Structures of the Shigella flexneri type 3 secretion system protein MxiC reveal conformational variability amongst homologues.
This negative regulation is mediated, in part, by a family of proteins that are thought to physically block the entrance to the secretion apparatus until an appropriate signal is received following host cell contact.Interestingly, comparison of the Shigella and Yersinia structures reveals a significant structural change that results in substantial domain re-arrangement and opening of one face of the molecule.The conservation of a negatively charged patch on this face suggests it may have a role in binding other components of the T3SS.
Affiliation: Sir William Dunn School of Pathology, South Parks Rd, University of Oxford, Oxford OX1 3RE, UK.
Many Gram-negative pathogenic bacteria use a complex macromolecular machine, known as the type 3 secretion system (T3SS), to transfer virulence proteins into host cells. The T3SS is composed of a cytoplasmic bulb, a basal body spanning the inner and outer bacterial membranes, and an extracellular needle. Secretion is regulated by both cytoplasmic and inner membrane proteins that must respond to specific signals in order to ensure that virulence proteins are not secreted before contact with a eukaryotic cell. This negative regulation is mediated, in part, by a family of proteins that are thought to physically block the entrance to the secretion apparatus until an appropriate signal is received following host cell contact. Despite weak sequence homology between proteins of this family, the crystal structures of Shigella flexneri MxiC we present here confirm the conservation of domain topology with the homologue from Yersinia sp. Interestingly, comparison of the Shigella and Yersinia structures reveals a significant structural change that results in substantial domain re-arrangement and opening of one face of the molecule. The conservation of a negatively charged patch on this face suggests it may have a role in binding other components of the T3SS.
- Bacterial Outer Membrane Proteins/chemistry*/genetics/metabolism
- Protein Conformation*
- Shigella flexneri*
- Structural Homology, Protein*
- Amino Acid Sequence
- Bacterial Proteins/chemistry
- Carrier Proteins/chemistry
- Conserved Sequence
- Crystallography, X-Ray
- Membrane Proteins/chemistry
- Models, Molecular
- Molecular Sequence Data
- Protein Binding
- Sequence Homology, Amino Acid
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fig2: The structure and topology of MxiC. a, A ribbon diagram of MxiC, colored from blue at the N terminus to red at the C terminus. Views rotated by 90° about the long axis are shown. b, A diagram of the topology of MxiC illustrating the four-helix X-bundle of each domain colored as for a. c, Two molecules of MxiC from the P212121 crystal form (molecule B in magenta and molecule C in cyan), overlaid via their central domain (residues 154–265), illustrating the extremes of the movement seen for domains 1 and 3 (shown with cylindrical helices). Methods: Initial crystallization conditions were obtained by sparse-matrix screening,30 using the sitting drop vapor diffusion technique. Drops were prepared using an OryxNano crystallization robot (Douglas Instruments) by mixing 0.2 μl of protein (7 mg ml− 1 in 20 mM Tris (pH 7.5), 150 mM NaCl) with 0.2 μl of reservoir solution and were equilibrated against 100 μl of reservoir solution at 20 °C. Initial, low-resolution diffracting crystals of MxiCFL grew within two weeks in condition P2-26 of the PACT Premier screen (0.2 M NaBr, 0.1 M BisTris–propane (pH 7.5), 20% (w/v) PEG3350: space group P43212 with one molecule in the asymmetric unit) and condition 3 of Molecular Dimensions Structure Screen II (2% (v/v) dioxane, 0.1 M bicine (pH 9.0), 10% (w/v) PEG20000: two different, related P21 forms with two molecules in the asymmetric unit). The former condition yielded diffraction-quality crystals of SeMet-labeled MxiCFL†. Crystals of native MxiCFL diffracting to 3.0 Å resolution grew in 0.2 M Na2SO4, 0.1 M BisTris–propane (pH 6.5), 20% (w/v) PEG3350, again in P43212 but with a longer c axis and two molecules in the asymmetric unit. The methylation reaction was performed as described in Refs. 31 and 32 on purified MxiCFL and MxiCNΔ73 each at 1 mg ml− 1 in 50 mM Hepes (pH 7.5), 250 mM NaCl. Samples were centrifuged (5 min, 13,000 rpm, 10,000g 4 °C) before purification of soluble methylated protein by size-exclusion chromatography (as described above). Methylation of all lysine side chains and the N terminus was verified by mass spectrometry (42,952 Da for MxiCFL and 35,106 Da for MxiCNΔ73). The P222 crystal form grew in 1.0 M succinic acid, 0.1 M Hepes (pH 7.0), 1% (w/v) PEG2000MME. The P212121 crystal form grew in 0.2 M sodium acetate, 0.1 M BisTris–propane (pH 7.5), 20% (w/v) PEG3350. Crystals of MxiC were cryoprotected in reservoir solution containing 25% (v/v) glycerol for 15 s and flash cryocooled in liquid nitrogen for data collection. Diffraction data were recorded at 100 K. Data were indexed and integrated in MOSFLM,33 and scaled with Scala,34 within the CCP4 program suite,35 except for the native MxiCFLP43212 3.0 Å dataset, which was indexed in Labelit36 and integrated in XDS,37 both run from the processing suite Xia2 (G. Winter et al., unpublished program). Initial phases were computed using SHARP:38 five sites were found by SHELXD39 run from the suite of programs autoSHARP40 against FAs calculated from the peak, inflexion and low-energy remote wavelengths of a SeMet-labeled P43212 MxiCFL crystal. The coordinates and B-factors of these sites were refined in SHARP against the above data plus the second remote wavelength from the same SeMet crystal. Solvent flattening was performed using CCP4-DM41 and SOLOMON,42 yielding a 3.5 Å map that was used for initial model building guided by the YopN–TyeA structure (PDB ID 1xl3).5 After alternate cycles of model building in Coot,43 refinement in Buster-TNT,44 and simulated annealing in PHENIX,45 this initial model was used for molecular replacement, using CCP4 PHASER,46 into the higher resolution P212121 form. The resultant model was used for molecular replacement against the MxiCNΔ73P222 and native MxiCFLP43212 crystal forms. The final Buster-TNT refinements in the latter forms used NCS restraints throughout, and extra geometry restraints tying the geometry to Refmac47-refined models, to improve the stereochemistry (as Refmac5 implements torsion angle restraints and can refine riding H atoms), a refinement strategy devised by Dr. Stephen Graham (University of Oxford).
Reductive methylation of MxiCNΔ73 yielded crystals that diffracted to higher resolution: 2.85 Å and 2.5 Å in space groups P212121 and P222, respectively (Table 1). MxiC is an elongated rod-shaped molecule with a long axis of 86 Å (Fig. 2a). It is composed of three domains, each possessing a four-helix X-bundle fold (Fig. 2b).19 The first and last domains consist only of the X-bundle motif while the central domain also possesses a bent helix (α5) that is packed against domain 1. It is the first two domains of MxiC that are equivalent to YopN while the third domain of MxiC, equivalent to TyeA, is connected to domain 2 via a ten-residue linker. This linker acts only to tether the domains and does not have any major structural role, allowing the equivalent regions of MxiC and YopN–TyeA to adopt similar folds. There are a total of seven independent MxiC molecules in the crystallographic asymmetric units of the three refined crystal forms (see Table 1). These structures reveal that, although the fold of each domain is maintained in all structures (rmsd over Cα atoms of domains 1, 2 and 3 are 0.5 Å, 0.9 Å and 0.8 Å, respectively), there is some flexibility at the interfaces between domains resulting in a “wobble” of the terminal domains about the central domain (rmsd over all Cα atoms of 1.4 Å; Fig. 2c). The elongated shape of MxiC means that the most distal regions undergo the greatest displacement while the more central interdomain interfaces undergo minimal change.