OmpF and TolA are involved in ColN translocation (Y. C. Kim et al., 2014). It is proposed that the T-domain first binds to OmpF and then TolA (Housden et al., 2013; Johnson et al., 2013). The binding to TolA then triggers the entry of ColN into bacterial cells. TolA and OmpF have been shown to form complexes in vitro (Derouiche et al., 1996; Dover et al., 2000) but no evidence yet exists for in vivo interaction. In order to understand how these two interaction partners of ColN could form complexes prior to the translocation, the structure of in vitro OmpF/TolA complexes previously observed in SDS (Derouiche et al., 1996) (Dover et al., 2000) was further studied by small-angle solution scattering. The truncated TolA consisting of domain II and III was used in these experiments because the full length TolA is poorly expressed. The complexes were formed in the presence of 0.5% (w/v) SDS. Firstly, the complexes in SDS were assessed by SDS-PAGE as in Figure 3.19. The native sample of complexes were run along with the purified TolA II-III and OmpF without boiling hence all proteins in the sample were in their native structure. Results showed that the complex band shifted towards higher molecular weight and confirmed that TolA II-III can interact with OmpF in vitro. This is in good agreement with Derouiche
et al and Dover et al (Derouiche et al., 1996; Dover et al., 2000). Interestingly,
the characteristic OmpF ladder on SDS-PAGE still existed when OmpF formed complexes with TolA II-III. The ladder is caused by LPS tightly bound to the extracellular half of the membrane face of OmpF. It has been previously demonstrated that ColN binding displaces LPS from OmpF at this extracellular part since these complexes do not show a ladder on SDS-PAGE (Baboolal et
al., 2008). On the other hand, the existence of a ladder in OmpF/TolA
complexes on SDS-PAGE suggested that TolA II-III bound to OmpF elsewhere, perhaps at the periplasmic side. Interestingly, ColN and TolA II also compete for a binding site so this must overlap (Dover et al., 2000).
Figure 3.19 SDS-PAGE of OmpF/TolA II-III complex. All samples were prepared without boiling thus proteins were in their native state. Both OmpF and complexes had the ladder features due to the binding of LPS to OmpF. The LPS came from the outer membrane of E. coli when purifying OmpF.
The SANS experiments on OmpF/TolA II-III complexes were carried out on SANS2D beamline at ISIS, UK. Using the same technique as the study of OmpF/ColN-TR complex, the dOmpF with the CMP of 87% D2O formed
complexes with TolA II-III at a 1:1 molecular ratio (3 molecules of TolA II-III per 1 molecule of OmpF trimer) in 50 mM sodium phosphate, pH 7.4, 300 mM NaCl and the mixture of h/d SDS. This was done to ensure that the scatter from SDS is matched to all D2O solutions. The samples were prepared in four D2O
solutions; 13%, 41%, 90% and 100% D2O. Varying the H2O/D2O content in the
sample allowed us to observe either the individual component or the whole complex. The whole complex can be seen in 13% and 100% D2O solutions
whereas the dOmpF and TolA II-III were observed at 41% and 90% D2O,
respectively. The scattering curves were recorded and evaluated by the indirect Fourier transform using GNOM (Figure 3.20a). The P(r) distribution function generated by GNOM revealed the smallest P(r) at 41% D O when observing
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only dOmpF and the largest P(r) at 100% D2O when observing the entire
complex (Figure 3.20b). At 90% D2O where only TolA II-III is visible, the
maximum peak was shifted towards the short distance and the shape of P(r) function was changed. Therefore, it was an obvious indication of TolA II-III forming complexes with dOmpF because the P(r) of complex (at 13% and 100% D2O) is larger than that of only OmpF (at 41% D2O). The Guinier plots in Figure
3.20c were linear and confirmed the lack of aggregation in samples.
Figure 3.20 SANS data for dOmpF/TolA II-III complexes in 1% h/d SDS. a) SANS data (symbols) and fitting (lines) generated by GNOM. b) Distance distribution function, P(r), calculated by GNOM. c) Guinier analysis
The size parameters, Rg and Dmax, derived from Guinier and P(r) plots were
summarized in Table 3.6. The consistency of the Rg value determined from
these two analyses also pointed out that the complexes are monodisperse and the assigned Dmax values were accurate. When comparing the Rg value of
complexes in 90% D2O (only TolA II-III is visible) to that of TolA II-III in normal
experiment were inconsistent due to the flexibility of TolA II-III, however the Rg
values of TolA II-III in complex with OmpF in the SANS experiment were consistent. Dmax of TolA II-III also substantially changed when TolA II-III bound
to dOmpF. This indicated that the structure of TolA II-III in complex becomes more compact.
Table 3.6 Summary of all parameters derived from Guinier plot and P(r) function for OmpF/TolA II-III complexes. PRIMUS is used for Guinier analysis and GNOM calculated P(r) function.
The Rg of complexes derived from the initial analysis of SANS data were then
utilised for the Stuhrmann analysis. The change of Rg as a function of contrast
was evaluated by plotting of against
and fitting by a parabola curve.
Rg and I(0) from the Guinier plot were used as the input in MULCh program in
order to calculated . This analysis shows the position where TolA II-III is in complex with OmpF. Figure 3.21 shows the Stuhrmann plot for dOmpF/TolA II- III complexes along with the parabola fit with a 2 of 0.78. The α value obtained from a fit has a negative sign. The sign of α suggested that TolA II-III is located on the outside of OmpF trimer.
Sample Guinier Analysis 0.4<RgQ<1.3 P(r) function Rg (Å) Rg (Å) Dmax(Å) dOmpF:hTolA in 13% D2O (entire complex) 41.53±2.792 42.11±0.383 170 dOmpF:hTolA in 41% D2O (dOmpF only) 35.00±3.236 37.36±0.324 112 dOmpF:hTolA in 90% D2O (hTolA only) 48.70±3.530 50.50±1.523 167 dOmpF:hTolA in 100% D2O
(the entire complex) 54.70±2.410 56.66±0.964 175.5 hTolA in H2O 45.18 ± 2.93 118.70 ± 2.09 410.0
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According to the findings from SANS and SDS-PAGE studies, the proposed model of OmpF/TolA II-III complexes is shown in Figure 3.22. TolA II-III is located at the periplasmic side of the OmpF trimer. The length of TolA II-III is derived from the P(r) function at 90% D2O where only TolA II-III is visible to
neutrons.
Figure 3.21 Stuhrmann analysis of dOmpF/TolA II-III complex. The reciprocal of the contrast is plotted against the square of the radius of gyration (Rg). The experimental data points are shown as symbols. A parabola fitted to
the data is shown in red.
Figure 3.22 Proposed model for OmpF/TolA II-III complex in solution. Three blue cylinders represent the OmpF trimer whose height and diameter are obtained from its crystallographic structure (PDB code: 2OMF). Three red rectangles display TolA II-III with a length of ≈110 Å interacting with OmpF at the periplasmic side.