High resolution solid-state NMR spectroscopy of the Yersinia pestis outer membrane protein Ail in lipid membranes

Abstract

The outer membrane protein Ail (Adhesion invasion locus) is one of the most abundant proteins on the cell surface of Yersinia pestis during human infection. Its functions are expressed through interactions with a variety of human host proteins, and are essential for microbial virulence. Structures of Ail have been determined by X-ray diffraction and solution NMR spectroscopy, but those samples contained detergents that interfere with functionality, thus, precluding analysis of the structural basis for Ail’s biological activity. Here, we demonstrate that high-resolution solid-state NMR spectra can be obtained from samples of Ail in detergent-free phospholipid liposomes, prepared with a lipid to protein molar ratio of 100. The spectra, obtained with 13C or 1H detection, have very narrow line widths (0.40–0.60 ppm for 13C, 0.11–0.15 ppm for 1H, and 0.46–0.64 ppm for 15N) that are consistent with a high level of sample homogeneity. The spectra enable resonance assignments to be obtained for N, CO, CA and CB atomic sites from 75 out of 156 residues in the sequence of Ail, including 80% of the transmembrane region. The 1H-detected solid-state NMR 1H/15N correlation spectra obtained for Ail in liposomes compare very favorably with the solution NMR 1H/15N TROSY spectra obtained for Ail in nanodiscs prepared with a similar lipid to protein molar ratio. These results set the stage for studies of the molecular basis of the functional interactions of Ail with its protein partners from human host cells, as well as the development of drugs targeting Ail.

Structural Insights into the Yersinia pestis Outer Membrane Protein Ail in Lipid Bilayers

“…DSC analysis shows that the KLA-containing, KLA(+), Ail lipid bilayers undergo a main endothermic phase transition ( T m ) at 24 °C (Figure 4B, black), very close to the transitions observed 36 for KLA(−) Ail liposomes at 24.7 °C (Figure 4B, red), and for pure lipids at 24.2 °C (Figure 4B, blue). The DSC endotherms reflect the cooperative transformation from the lamellar gel phase (L β ′), where the lipid acyl chains are tilted and all-trans, to the lamellar liquid crystalline phase (L α ), where the lipid chains are disordered and fluid.…”

“…Both preparations proceeded smoothly, with no evidence of either precipitation or the presence of soluble Ail in the liposome supernatant. Moreover, the sample preparation has been optimized 36 to minimize lipid loss during reconstitution, as verified by 1 H NMR analysis of the lipids, and sample transfer from the centrifuge tube to the MAS rotor was quantitative in both cases.…”

“…The samples contain ∼3 mg of Ail and have a protein/lipid molar ratio of 1:100, as evidenced by measuring the 1 H NMR signal intensity at 1.2 ppm from the lipid acyl CH 2 groups. 36 Proteoliposomes were harvested by ultracentrifugation at 168000g, 4 °C, for 16 h, and then packed in a 4 mm MAS rotor with a 50 μ L insert (Bruker). Two samples were prepared, one containing Ail, DMPC, and DMPG at a molar ratio of 1:74:25, and another containing Ail, DMPC, DMPG, and di(3-deoxy-D-manno-octulosonyl)-lipid A (Kdo 2 -Lipid A or KLA; Avanti) at a molar ratio of 1:68:23:8.…”

“…2D heteronuclear 13 C– 15 N correlation spectra were acquired using SPECIFIC-CP 41 for band selective polarization transfer, during which continuous wave (CW) 1 H decoupling (100 kHz RF field strength) was applied, and a tangent ramp was applied on the 15 N channel with contact times of 3 ms for N–CA transfer. Resonance assignments were described in a previous study; 36 they were obtained with 2D and 3D 13C – 15 N correlation spectra and assisted by direct comparison with the solution NMR data obtained for Ail in nanodiscs.…”

Applications of NMR to membrane proteins

“…5) illustrates the spectral resolution attainable for homogeneous liposome preparations [144]. In this sample, the protein was uniformly labeled with 15 N and 13 C, and fractionally (∼70%) 2 H labeled, with amides back exchanged to 1 H during protein purification, to enable NMR detection.…”