Structural Insight Into Conformational Changes Induced by ATP Binding in a Type III Secretion-Associated ATPase From Shigella flexneri

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.

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

“…Similarly, the α‐helical content of the ATPγS‐structure was scarcely affected (Figure ), confirming the FTIR and CD spectroscopy results obtained in solution. Structural changes of the β9‐α7 and α10‐α11 loops upon ligand recognition were previously reported for the Shigella Spa47 and for the E. coli EscN ATPases . Additionally, the integrity of the loops β9‐α7 and α10‐α11 has been shown to be essential for type III secretion .…”

“…The adenine group is stabilized by π‐π stacking with Y338. The interaction of ligands with InvCΔ79 resembles the ATP‐analogue binding observed for orthologue T3SS ATPases (Figure ) . Additionally, single mutations of G164 and K165 in the InvC P‐loop result in loss of ATP‐hydrolysis function .…”

“…Point mutations in this region of InvC, including E306A, E308A, E309A, E310A, and D312A, were reported to affect secretion of late effectors by the T3SS . 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 …”

“…In addition, the loop including V411 (α11‐α12) is located closer to M167 surrounding and further defining the open hydrophobic pocket (Figure ). A similar pocket is formed in the ligand‐binding site of other T3SS ATPases, although the amino acid composition in this region has little sequence similarity . A comparison with the nucleotide‐binding site of the Shigella Spa47 and E. coli EscN T3SS ATPases shows that the adenine double‐ring from the ADP‐InvCΔ79 structure is rather displaced from the inner region of the hydrophobic pocket but still captured on its surface (Figure ).…”

“…The ligand density found in the ADP‐InvCΔ79 co‐crystal structure was not continuous, suggesting some flexibility or partial occupancy and posing a limit on the conclusions that can be drawn from this. Noteworthy, ligand densities found in E. coli ADP‐EscN, Salmonella Flagellar ADP‐FliI, and Shigella AMP‐PNP‐Spa47 crystal structures were also fragmented . Gao and coworkers have shown that ATP‐analogues can bind Spa47 with low affinity ( K d ~ 21–322 μM) .…”