PDBsum entry 2q1k

Chaperone

PDB id

2q1k

Contents

			Protein chains

					52 a.a.

References listed in PDB file

Key reference

Title

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.

Authors

Y.W.Tan, H.B.Yu, K.Y.Leung, J.Sivaraman, Y.K.Mok.

Ref.

Protein Sci, 2008, 17, 1748-1760. [DOI no: 10.1110/ps.036798.108]

PubMed id

18662905

Abstract

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 A 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 is comprised of two helix-turn-helix monomers packed in an antiparallel fashion. The N-terminal 13 residues of AscE are buried only upon binding with AscG, but this region is found to be nonessential for the interaction. AscE functions as a monomer and can be coexpressed 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 remain 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-terminal hydrophobic residues, Ile83 and Leu84, of AscF are essential for chaperone binding.



	Figure 1. Crystal structure and the simulated-annealing F [o] --F [c] omit map in the conserved region of AscE. (A) The map is contoured at a level of 2.0 [sigma]. Residues Leu24 to Ala27 and all atoms within 3.0 A of Leu24 to Ala27 were omitted prior to refinement. (B) Ribbon representation of the crystal structure of the dimeric AscE from residue Pro14 to Glu65 at two different angles. The hydrophobic residues (Leu20, Leu24, Ala27, Val31, Trp47, Ala53, Ile60, and Ile64) that form an interlocking network at the dimeric interface of the protein are shown in a ball-and-stick model. The figure was generated with the program Chimera (Pettersen et al. 2004). (C) Overlay of the crystal structures of AscE (purple) and YscE (chain A and B) (cyan) viewed at two different angles. The dimers of AscE and YscE overlay with an RMSD of 2.2 A for 101 C[[alpha]] atoms using DaliLite pairwise comparison of protein structure. (D) Ribbon representation of the structures of PscE (green) and YscE (yellow) as in the crystal structures of the complexes PscE-PscF^55 --85-PscG and YscEFG viewed at two different angles (Quinaud et al. 2007; Sun et al. 2008). Figure 1A Figure 1.-was prepared using the program PyMOL (DeLano Scientific). Figure 1B --D Figure 1.-was prepared using the program Chimera (Pettersen et al. 2004).		Figure 7. The C-terminal hydrophobic residues (Ile83 and Leu84) of AscF are essential for chaperone binding. (Lane 1) Protein molecular weight marker; (lane 2) coexpression of full-length His-AscF with AscE and AscG. The formation of the ternary complex is indicated by copurification of AscE (7.56 kDa). (Lane 3) Coexpression of the His-AscF^[Delta]81 --87 C-terminal truncation mutant with AscE and AscG. No ternary complex can be formed, as indicated by the absence of the band corresponding to AscE. (Lanes 4 --7) Coexpression of His-AscF I83A_L84A, His-AscF I83A_I87A, His-AscF L84A_I87A, and His-AscF I83A_L84A_I87A, respectively, with AscE and AscG. Mutation of both residues Ile83 and Leu84 are necessary to reduce the chaperone binding of AscF significantly. Residue Ile87 is also involved in the interaction but not to the same extent as residues Ile83 and Leu84.

PROCHECK

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