Gram-negative trimeric porins have specific LPS binding sites that are essential for porin biogenesis

Abstract

The outer membrane (OM) of gram-negative bacteria is an unusual asymmetric bilayer with an external monolayer of lipopolysaccharide (LPS) and an inner layer of phospholipids. The LPS layer is rigid and stabilized by divalent cation cross-links between phosphate groups on the core oligosaccharide regions. This means that the OM is robust and highly impermeable to toxins and antibiotics. During their biogenesis, OM proteins (OMPs), which function as transporters and receptors, must integrate into this ordered monolayer while preserving its impermeability. Here we reveal the specific interactions between the trimeric porins of Enterobacteriaceae and LPS. Isolated porins form complexes with variable numbers of LPS molecules, which are stabilized by calcium ions. In earlier studies, two high-affinity sites were predicted to contain groups of positively charged side chains. Mutation of these residues led to the loss of LPS binding and, in one site, also prevented trimerization of the porin, explaining the previously observed effect of LPS mutants on porin folding. The high-resolution X-ray crystal structure of a trimeric porin-LPS complex not only helps to explain the mutagenesis results but also reveals more complex, subtle porin-LPS interactions and a bridging calcium ion.

Keywords: X-ray crystal structure; gram-negative bacteria; lipopolysaccharide; outer membrane; porin.

Fig. 1.

Binding of LPS to OmpF causes slower mobility of complexes on SDS/PAGE. (A) Structure of LPS from E. coli with the R3 core structure, including that of the Rc-LPS from E. coli J5 used in this study (63) (nonstoichiometric additions are shown with dotted lines). In lipid A, GlcN_I and GlcN_II are, respectively, the reducing and nonreducing glucosamine residues. The inner core usually comprises two or three Kdo and three Hep (l-glycero-d-manno-heptose) molecules. This region is phosphorylated at several sites. The variable trihexose backbone forms the outer core with varying side chains; shown here as present in the R3 form are glucose (Glc), N-acetylglucosamine (GlcNAc), and galactose (Gal). These link to the long O-antigen polysaccharide (O-PS) region found only in smooth strains. The depiction of the rough Ra-to-Re mutants that define the different chemotypes is based upon the original classification in S. minnesota (64). The Rd-LPS used here is from an Rd2 mutant, as shown. (B) Characteristic ladder, on 10% SDS/PAGE, of OmpF resulting from LPS binding. In vitro folded LPS-free OmpF (Left) and an identical sample mixed with a fivefold molar excess of Ra-LPS (Right). (C) Samples of in vitro folded OmpF + Ra-LPS as in B mixed with 5 mM MgCl₂, CaCl₂, or EDTA. (D) In vitro folded OmpF mixed with a fivefold molar ratio of LPS variants of increasing size. Sm, smooth LPS. (E) In vitro folded OmpF mixed with increasing molar ratios of Ra-LPS.

Fig. 2.

Removing positively charged residues decreases the amount of LPS bound to OmpF. (A) Localization of positively charged residues on the extracellular side of an OmpF trimer (PDB ID code 2OMF). We divided these residues into group A in the cleft (magenta) and group B at the perimeter (yellow) separated by the red dashed line. This and other structural images were created using PyMOL (65). (B) Native OmpF, WT, and site A mutant proteins, purified from the OM with bound LPS, analyzed on 10% SDS/PAGE stained with Coomassie blue. The double- and triple-glutamine mutations retain a tail of LPS-bound forms, which contrasts with the quadruple-glutamate mutations (Fig. 3F).

Fig. 3.

LPS binding site B is essential for OmpF trimerization in vivo but not in vitro. The abbreviations describe the mutations applied at each site; for example, A-Gln, all basic residues in site A replaced by glutamine; AB-Glu, all basic residues in sites A and B replaced by glutamate. (A) Purification of A-Gln mutant protein. BEX and EX, boiled and native OmpF extraction samples showing monomers and trimers, respectively; BOF and NOF, boiled and native WT OmpF control samples from in vitro folded stock; M, position of the monomer band on the gel; MM, molecular weight markers; P, membrane pellet; S, supernatant; T, position of the trimer band on the gel; W1 and W2, supernatants after washes 1 and 2. (B) Purification of B-Gln mutant protein. B W1/2 and W1/2, boiled and native supernatants from wash 1/2; B Ext and Ext, as above, showing lack of trimeric OmpF extracted with SDS and NaCl; Pre I, whole-cell pellet before induction; Post I, whole-cell pellet after 1-h induction showing an OmpF band at 37 kDa; Sol, supernatant after cell breakage. (C) Summary table of results for mutants. “Expression” indicates a band observed at 37 kDa after induction; “maturation” indicates that intact trimers were purified from the OM. (D) Solubilization of A-Glu, B-Glu, and AB-Glu inclusion bodies in urea, showing the different migration of unfolded monomers on SDS/PAGE. (E) In vitro folding of trimeric porins. N WT, native WT purified from the OM; R A, R B, and R AB, in vitro folded A-Glu, B-Glu, and AB-Glu mutants with and without boiling; R WT, in vitro folded WT. A-Glu and B-Glu fold fully in vitro, but no heat-modifiable trimer is evident in AB-Glu samples. (F) LPS binding to in vitro folded mutants. WT, A-Glu (site B intact), and B-Glu (site A intact) without added calcium or plus 5 mM CaCl₂ or 5 mM EDTA. Note the clear effect of calcium removal on the A-Glu mutant, indicating a role for the calcium ion(s) in site B.

Fig. 4.

SANS data indicate that LPS binds at the periphery of OmpF in SDS solution. (A) Log/log plot of scattering data for deuterated (d-)OmpF in complex with hydrogenous Ra-LPS after size-exclusion chromatography. Q (momentum transfer) is a product of the scattering angle and neutron wavelength (6 Å) (Methods). Fitted lines were generated by the program BayesApp to calculate the P(r) vs. distance (r) plots (see Table 1 for fitting parameters and SI Appendix, Fig. S8 for an enlargement of the panel). P(r) is the real-space pair-distance function, which describes the distribution of pairs of scattering centers within the complex (Methods). The error bars in A represent the range of intensity values about the plotted data point and are the result of data reduction and averaging procedures within the program GRASP. (B) P(r) plot calculated from data in A using the same color scheme shows that at 13%, 27%, and 41% D₂O, where LPS scattering is minimal, the plots resemble free OmpF (32). At 77% D₂O, when the d-OmpF scattering is minimal, the plot describes small groups of scattering centers separated by about 90 Å. This corresponds to groups of LPS arranged at fixed sites around the trimer, as in the case of OmpF–colicin complexes (32). (C) The line of best fit to the Stuhrmann plot, the square of the radius of gyration (${\mathrm{R}}_{\text{g}}^2$) versus the inverse of the contrast (1/Δρ) (Methods), has a negative value of α (Results), a result most easily appreciated from the apex of the parabola being at negative values of 1/Δρ. This indicates that the low-nSLD LPS has a larger R_g than the high-nSLD OmpF and is likely to be situated at the periphery of the complex.

Fig. 5.

X-ray crystal structure of the E. cloacae OmpE36–LPS complex. (A) Cartoon view of the OmpE36 trimer from the top, with the bound LPS molecules indicated by stick models. The bound calcium ion is shown as a green sphere. (B) Side view showing LPS A and LPS B binding. The approximate boundaries of the hydrophobic core of the OM are indicated by horizontal lines. (C) Close-up of LPS B, with polar interactions between the LPS and OmpE36 indicated by dashed lines. (D) Close-up of the boxed region in B, showing the interactions of the central Arg213 with both LPS A and LPS B. For clarity, some LPS B acyl chains have been removed. (E) View from the extracellular side highlighting interactions between LPS B and OmpE36. (F) Close-up of the calcium ion bridging LPS B and OmpE36.

Fig. 6.

Two-dimensional map showing interactions between OmpE36 residues and LPS A and B. The figure was generated using PoseView (66). GlcN_I and GlcN_II are the reducing and nonreducing 2-amino-2-deoxy-α-d-glucopyranose residues of lipid A (glucosamine), respectively. Acyl chains of lipid A are indicated by asterisks.