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Structure of AscE and induced burial regions in AscE and AscG upon formation of the chaperone needle-subunit complex of type III secretion system in Aeromonas hydrophila

National University of Singapore

(RECEIVED June 4, 2008; ACCEPTED July 17, 2008)

In the type III secretion system (T3SS) of Aeromonas hydrophila, the putative needle complex subunit, AscF, requires both putative chaperones, AscE and AscG, for formation of a ternary complex to avoid premature assembly. Here we report the crystal structure of AscE at 2.7 Å resolution and the mapping of buried regions of AscE, AscG and AscF in the AscEG and AscEFG complexes using limited protease digestion. The dimeric AscE comprises of two helix-turn-helix monomers packed in an anti-parallel fashion. The N-terminal 13 residues of AscE are buried only upon binding with AscG but this region is found to be non-essential for the interaction. AscE functions as a monomer and can be co-expressed with AscG or with both AscG and AscF to form soluble complexes. The AscE binding region of AscG in the AscEG complex is identified to be within the N-terminal 61 residues of AscG. The exposed C-terminal substrate binding region of AscG in the AscEG complex is induced to be buried only upon binding to AscF. However, the N-terminal 52 residues of AscF remains exposed even in the ternary AscEFG complex. On the other hand, the 35 residue C-terminal region of AscF in the complex is resistant to protease digestion in the AscEFG complex. Site-directed mutagenesis showed that two C-terminus hydrophobic residues, Ile83 and Leu84, of AscF are essential for chaperone binding.

Keywords: Structure/function studies; Crystallography; Protein Structures - New; Limited protease digestion; Type III secretion system

¹ E-mail: dbsmokh{at}nus.edu.sg

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