Structural analysis of ligand‐bound states of the Salmonella type III secretion system ATPase InvC

Abstract

Translocation of virulence effector proteins through the type III secretion system (T3SS) is essential for the virulence of many medically relevant Gram‐negative bacteria. The T3SS ATPases are conserved components that specifically recognize chaperone–effector complexes and energize effector secretion through the system. It is thought that functional T3SS ATPases assemble into a cylindrical structure maintained by their N‐terminal domains. Using size‐exclusion chromatography coupled to multi‐angle light scattering and native mass spectrometry, we show that in the absence of the N‐terminal oligomerization domain the Salmonella T3SS ATPase InvC can form monomers and dimers in solution. We also present for the first time a 2.05 å resolution crystal structure of InvC lacking the oligomerization domain (InvCΔ79) and map the amino acids suggested for ATPase intersubunit interaction, binding to other T3SS proteins and chaperone–effector recognition. Furthermore, we validate the InvC ATP‐binding site by co‐crystallization of InvCΔ79 with ATPγS (2.65 å) and ADP (2.80 å). Upon ATP‐analogue recognition, these structures reveal remodeling of the ATP‐binding site and conformational changes of two loops located outside of the catalytic site. Both loops face the central pore of the predicted InvC cylinder and are essential for the function of the T3SS ATPase. Our results present a fine functional and structural correlation of InvC and provide further details of the homo‐oligomerization process and ATP‐dependent conformational changes underlying the T3SS ATPase activity.

The PopN gate-keeper complex acts on the ATPase PscN to regulate the T3SS secretion switch from early to middle substrates inPseudomonas aeruginosa

“…To date, only structures of the N-terminally truncated monomers of SctN were obtained by Xray crystallography (Allison et al, 2014;Bernal et al, 2019;Burgess et al, 2016;Zarivach et al, 2007), suggesting that the N-terminal domain of SctN is flexible and/or partially unfolded. Some groups were able to heterologously express and purify the His-tagged recombinant full-length protein from E. coli (Akeda and Galan, 2005;Andrade et al, 2007;Chatterjee et al, 2013;Yoshida et al, 2014).…”

“…Displaying a structural homology to the F1-ATPases family, the SctN ATPase is believed to assemble into a hexameric ring presenting a central cavity that is blocked by the stalk SctO, whose role and structure are similar to the γ subunit of F1-ATPase (Gao et al, 2018). The structure of the Nterminally truncated monomer of SctN was characterized by X-ray crystallography, showing two main domains (Allison et al, 2014;Bernal et al, 2019;Burgess et al, 2016;Zarivach et al, 2007). The Cterminal domain contains five α-helices and is thought to interact with the secreted T3SS proteins in the first step of the secretion process.…”

“…The Walker A and B motifs, which are crucial for the catalytic activity, are also found in this domain. Based on the similarity with the α/β subunit of the F1-ATPase, a model of the homo-hexameric ring SctN was built in silico with the ATP binding pocket interfaces located between two adjacent subunits (Allison et al, 2014;Bernal et al, 2019;Burgess et al, 2016;Demler et al, 2019). It was thus suggested that the oligomerization state is favorable to the catalytic activity.…”

Distinct cytosolic complexes containing the type III secretion system ATPase resolved by 3D single-molecule tracking in liveYersinia enterocolitica

“…There are numerous reports documenting interactions between SctN and SctL in both flagellar and virulence-associated T3SSs [28][29][30][31] . SctL binding prevents SctN hexamerization prior to injectisome binding and thereby negatively regulates SctN's ATPase activity, which is the one of the most well-established functional roles of the cytosolic injectisome proteins 28,30,[32][33][34][35] .…”

Type III secretion system effector proteins are mechanically labile

“…However, the T3SS ATPase does not belong to the AAA(+) family of ATPases. Instead, it is structurally similar to the catalytic β-subunit of the F 1 F 0 ATP synthase, a rotary motor that normally couples proton gradient dissipation to ATP synthesis but can also run in reverse and hydrolyze ATP to do work (15,(28)(29)(30). The T3SS ATPase is not as powerful an unfoldase as the AAA(+) family, as fusions of effector proteins with GFP, ubiquitin, or DHFR stall in the injectisome and are poorly secreted (20,22,31,32).…”