Abstract
Gram-negative bacteria utilize the type III secretion system (T3SS) to inject effector proteins into the host cell cytoplasm, where they subvert cellular functions and assist pathogen invasion. The conserved type III-associated ATPase is critical for the separation of chaperones from effector proteins, the unfolding of effector proteins and translocating them through the narrow channel of the secretion apparatus. However, how ATP hydrolysis is coupled to the mechanical work of the enzyme remains elusive. Herein, we present a complete description of nucleoside triphosphate binding by surface presentation antigens 47 (Spa47) from Shigella flexneri, based on crystal structures containing ATPγS, a catalytic magnesium ion and an ordered water molecule. Combining the crystal structures of Spa47-ATPγS and unliganded Spa47, we propose conformational changes in Spa47 associated with ATP binding, the binding of ATP induces a conformational change of a highly conserved luminal loop, facilitating ATP hydrolysis by the Spa47 ATPase. Additionally, we identified a specific hydrogen bond critical for ATP recognition and demonstrated that, while ATPγS is an ideal analog for probing ATP binding, AMPPNP is a poor ATP mimic. Our findings provide structural insight pertinent for inhibitor design.
Highlights
Shigellosis caused by shigella species is a leading cause of diarrheal disease worldwide and the second leading cause of death in children aged 1–4 years living in low-income and middle-income countries, there are roughly 1,64,000 annual deaths attributable to shigellosis (Kotloff et al, 2018)
T3SSs are evolutionarily related to flagella and many substructures and components that are involved in assembly are highly conserved, which is built from more than 20 unique components assembled into a 3.5 megadalton syringe-like complex, including a cytosolic ATPase complex, a cytoplasmic ring (C-ring), an inner membrane export apparatus, a basal body, and a translocation pore that is in the host cell membrane (Kubori et al, 1998; Galán et al, 2014; Deng et al, 2017; Galán and Waksman, 2018)
Kato and colleagues identified a two-helixfinger motif and a luminal loop located at the central pore in the predicted hexameric ring of InvC (Kato et al, 2015) that is reminiscent of similar features in ATP-powered unfoldase and translocase enzymes
Summary
Shigellosis caused by shigella species is a leading cause of diarrheal disease worldwide and the second leading cause of death in children aged 1–4 years living in low-income and middle-income countries, there are roughly 1,64,000 annual deaths attributable to shigellosis (Kotloff et al, 2018). The T3SS is a specialized apparatus employed by many Gram-negative bacteria and symbionts to inject effector proteins into host cells (Blaylock and Schneewind, 2005; Cornelis, 2006; Deng et al, 2017). T3SSs are evolutionarily related to flagella and many substructures and components that are involved in assembly are highly conserved, which is built from more than 20 unique components assembled into a 3.5 megadalton syringe-like complex, including a cytosolic ATPase complex, a cytoplasmic ring (C-ring), an inner membrane export apparatus, a basal body, and a translocation pore that is in the host cell membrane (Kubori et al, 1998; Galán et al, 2014; Deng et al, 2017; Galán and Waksman, 2018). Upon delivery into the cytoplasm, effector proteins engage in various activities that subvert cellular functions and promote pathogen invasion
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