Dynamic Nuclear Polarization Illuminates Key Protein-Lipid Interactions in the Native Bacterial Cell Envelope

Abstract

Elucidating the structure and interactions of proteins in native environments is a fundamental goal of structural biology. Nuclear magnetic resonance (NMR) spectroscopy is well suited for this task but often suffers from low sensitivity, especially in complex biological settings. Here, we use a sensitivity-enhancement technique called dynamic nuclear polarization (DNP) to overcome this challenge. We apply DNP to capture the membrane interactions of the outer membrane protein Ail, a key component of the host invasion pathway of Yersinia pestis. We show that the DNP-enhanced NMR spectra of Ail in native bacterial cell envelopes are well resolved and enriched in correlations that are invisible in conventional solid-state NMR experiments. Furthermore, we demonstrate the ability of DNP to capture elusive interactions between the protein and the surrounding lipopolysaccharide layer. Our results support a model where the extracellular loop arginine residues remodel the membrane environment, a process that is crucial for host invasion and pathogenesis.

Figure 1.. DNP signal enhancement of the ¹⁵N and ¹³C MAS solid-state NMR spectra of Ail in E. coli cell envelopes.

(A-C) One dimensional spectra acquired with (A) ¹H-¹⁵N CP, (B) ¹H-¹³C CP, or (C) ¹H-¹⁵N-¹³C double CP, and with (green) or without (black) microwave irradiation. Signal enhancement factors (ε_on/off) were measured as the ratio of signal intensity observed with and without microwave irradiation. (D, E) Two dimensional ¹³C/¹³C correlation spectrum of Ail in E. coli cell envelopes acquired with DNP (green). The spectra from Ail in E. coli cell envelopes (black) and Ail in liposomes (red), acquired without DNP are shown for comparison and were described previously. The yellow rectangle marks LPS-related correlations.

Figure 2.. Two-dimensional NCA spectrum of ¹⁵N,¹³C-Ail in E. coli cell envelopes acquired with DNP (green).

(A) Resolved assigned peaks are marked. Asterisks denote a new unassigned peak observed with DNP. The spectrum from Ail in E. coli cell envelopes (black) acquired without DNP is shown for comparison and was described previously. (B) Amino acid sequence of Ail. The eight β-strands (β1-β8), four extracellular loops (EL1-EL4) and three intracellular turns (IT1-IT3) are mapped above the sequence. There are 8 Arg (blue), 11 Lys (red), and 20 Gly residues. (C) Snapshot of the Ail β-barrel obtained after 1.5 μs of MD simulation in a Y. pestis outer membrane depicting the eight Arg residues.

Figure 3.. Two-dimensional NHHC spectra acquired with DNP.

(A-C) Spectra were acquired for (15N,13C)-Ail(+) (green) or (¹⁵N,¹³C)-Ail(–) (pink) E. coli cell envelopes. Spectral regions of correlations are marked (gold boxes). One-dimensional slices (B, C) were taken at specific ¹⁵N chemical shifts (dashed lines) of sidechain N from Arg and Lys. (D) Snapshot of Ail obtained after 1.5 μs of MD simulation in a Y. pestis outer membrane showing that Arg27 in the LPS-recognition motif establishes multiple interactions with lipid A of an LPS molecule. Characteristic ¹³C chemical shifts of the different LPS groups are marked. MD simulations were described previously.