Conformational flexibility of EptA driven by an interdomain helix provides insights for enzyme-substrate recognition

Abstract

Many pathogenic gram-negative bacteria have developed mechanisms to increase resistance to cationic antimicrobial peptides by modifying the lipid A moiety. One modification is the addition of phospho-ethano-lamine to lipid A by the enzyme phospho-ethano-lamine transferase (EptA). Previously we reported the structure of EptA from Neisseria, revealing a two-domain architecture consisting of a periplasmic facing soluble domain and a transmembrane domain, linked together by a bridging helix. Here, the conformational flexibility of EptA in different detergent environments is probed by solution scattering and intrinsic fluorescence-quenching studies. The solution scattering studies reveal the enzyme in a more compact state with the two domains positioned close together in an n-do-decyl-β-d-maltoside micelle environment and an open extended structure in an n-do-decyl-phospho-choline micelle environment. Intrinsic fluorescence quenching studies localize the domain movements to the bridging helix. These results provide important insights into substrate binding and the molecular mechanism of endotoxin modification by EptA.

Keywords: EptA; conformational flexibility; enzyme substrate recognition; phosphoethanolamine transferase; small-angle X-ray scattering; tryptophan fluorescence.

Figure 1

Catalytic reaction and structure of EptA. (a) Reaction catalysed by EptA. (b) Secondary structure representation of EptA from N. meningitidis (PDB entry 5fgn). The N-terminal transmembrane domain is represented in dark blue and the C-terminal soluble domain is represented in red. The bridging helix that connects the transmembrane domain and soluble domain is represented in pale blue. The tryptophan residues in the protein are depicted as cyan balls and sticks.

Figure 2

SEC-SAXS results for the EptA:DDM complex and EptA:DPC complex. (a) I(s) versus s, as log–linear plots with the inset showing the Guinier fits (black line) laid on the experimental data (open symbols). (b) Profiles for the distance distribution function, P(r), in arbitrary units versus r in ångströms for the data shown in (a), normalized for ease of comparison. (c) Dimensionless Kratky plots for the data in (a).

Figure 3

MEMPROT and DADIMODO modelling results. (a) Model and scattering fit for the EptA:DDM complex and (b) the EptA:DPC complex. (a) (i) Representative MEMPROT generated model for the EptA:DDM complex. The atomic structure of EptA is represented in cartoon mode and coloured grey. The DDM corona around EptA is represented as pale blue spheres. (a) (ii) Comparison of the SAXS experimental result (red dots) with the MEMPROT calculated scattering curve (black solid line). The lower inset shows the error-weighted residual difference plot for the experimental SAXS model and the MEMPROT generated model (red dots). (b) (i) Representative MEMPROT generated model for the EptA:DPC complex. The atomic structure of EptA is represented in cartoon mode and coloured grey. The DPC corona around EptA is represented as orange spheres. (b) (ii) Comparison of the SAXS experimental result (blue dots) with the MEMPROT calculated scattering curve (black solid line). The lower inset shows the error-weighted residual difference plot for the experimental SAXS data and the MEMPROT generated model (blue dots). (b) (iii) Model of the EptA:DPC complex obtained with DADIMODO, assigning the soluble domain and transmembrane domain as rigid bodies connected by a flexible bridging helix and linker region. The atomic structure of EptA is represented in cartoon mode and coloured grey and the DPC corona around EptA is represented as orange spheres. (b) (iv) The lower inset shows the error-weighted residual difference plot for the experimental SAXS data and the DADIMODO generated model (blue dots).

Figure 4

Hybrid SAXS modelling results and flexibility analysis. Results for the (a) EptA:DDM complex and (b) EptA:DPC complex. (i) Hybrid SAXS models generated for each of the complexes using the combined MPBuilder/PACKMOL/CORAL approach. The atomic structure of EptA is represented in cartoon mode and coloured grey. The detergent corona around EptA is represented as pale blue and orange coloured lines for DDM and DPC, respectively. (ii) Comparison of the SAXS experimental data (coloured red and blue for EptA:DDM and EptA:DPC, respectively) with the scattering curves computed from the best fitting hybrid models (black solid line). The lower insets show the error-weighted residual difference plot for the fits. (iii) Flexibility analysis of the complexes using the EOM. Distance profiles (R _g) of the random pools of PDC conformations generated (grey histograms) and of the selected ensembles of conformations that best fit the SAXS data (red and blue histograms for EptA:DDM and EptA:DPC, respectively). The fits of the selected ensembles to the experimental data and residuals are shown in the inset. For EptA:DDM the selected ensemble consists of a narrow population of similar compact conformations, and two wide populations of both compact and extended conformations are selected for EptA:DPC, indicating significant conformational flexibility.

Figure 5

Intrinsic fluorescence spectra and quenching profiles for EptA in different detergent micelles. (a) Fluorescence spectra and Stern–Volmer plots for native folded enzyme and (b) fluorescence spectra for the denatured enzyme. The emission wavelength maxima (λ_max) for each condition are indicated above each curve. The red and blue coloured lines represent enzyme in DDM and DPC micelles, respectively. The solid lines represent EptA_WT, the dashed lines represent EptA_{Trp126/148Phe} and the dotted lines represent EptA_{Trp126/148/207Phe}. Panels (i) and (iii) show the emission spectra of the enzymes and panels (ii) and (iv) in (a) show the Stern–Volmer plot for quenching using iodide. A linear regression was fitted using GraphPad prism with an R ² value above 0.95. Where a non-linear relationship was observed, only the initial linear slope was fitted.