The structure of the Slrp–Trx1 complex sheds light on the autoinhibition mechanism of the type III secretion system effectors of the NEL family

Abstract

Salmonella infections are a leading cause of bacterial foodborne illness in the United States and the European Union. Antimicrobial therapy is often administered to treat the infection but increasing isolates are being detected that demonstrate resistance to multiple antibiotics. Salmonella enterica contains two virulence related type-III secretion systems (T3SS): one promotes invasion of the intestine and the other one mediates systemic disease. Both of them secrete the SlrP protein acting as E3 ubiquitin ligase in human host cells where it targets thioredoxin-1 (Trx1). SlrP belongs to the NEL family of bacterial E3 ubiquitin ligases that have been observed in two distinct autoinhibitory conformations. We solved the 3D structure of the SlrP/Trx1 complex and determined the Trx1 ubiquitination site. The description of the substrate-binding mode sheds light on the first step of the activation mechanism of SlrP. Comparison with the available structural data of other NEL effectors allowed us to gain new insights into their autoinhibitory mechanism. We propose a molecular mechanism for the regulation of SlrP in which structural constraints sequestrating the NEL domain would be sequentially released. This work thus constitutes a new milestone in the understanding of how these T3SS effectors influence pathogen virulence. It also provides the fundamental basis for future development of new antimicrobials.

The ubiquitin ligase SspH1 from Salmonella uses a modular and dynamic E3 domain to catalyze substrate ubiquitylation

“…Much of the recent literature on SspH/IpaH E3s has focused on the large reorientation of the LRR and E3 domains observed in various crystal structures (16,19,21,32). Our further analysis of the same structures revealed that the E3 domains themselves can also adopt multiple conformations.…”

“…1B). Subsequent structures all exhibit the same overall ␣-helical fold (12)(13)(14)21). Despite this similarity, close inspection reveals conformational heterogeneity within the E3 domain.…”

“…15 N-Labeled proteins were grown in minimal MOPS medium supplemented with 15 NH 4 Cl. His-tagged proteins were purified by Ni 2ϩ affinity purification; Ube2D3 variants without affinity tags were purified by strong cation-exchange chromatography on an SP column (GE Healthcare) via a 0 -1 M salt gradient in 25 mM MES, pH 6, at 4°C (21). The final step of purification for all proteins was size-exclusion chromatography over Superdex75 resin equilibrated in 25 mM sodium phosphate, 150 mM NaCl, pH 7, at 4°C or room temperature.…”

The species-spanning family of LPX-motif harbouring effector proteins

“…The E3 ubiquitin ligase SlrP is translocated both via SPI‐1‐ and SPI‐2‐encoded T3SS (Cordero‐Alba & Ramos‐Morales, ; Ellermeier & Slauch, ). The host reduction/oxidation‐regulatory protein thioredoxin‐1 (Trx1) was identified to undergo SlrP‐mediated ubiquitinylation, which results, through a proteolytic or a nonproteolytic pathway, in decreased activity of Trx1 and an increase in host cell death (Bernal‐Bayard & Ramos‐Morales, ; Zouhir et al, ). Additionally, SlrP interacts with ERdj3 thereby interfering with the function of the host endoplasmic reticulum lumenal chaperone.…”

Exploitation of the host cell ubiquitin machinery by microbial effector proteins

“…The LRR domain is connected to the NEL domain through a flexible linker and inhibits its ubiquitin ligase activity in the absence of substrate proteins (Chou et al, 2012;Quezada et al, 2009;Keszei and Sicheri, 2017). Removal of the LRR domain from SspH1, SspH2, SlrP or IpaH9.8 increases their E3 ligase activity (Quezada et al, 2009;Chou et al, 2012;Zouhir et al, 2014). A structural study of SspH1 showed that the binding of the host protein kinase N1 (PKN1) to the LRR domain releases its inhibitory effect on the NEL domain, which promotes the ubiquitylation and degradation of PKN1, and leads to the attenuation of the androgen response (Haraga and Miller, 2006;Keszei et al, 2014).…”