Structural Insights of Shigella Translocator IpaB and Its Chaperone IpgC in Solution

Abstract

Bacterial Type III Secretion Systems (T3SSs) are specialized multicomponent nanomachines that mediate the transport of proteins either to extracellular locations or deliver Type III Secretion effectors directly into eukaryotic host cell cytoplasm. Shigella, the causing agent of bacillary dysentery or shigellosis, bears a set of T3SS proteins termed translocators that form a pore in the host cell membrane. IpaB, the major translocator of the system, is a key factor in promoting Shigella pathogenicity. Prior to secretion, IpaB is maintained inside the bacterial cytoplasm in a secretion competent folding state thanks to its cognate chaperone IpgC. IpgC couples T3SS activation to transcription of effector genes through its binding to MxiE, probably after the delivery of IpaB to the secretion export gate. Small Angle X-ray Scattering experiments and modeling reveal that IpgC is found in different oligomeric states in solution, as it forms a stable heterodimer with full-length IpaB in contrast to an aggregation-prone homodimer in the absence of the translocator. These results support a stoichiometry of interaction 1:1 in the IpgC/IpaB complex and the multi-functional nature of IpgC under different T3SS states.

Keywords: IpaB translocator; IpgC chaperone; Shigella flexneri; small angle x-ray scattering; type III secretion (T3S); type III translocator.

Figure 1

IpgC/IpaB available structural information. (A) A schematic representation of the IpaB full-length protein and the available structural information. The long coiled-coil domain of IpaB (green rectangle, residues 74 - 239) is the only known domain in atomic resolution along with the Chaperone Binding Domain (CBD, magenta rectangle, residues 60-72). The CBD domain was found in extended conformation interacting with the major groove of one IpgC molecule (cartoon representation, blue monomer). See text for details. Other segments of IpaB were proposed as being implicated in the interaction with IpgC: a second CBD (red rectangle) at the N-terminal of IpaB; and an extension of the first CBD (pink rectangle). Grey rectangles on bottom of IpaB sequence represent predicted α-helices. (B) Constraint-based Multiple Alignment, COBALT (Papadopoulos and Agarwala, 2007). Part of the alignment, focusing on the C-terminal part of major translocators from T3S systems of different origin, is presented. Multiple sequence alignment columns with no gaps are colored. Higher conserved columns in red, less conserved ones in blue and non-conserved in grey. Accession numbers for the protein sequences used are provided in Materials and Methods section Modeling of the IpgC/IpaB Complex.

Figure 2

IpgC/IpaB available structural information and hydrodynamic parameters. (A) Chromatograph of Size Exclusion analysis on the elution fractions of the IpgC/IpaB complex collected following the metal affinity purification step. Four peaks are observed; V: Void Volume; I: The IpgC/IpaB peak as judged by the SDS-PAGE analysis in (B); II: A proteolytic form of IpaB in complex with IpgC; and III: IpgC alone. On top of the graph the apparent Molecular Weights (MW) and the Hydrodynamic Radii (R_h) of the molecular markers used to calibrate the size exclusion chromatography column are shown. (B) SDS-PAGE analysis of the various peaks in A after their collection and concentration. (C) Chromatograph of Size Exclusion analysis on the elution fractions of the IpgC collected following the metal affinity purification step. (D) SDS-PAGE analysis of the IpgC peak in (C).

Figure 3

SAXS analysis of the IpgC (A, B, E, F) and IpgC/IpaB complex (C–F). (A, C) SAXS Intensity profiles (in logarithmic scale) for six concentrations of IpgC (A) and four concentrations of the IpgC/IpaB complex (C). (B, D) Guinier plots linearity indicates monodispersity for the IpgC/IpaB complex (D) and aggregation for the higher concentrations of IpgC (B). (E) Normalized pair distance distribution functions P(r) for IpgC and IpgC/IpaB. (F) Normalized Kratky plots of IpgC and the IpgC/IpaB complex (a globular, well folder protein, BSA, is also shown for comparison).

Figure 4

Models of IpgC dimers (right panels) and the corresponding fits to solution SAXS data (left panels). Model (A) is the asymmetric IpgC dimer (PDB ID: 3GZ1). Model (B) is the symmetric IpgC dimer (PDB ID: 3KS2). Model (C) is the symmetric IpgC dimer where the residues not present in the crystal structure were modeled to be compatible with the SAXS pattern.

Figure 5

IpgC/IpaB SAXS model. (A) Fit of the theoretical SAXS pattern calculated from the model in (B) to the experimental SAXS data. (B) Three perpendicular representations of the IpgC/IpaB SAXS model. IpaB is depicted in yellow-gold and IpgC in turquoise. Residues where high resolution information is available (crystal structure or homology model) are shown in cartoon representation while spheres represent dummy residues.