMBL‐EBI (Blum et al., 2021), as well as through protein sequence alignment and comparison to SspH1 and SspH2.…”

Shigella ubiquitin ligase IpaH7.8 targets gasdermin D for degradation to prevent pyroptosis and enable infection

“…regulated by substrate binding (Keszei and Sicheri, 2017). For example, the LRR of IpaH9.8 auto-inhibits the NEL domain until the substrate human guanylate binding protein 1 (hGBP1) binds to and releases it (Ye et al, 2020). Consistent with this model, the LRR of IpaH7.8 is sufficient to mediate interactions with human and mouse GSDMD, but the consequence of these interactions may be distinguished by their ability to promote subsequent conformational changes necessary for regulating enzymatic activity.…”

Chromosome-encoded IpaH ubiquitin ligases indicate non-human enteroinvasive Escherichia

“…The first strategy is to target the C-terminal domain as it is highly conserved among Shigella IpaH effectors 37 . However, IpaH can affect the antimicrobial activity of host proteins even in the absence of catalytic activity 38 . Thus, targeting N-domains may be more effective but this strategy requires understanding of the ipaH repertoire in specific strains.…”