Structural And Functional Study Of The Highly Conserved ATPase From Type Ⅲ Secretion System Of Bacterial Pathogens

Type III secretion systems (T3SS) play an central role in bacteria pathogenicity. Proper environment signals can activate the transcription of virulence gene. All T3SS is highly conserved and forms a super macro molecular complex known as the needle complex composed 20-25 different proteins that spanning the inner and outer bacterial membranes and cross the eukaryotic cell membrane. Secreted proteins are traversed the central channel of syringe-like apparatus and target host cells for pathogenicity. Therefore, T3 S S serve as a important target for drug design.An essential component of all T3SS is highly conserved ATPase, which is thought to energize the secretion process. These ATPases have also been demonstrated to form hexamer and exert function in the recognition and unfolding of effectors destined to through a channel at the centre of the hexamer ring by harnessing energy released from ATP. Previous research reported F-Type/V-Type ATPase have highly similarity 3-D structure. Which indicative of these ATPases have similar molecular mechanism for effectors transport. Flagellar ATPase FliI and prototypical T3SS ATPase EscN crystal structure show that T3SS ATPase monomer have similar structure with F1F0-ATPase β subunit. However, both structure did not form hexamer owing to T3SS ATPase highly instability. To date, the unique experimental evidence for T3SS ATPase hexamer assembly are HrcN low resolution electron microscopy structure.Which is not enough for understanding how T3SS hexamer assembly, NTP binding and effectors transport.To understand molecular basis underlying recognition of the substrate by ATPase and its role in effector transport, we employed the methods of structural biology, and biochemistry and bacteriology to investigate the mode of ATPase and substrate interaction.First, this study performed isothermal titration calorimetry (ITC) experiment to investigate the ATP binding specificity of Spa47 in a near physiological solution, our data firstly suggests that suggesting ATPyS works an ideal analogue with the regard of nucleotide binding specificity, whereas AMPPNP is not ideal for probing nucleotide binding specificity of Spa47. To reveal the structural basis for nucleotide binding specificity of ATPase, we determined the crystal structure of apo Spa47/InvC Δ1-83 and its nucleotide bound forms, namely Spa47Δ1-83-ATPγS, Spa47Δ1-83-AMP-PNP, Spa47Δ1-83-ADP, InvCΔ1-83-ATPγS, InvCΔ1-83-A MP-PNP, InvCΔ1-83-ADP. The structures analysis showed that both proteins were folded liking classic α/β Rossmann folding and C terminal helical bundle domain. The crystal structure of Spa47Δ1-N83 complexed by Mg-ATPγS provides the first detailed scenario for nucleotide binding by T3SS ATPase and further indicate that AMPPNP did not fully settle the nucleotide binding pocket. Comparing the structure of apo Spa47Δ1-83 and its nucleotide bound structure show that the critical function for ATP recognition is a hydrogen bond donated by NH group of G162 to the oxygen between β-and γ-phosphates. Because the alteration of this atom can lead to dramatic loses of binding affinity. Evidenced by AMPPNP, the oxygen between β- andγ-phosphates is replaced by a nitrogen atom, our discovery explain AMPPNP and ADP have similar binding ability. Further analysis the structure of apo Spa47Δ1-83/InvCΔ1-83 and its nucleotide bound structure show that conformational changes induced by nucleotide binding. Collectively, our data suggests a channel in Spa47 that connects the nucleotide binding event at the active site to a chain of conformational change, which eventually leads to the conformational dynamics of the β9-α8 loop that makes direct contacts to T3SS substrate.To sum up, we firstly solved T3SS ATPase complexed by catalytically important Mg-ATPγS crystal structure, which provides the first detailed scenario for nucleotide binding by T3SS ATPase. Serving a important target for NTP analogue inhibitor. We found conformational changes induced by ATP binding can lead to conformational dynamics of the β9-α8 loop that makes direct contacts to T3SS substrate. Which indicative of the relationship between ATP hydrolysis and effector transport. Our studies provide a clue for understanding the T3SS ATPase function. T3SS is one of the key question to be addressed in the field of pathogenic bacteria, and ATPase also a important target for drug design. Our works provided the basis for the drug design targeted for T3SS.