Cryo-EM structure of the homohexameric T3SS ATPase-central stalk complex reveals rotary ATPase-like asymmetry

Majewski, D.D.; Worrall, L.J.; Hong, Chuan; Atkinson, Claire; Vuckovic, M.; Watanabe, Nobuhiko; Yu, Zhiheng; Strynadka, N.C.J.

doi:10.1038/s41467-019-08477-7

Cited by 61 publications

(105 citation statements)

References 77 publications

(117 reference statements)

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“…The E. coli T3SS ATPase forms a homo‐hexameric cylinder with ATP‐binding sites located at the interface of adjacent subunits, similar to the F 1 ATPases. We reasoned that interaction between ATPase subunits might occur also in the absence of its N‐terminal region and characterized the molecular size of InvCΔ79 in solution.…”

Section: Resultssupporting

confidence: 86%

“…However, we cannot discard the possibility of a structural change around this amino acid upon ATP binding or chaperone–effector interaction. The amino acid E384 was reported to participate in the interaction with the stalk protein in Escherichia coli . E384 is surface exposed in our structure, and as Y385, is located in the α10‐α11 mobile loop (Figure a).…”

Section: Resultsmentioning

confidence: 99%

“…R189 and R191 are related with intersubunit interaction (lilac) . Y385 is essential for full type III secretion and the conserved E384 interacts with the stalk protein in EscN (raspberry) . L376 interacts with chaperone–effector complexes (green)…”

Section: Resultsmentioning

confidence: 99%

“…Similarly, the corresponding β9‐α7 loop of the Shigella Spa47Δ83 was shown to increase its flexibility upon interaction with ATP‐analogues . Interestingly, cryo‐EM analysis of the E. coli EscN homo‐hexameric cylinder showed differences in the α10‐α11 loop conformation for each ligand‐bound state and differences in the β9‐α7 loop upon ligand recognition …”

Section: Resultsmentioning

confidence: 99%

“…However, the T3SS ATPases share high sequence homology and three‐dimensional structural similarities with the β subunit of the F 1 F O ATPases . Recently, the E. coli ATPase EscN has been shown to form a homo‐hexameric cylinder with six ATP‐binding sites located at the interface of adjacent dimer pairs, suggesting cooperativity between subunits . EscN binds to its central stalk protein and presents different functional states supporting a rotary catalytic mechanism homologous to the F 1 F O ATPases.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2019

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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.

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Section: Resultssupporting

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2019

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The sorting platform in the type III secretion pathway: From assembly to function

Soto

Lara‐Tejero

2023

BioEssays

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The type III secretion system (T3SS) is a specialized nanomachine that enables bacteria to secrete proteins in a specific order and directly deliver a specific set of them, collectively known as effectors, into eukaryotic organisms. The core structure of the T3SS is a syringe‐like apparatus composed of multiple building blocks, including both membrane‐associated and soluble proteins. The cytosolic components organize together in a chamber‐like structure known as the sorting platform (SP), responsible for recruiting, sorting, and initiating the substrates destined to engage this secretion pathway. In this article, we provide an overview of recent findings on the SP's structure and function, with a particular focus on its assembly pathway. Furthermore, we discuss the molecular mechanisms behind the recruitment and hierarchical sorting of substrates by this cytosolic complex. Overall, the T3SS is a highly specialized and complex system that requires precise coordination to function properly. A deeper understanding of how the SP orchestrates T3S could enhance our comprehension of this complex nanomachine, which is central to the host‐pathogen interface, and could aid in the development of novel strategies to fight bacterial infections.

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Interfacial amino acids support Spa47 oligomerization and shigella type three secretion system activation

et al. 2019

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Like many Gram-negative pathogens, Shigella rely on a type three secretion system (T3SS) for injection of effector proteins directly into eukaryotic host cells to initiate and sustain infection. Protein secretion through the needle-like type three secretion apparatus (T3SA) requires ATP hydrolysis by the T3SS ATPase Spa47, making it a likely target for in vivo regulation of T3SS activity and an attractive target for small molecule therapeutics against shigellosis. Here, we developed a model of an activated Spa47 homo-hexamer, identifying two distinct regions at each protomer interface that we hypothesized to provide intermolecular interactions supporting Spa47 oligomerization and enzymatic activation. Mutational analysis and a series of high-resolution crystal structures confirm the importance of these residues, as many of the engineered mutants are unable to form oligomers and efficiently hydrolyze ATP in vitro. Furthermore, in vivo evaluation of Shigella virulence phenotype uncovered a strong correlation between T3SS effector protein secretion, host cell membrane disruption, and cellular invasion by the tested mutant strains, suggesting that perturbation of the identified interfacial residues/interactions influences Spa47 activity through preventing oligomer formation, which in turn regulates Shigella virulence. The most impactful mutations are observed within the conserved Site 2 interface where the native residues support oligomerization and likely contribute to a complex hydrogen bonding network that organizes the active site and supports catalysis. The critical reliance on these conserved residues suggests that aspects of T3SS regulation may also be conserved, providing promise for the development of a cross-species therapeutic that broadly targets T3SS ATPase oligomerization and activation.

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Cryo-EM structure of the homohexameric T3SS ATPase-central stalk complex reveals rotary ATPase-like asymmetry

Cited by 61 publications

References 77 publications

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

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

The sorting platform in the type III secretion pathway: From assembly to function

Interfacial amino acids support Spa47 oligomerization and shigella type three secretion system activation

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