Conformational analysis of the full-length M2 protein of the influenza A virus using solid-state NMR

Abstract

The influenza A M2 protein forms a proton channel for virus infection and mediates virus assembly and budding. While extensive structural information is known about the transmembrane helix and an adjacent amphipathic helix, the conformation of the N-terminal ectodomain and the C-terminal cytoplasmic tail remains largely unknown. Using two-dimensional (2D) magic-angle-spinning solid-state NMR, we have investigated the secondary structure and dynamics of full-length M2 (M2FL) and found them to depend on the membrane composition. In 2D (13)C DARR correlation spectra, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-bound M2FL exhibits several peaks at β-sheet chemical shifts, which result from water-exposed extramembrane residues. In contrast, M2FL bound to cholesterol-containing membranes gives predominantly α-helical chemical shifts. Two-dimensional J-INADEQUATE spectra and variable-temperature (13)C spectra indicate that DMPC-bound M2FL is highly dynamic while the cholesterol-containing membranes significantly immobilize the protein at physiological temperature. Chemical-shift prediction for various secondary-structure models suggests that the β-strand is located at the N-terminus of the DMPC-bound protein, while the cytoplasmic domain is unstructured. This prediction is confirmed by the 2D DARR spectrum of the ectodomain-truncated M2(21-97), which no longer exhibits β-sheet chemical shifts in the DMPC-bound state. We propose that the M2 conformational change results from the influence of cholesterol, and the increased helicity of M2FL in cholesterol-rich membranes may be relevant for M2 interaction with the matrix protein M1 during virus assembly and budding. The successful determination of the β-strand location suggests that chemical-shift prediction is a promising approach for obtaining structural information of disordered proteins before resonance assignment.

Keywords: chemical shift prediction; conformational change; influenza M2; membrane protein.

Figure 1

One-dimensional C CP-MAS spectra of uniformly C, N-labeled M2FL in (a) the DMPC membrane, (b) the POPC:POPG:cholesterol membrane, and (c) the VM+ membrane. The spectra were measured at 303, 273, and 243 K from top to bottom. The integrated intensities of the Cα region relative to the 243 K spectra are indicated. DMPC-bound M2FL shows much lower intensities at 303 K than at 243 K, indicating large-amplitude motion of the protein at high temperature when bound to this membrane.

Figure 2

Two-dimensional C–¹³C correlation spectra of DMPC-bound M2FL. (a) Full DARR spectrum with 15-ms C spin diffusion. Spectra measured at 253 and 273 K did not show significant differences and were thus co-added to give higher sensitivity. Residue-type assignments for peaks with characteristic chemical shifts are shown in blue for β-sheet, red for α-helix, and green for random coil. Superimposed in orange is the sheared J-INADEQUATE 2D spectrum in Figure 3, indicating the chemical shifts of mobile residues. (b) Water-edited 2D DARR spectrum, measured at 253 K. Shaded bars guide the eye for the preferential retention of the β-sheet signals and suppression of the α-helical peaks. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]

Figure 3

Two-dimensional C refocused J-INADEQUATE spectrum of DMPC-bound M2FL, measured at 303 K under 12 kHz MAS. Amino-acid type assignments indicate random coil chemical shifts for most detected residues (Supporting Information Table S2). [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]

Figure 4

(a) Two-dimensional C DARR correlation spectrum of VM+ bound M2FL, measured at 273K with a 20-ms mixing time. (b) Two-dimensional spectrum of DMPC-bound M2FL reproduced from Figure 2(a) for comparison. Yellow rectangles highlight regions where β-sheet signals are absent in the VM+ membrane but present in the DMPC membrane. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]

Figure 5

Ideal secondary-structure models and the predicted chemical shifts, superimposed with the measured 2D DARR spectrum (gray) and sheared J-INADEQUATE spectrum (orange) of DMPC-bound M2FL. (a) All-coil model. (b) All-helix model. (c) All-strand model. (d) A coil–helix–coil model. Coil, strand, and helix are denoted by a thin line, a thick line with an arrow, and an oscillation, respectively. The predicted chemical shifts are colored in green for the N-terminal ectodomain, red for the TM-AH domain, and blue for the cytoplasmic tail. Boxes show a few peaks of interest. The simulated spectra plot correlation peaks up to three bonds.

Figure 6

Predicted 2D spectra of mixed secondary-structure models, superimposed with the experimental spectra of DMPC-bound M2FL. (a) The cytoplasmic tail as strand while the rest of the protein as helix. (b) The ectodomain as strand while the rest of the protein as helix. (c) PSIPRED-predicted secondary structure. Blue arrows indicate the positions of β-strand Ile peaks. (d) A model with a β-strand at the N-terminus, coil for the rest of the ectodomain, helix for TM-AH, and coil for the cytoplasmic tail.

Figure 7

Predicted coil chemical shifts and one-bond cross peaks for several structural models, superimposed with the sheared 2D J-INADEQUATE spectrum (dark orange) of DMPC-bound M2FL. A solid orange line represents the coil segment, while dotted black lines indicate residues that are not simulated. (a) Coil for the full ectodomain. (b) Coil for the cytoplasmic tail. (c) Coil for the ectodomain and two segments in the cytoplasmic tail. (d) Coil for a part of the ectodomain and the entire cytoplasmic tail. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]

Figure 8

Experimental 2D C DARR spectrum (gray contours) of DMPC-bound M2(21–97), superimposed with the predicted chemical shifts using the secondary-structure model of Figure 6(d) except that the ectodomain is removed. The SNA triplet is assumed to be random coil, the TM-AH domain is kept α-helical, and the cytoplasmic tail is kept as random coil. Yellow rectangles highlight the frequency positions of β-strand peaks that are present in the DMPC-M2FL spectrum but absent in the M2(21–97) spectrum. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]