Biochemical and structural analysis of bacterial O-antigen chain length regulator proteins reveals a conserved quaternary structure - PubMed (original) (raw)
Biochemical and structural analysis of bacterial O-antigen chain length regulator proteins reveals a conserved quaternary structure
Kane Larue et al. J Biol Chem. 2009.
Abstract
Lipopolysaccharide (LPS) is a major component of the Gram-negative outer membrane and is an important virulence determinant. The O-antigen polysaccharide of the LPS molecule provides protection from host defenses, and the length of O-antigen chains plays a pivotal role. In the Wzy-dependent O-antigen biosynthesis pathway, the integral inner membrane protein Wzz determines the O-antigen chain length. How these proteins function is currently unknown, but the hypothesis includes activities such as a "molecular ruler" or a "molecular stopwatch," and other possibilities may exist. Wzz homologs are membrane proteins with two transmembrane helices that flank a large periplasmic domain. Recent x-ray crystallographic studies of the periplasmic portions of Wzz proteins found multiple oligomeric forms, with quaternary structures favoring the "molecular ruler" interpretation. Here, we have studied full-length Wzz proteins with the transmembrane portions embedded in lipid membranes. Using electron microscopy and image analysis we find a unique hexameric state rather than differing oligomeric forms. The data suggest that in vivo Wzz proteins determine O-antigen chain length via subtle structure-function relationships at the level of primary, secondary, or tertiary structure within the context of a hexameric complex.
Figures
FIGURE 1.
O-antigen chain length is regulated by Wzz. LPS preparations from_S. enterica_ serovar Typhimurium LT2 (WT) and its derivative_wzz_-null strains (CWG859 and CWG860) were analyzed by SDS-PAGE on a 4-12% Bis-Tris gradient gel and silver-stained. wzz genes were expressed in trans from pBAD-based plasmids in CWG860 and result in the production of long (L) and very long (VL) mode LPS as previously reported (16, 23, 29, 34, 44).
FIGURE 2.
Wzz oligomerization in time course cross-linking experiments. Silver-stained SDS-PAGE gels of polyhistidine-tagged Wzz proteins incubated at 37 °C with 1% formaldehyde. Aliquots were removed from cross-linking reactions at time intervals and quenched in 100 m
m
Tris-Cl, pH 7.5. An untreated sample of each homolog was included as a control (lane c).
FIGURE 3.
Membrane-extracted Wzz exists in several oligomeric states. PFO-PAGE analysis, visualized with silver-staining, shows that DDM-solubilized Wzz preparations contain a heterogeneous population of oligomers. Formaldehyde cross-linking does not produce any novel protein bands that are not evident in untreated samples.
FIGURE 4.
Transmission electron microscopy of negatively stained Wzz proteoliposomes. A, reconstituted WzzST forms large two-dimensional crystalline arrays. The inset area (white box) is a 512 × 512 pixel region that is shown at higher magnification in_panel B_, and was used to determine the reciprocal lattice parameters (panel C) and for generation of a projection map (panel D). The dimensions of the unit cell are a = b = 95.2 Å and γ = 120°. E, WzzFepE proteoliposome and the 512 × 512 pixel region (white box) shown at higher magnification in_panel F_ that was used for reciprocal lattice refinement (panel G) and for generation of a projection map (panel H). The dimensions of the unit cell are a = b = 107.0 Å and γ = 120°. I, WzzK40 proteoliposomes and the 512 × 512 pixel region (white box) shown at higher magnification in_panel J_ that was used for reciprocal lattice refinement (panel K) and for generation of a projection map (panel L). The dimensions of the unit cell are a = b = 98.8 Å and γ = 120°.
FIGURE 5.
Cryo-electron microscopy of WzzST proteoliposomes embedded in vitreous buffer. A, large ∼2-μm diameter vesicle containing a two-dimensional Wzz crystalline array. The inset area (white box) is shown at higher magnification in panel B. C, projection map of the Wzz complex after patch averaging without the application of symmetry (p1). D, magnified few of the edge of a flattened vesicle with side views of Wzz molecules emerging from the membrane (arrowheads). E, average of side views generated with EMAN (43), showing the membrane-embedded region (band of density) at the bottom.
FIGURE 6.
The projection map of WzzST is consistent with a hexameric quaternary structure. A, left, projection map of the WzzST crystalline array from cryo-EM after image processing with the application of p622 symmetry. The unit cell dimensions are a =b = 93 Å. Right, model of a p622 crystalline array of WzzEPD hexamers containing two stacked layers. A superimposed region of WzzST and WzzEPD is highlighted in red. B, model of a hexameric Wzz complex generated using the WzzEPD protomer (PDB: 3B8O) shown as molecular surface representations from side and top views. A single protomer is shown in cyan.
FIGURE 7.
The periplasmic region of PCP proteins and the periplasmic component of the TolC multi-drug efflux pump share a similar secondary structure. Ribbon representations of the FepEPD protomer (PDB 3B8N) from E. coli O157 and the MexA protomer (PDB 1T5E) from P. aeruginosa. Both proteins possess extended α-helical domains that are proposed to self-assemble into ring-shaped periplasmic complexes (27, 48, 49).
References
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources