Structural and biochemical characterization of the type III secretion chaperones CesT and SigE

Several Gram-negative bacterial pathogens have evolved a type III secretion system to deliver virulence effector proteins directly into eukaryotic cells, a process essential for disease. This specialized secretion process requires customized chaperones specific for particular effector proteins. The crystal structures of the enterohemorrhagic Escherichia coli O157:H7 Tir-specific chaperone CesT and the Salmonella enterica SigD-specific chaperone SigE reveal a common overall fold and formation of homodimers. Site-directed mutagenesis suggests that variable, delocalized hydrophobic surfaces observed on the chaperone homodimers are responsible for specific binding to a particular effector protein. Isothermal titration calorimetry studies of Tir–CesT and enzymatic activity profiles of SigD–SigE indicate that the effector proteins are not globally unfolded in the presence of their cognate chaperones.

[1]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[2]  D. Bacon,et al.  A fast algorithm for rendering space-filling molecule pictures , 1988 .

[3]  J M Thornton,et al.  Validation of protein models derived from experiment. , 1998, Current opinion in structural biology.

[4]  B. Finlay,et al.  Identification of the intimin‐binding domain of Tir of enteropathogenic Escherichia coli , 1999, Cellular microbiology.

[5]  D Eisenberg,et al.  3D domain swapping: A mechanism for oligomer assembly , 1995, Protein science : a publication of the Protein Society.

[6]  U. Jakob,et al.  The 2.2 A crystal structure of Hsp33: a heat shock protein with redox-regulated chaperone activity. , 2001, Structure.

[7]  C. Hughes,et al.  From flagellum assembly to virulence: the extended family of type III export chaperones. , 2000, Trends in microbiology.

[8]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[9]  S. Doublié.  [29] Preparation of selenomethionyl proteins for phase determination. , 1997, Methods in enzymology.

[10]  B. Finlay,et al.  A synaptojanin‐homologous region of Salmonella typhimurium SigD is essential for inositol phosphatase activity and Akt activation , 2001, FEBS letters.

[11]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[12]  D E McRee,et al.  XtalView/Xfit--A versatile program for manipulating atomic coordinates and electron density. , 1999, Journal of structural biology.

[13]  V. L. Miller,et al.  SigE Is a Chaperone for the Salmonella enterica Serovar Typhimurium Invasion Protein SigD , 2001, Journal of bacteriology.

[14]  Zhaohui Xu,et al.  Crystal structure of the bacterial protein export chaperone SecB , 2000, Nature Structural Biology.

[15]  R. Pfuetzner,et al.  Enteropathogenic Escherichia coli translocated intimin receptor, Tir, requires a specific chaperone for stable secretion , 1999, Molecular microbiology.

[16]  B. Finlay,et al.  Mechanical Fractionation Reveals Structural Requirements for Enteropathogenic Escherichia coli Tir Insertion into Host Membranes , 2000, Infection and Immunity.

[17]  M L Johnson,et al.  Analysis of data from the analytical ultracentrifuge by nonlinear least-squares techniques. , 1981, Biophysical journal.

[18]  G. Cornelis,et al.  Customized secretion chaperones in pathogenic bacteria , 1996, Molecular microbiology.

[19]  A. Abe,et al.  Functional Analysis of the Type III Secretion System in Enteropathogenic Escherichia coli O157: H45 , 2000, Microbiology and immunology.

[20]  R. Pfuetzner,et al.  Enteropathogenic E. coli translocated intimin receptor, Tir, interacts directly with α-actinin , 2000, Current Biology.

[21]  C Chothia,et al.  Surface, subunit interfaces and interior of oligomeric proteins. , 1988, Journal of molecular biology.

[22]  B. Finlay,et al.  Recruitment of Cytoskeletal and Signaling Proteins to Enteropathogenic and Enterohemorrhagic Escherichia coli Pedestals , 2001, Infection and Immunity.

[23]  C. Lee,et al.  Type III secretion systems: machines to deliver bacterial proteins into eukaryotic cells? , 1997, Trends in microbiology.

[24]  Thomas C. Terwilliger,et al.  Automated MAD and MIR structure solution , 1999, Acta crystallographica. Section D, Biological crystallography.

[25]  C. Hueck,et al.  Type III Protein Secretion Systems in Bacterial Pathogens of Animals and Plants , 1998, Microbiology and Molecular Biology Reviews.

[26]  Thomas C. Terwilliger,et al.  Reciprocal-space solvent flattening , 1999, Acta crystallographica. Section D, Biological crystallography.

[27]  Craig M. Ogata,et al.  Structural Analysis of Substrate Binding by the Molecular Chaperone DnaK , 1996, Science.

[28]  G. Cornelis,et al.  The cytosolic SycE and SycH chaperones of Yersinia protect the region of YopE and YopH involved in translocation across eukaryotic cell membranes , 1996, Molecular microbiology.

[29]  J. Kaper,et al.  Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenic Escherichia coli , 1999, Molecular microbiology.

[30]  J. Galán,et al.  Supramolecular structure of the Salmonella typhimurium type III protein secretion system. , 1998, Science.

[31]  Zbyszek Otwinowski,et al.  The crystal structure of the bacterial chaperonln GroEL at 2.8 Å , 1994, Nature.

[32]  W. Hendrickson.  Determination of macromolecular structures from anomalous diffraction of synchrotron radiation. , 1991, Science.

[33]  J. Abrahams,et al.  Methods used in the structure determination of bovine mitochondrial F1 ATPase. , 1996, Acta crystallographica. Section D, Biological crystallography.

[34]  B. Honig,et al.  Classical electrostatics in biology and chemistry. , 1995, Science.

[35]  I. Connerton,et al.  Binding of intimin from enteropathogenic Escherichia coli to Tir and to host cells , 1999, Molecular microbiology.