Conformational flexibility and strand arrangements of the membrane-associated HIV fusion peptide trimer probed by solid-state NMR spectroscopy - PubMed (original) (raw)

Conformational flexibility and strand arrangements of the membrane-associated HIV fusion peptide trimer probed by solid-state NMR spectroscopy

Zhaoxiong Zheng et al. Biochemistry. 2006.

Abstract

The human immunodeficiency virus (HIV) fusion peptide (HFP) is the N-terminal apolar region of the HIV gp41 fusion protein and interacts with target cell membranes and promotes membrane fusion. The free peptide catalyzes vesicle fusion at least to the lipid mixing stage and serves as a useful model fusion system. For gp41 constructs which lack the HFP, high-resolution structures show trimeric protein and suggest that at least three HFPs interact with the membrane with their C-termini in close proximity. In addition, previous studies have demonstrated that HFPs which are cross-linked at their C-termini to form trimers (HFPtr) catalyze fusion at a rate which is 15-40 times greater than that of non-cross-linked HFP. In the present study, the structure of membrane-associated HFPtr was probed with solid-state nuclear magnetic resonance (NMR) methods. Chemical shift and intramolecular (13)CO-(15)N distance measurements show that the conformation of the Leu-7 to Phe-11 region of HFPtr has predominant helical conformation in membranes without cholesterol and beta strand conformation in membranes containing approximately 30 mol % cholesterol. Interstrand (13)CO-(13)CO and (13)CO-(15)N distance measurements were not consistent with an in-register parallel strand arrangement but were consistent with either (1) parallel arrangement with adjacent strands two residues out-of-register or (2) antiparallel arrangement with adjacent strand crossing between Phe-8 and Leu-9. Arrangement 1 could support the rapid fusion rate of HFPtr because of placement of the apolar N-terminal regions of all strands on the same side of the oligomer while arrangement 2 could support the assembly of multiple fusion protein trimers.

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Figures

Figure 1

13C spectra of HFPtr-F8CL9N associated with (a) PC-PG and (b) LM3 membranes and of HFPtr-L7CF11N associated with (c, d) PC-PG, (e) LM3, and (f) LM3e membranes. The HFPtr:lipid mol ratio was ∼0.003 in the samples used to obtain spectra a and b and ∼0.007 in the samples used to obtain spectra c-f. Spectra a, b, and c are REDOR-filtered with τ_i_ = 1.0, 1.0, and 32.25 ms, respectively, and have 13CO peak chemical shifts of 178.4 (Phe-8), 172.5 (Phe-8), and 178.8 ppm (Leu-7), respectively. Spectra d, e, and f are REDOR _S_0 spectra with 32.25 ms dephasing period and have 13CO peak chemical shifts of 178.6 ppm, 173.8 ppm, and 173.4 ppm, respectively. Each spectrum was processed with 100 Hz Gaussian line broadening and baseline correction. The MAS frequency was 8000 Hz and the numbers of scans used to obtain spectra a, b, c, d, e, and f are 118784, 132864, 46048, 23024, 21760, and 98720, respectively.

Figure 2

13C REDOR spectra of the (a, b) I4 peptide and HFPtr-L7CF11N associated with (c, d) PC-PG and (e, f) LM3e membranes. For each lettered pair of spectra, the S_0 spectrum is on the left and the S_1 spectrum is on the right. The MAS frequency = 8000 Hz, τ_R = 125 μs, and τ_i = 8.25 ms (spectra a, c, e) or τ_i_ = 32.25 ms (spectra b, d, f). Dotted lines are drawn at the peak _S_0 intensities. The I4 spectra were processed with 50 Hz Gaussian line broadening, the HFPtr spectra were processed with 100 Hz Gaussian line broadening, and baseline correction was applied to all spectra. The numbers of scans used to obtain each spectrum in panels a, b, c, d, e, and f are 32, 32, 37710, 23024, 40528, and 98720, respectively.

Figure 3

(a) REDOR (Δ_S_/S_0)i cor (filled squares) and (Δ_S/S_0)i sim (crosses) vs. dephasing time for the I4 peptide. Each (Δ_S/S_0)i cor was based on a (Δ_S/_S_0)i exp determined by integrations of 1 ppm regions in the _S_0 and _S_1 spectra. The integration region was centered at 178.8 ppm which is the peak shift in the S_0 spectra. For each τ_i, there were 64 total (_S_0 + _S_1) scans. The values of σ cori are ∼0.005 and the heights of the black squares are approximately equal to the average value of 2 × σ cori. (b) A plot of χ_2 vs. dCN yields dCN = 44.78 ± 0.22 Hz which corresponds to rCN = 4.110 ± 0.007 Å for the Ala-9 13CO/Ala-13 15N labeled pair. The uncertainty was determined using the approach described in the Materials and Methods section. The (Δ_S/_S_0)i sim values in plot a were calculated with the best-fit dCN.

Figure 4

(a) REDOR (Δ_S_/S_0)i cor (open squares with error bars) and (Δ_S/S_0)i sim (crosses) vs. dephasing time for the HFPtr-L7CF11N/PC-PG sample. Each (Δ_S/S_0)i cor was based on a (Δ_S/_S_0)i exp determined from spectral integrations over a 1 ppm region centered at 178.6 ppm, the Leu-7 13CO peak chemical shift. The total (_S_0 + S_1) numbers of scans used to obtain the (Δ_S/S_0)i cor values for τ_i = 8.25, 16.25, 24.25, and 32.25 ms were 75420, 52832, 41568, and 46048, respectively. (b) A plot of χ_2 vs. dCN yields dCN = 44.8 ± 2.4 Hz which corresponds to r = 4.11 ± 0.08 Å for the Leu-7 13CO/Phe-11 15N labeled pair. The (Δ_S/_S_0)i sim values in plot a were calculated with the best-fit dCN.

Figure 5

(a) REDOR (Δ_S_/S_0)i cor (open squares with error bars) and (Δ_S/S_0)i sim (crosses) vs. dephasing time for the HFPtr-L7CF11N/LM3e sample. Each (Δ_S/S_0)i cor was based on a (Δ_S/_S_0)i exp determined from spectral integrations over a 1 ppm region centered at 173.4 ppm, the peak shift in the _S_0 spectra. The total (_S_0 + S_1) numbers of scans used to obtain the (Δ_S/S_0)0 cor values for τ_i = 8.25, 16.25, 24.25, and 32.25 ms were 81056, 76288, 172512, and 197440, respectively. (b) A plot of χ_2 vs. dCN yields dCN = 15.8 ± 1.8 Hz which corresponds to r CN = 5.8 ± 0.3 Å for the Leu-7 13CO/Phe-11 15N labeled pair. The (Δ_S/_S_0)i sim values in plot a were calculated with dCN = 15.8 Hz.

Figure 6

fpCTDQBU spectra of (a-d) the GFF sample and (e-h) the HFPtr-L7CF11N/LM3e sample. For each lettered pair of spectra, the S_0 spectrum is on the left and the S_1 spectrum is on the right. The MAS frequency = 8000 Hz, τ_R = 125 μs, the 13C π pulse rf field = 10 kHz, M = 336, and the total constant-time = (L + M + N) × τR = 84 ms. The values of L, N, and τi are: 128, 208, 32 ms (spectra a, e); 192, 144, 48 ms (spectra b, f); 256, 80, 64 ms (spectra c, g); 320, 16, 80 ms (spectra d, h). From left-to-right, each GFF spectrum has Phe-3, Phe-2 and Gly-1 13CO peaks at 180.3, 176.4, and 170.9 ppm, respectively. For each set of GFF and HFPtr spectra with the same τ_i, a dotted line is drawn at the peak _S_0 intensities of Gly-1 (GFF) and Leu-7 (HFPtr). The GFF spectra were processed with 50 Hz Gaussian line broadening, the HFPtr spectra were processed with 250 Hz Gaussian line broadening, and baseline correction was applied to all spectra. For some of the HFPtr spectra, there is a small glitch at ∼178 ppm which is due to DC offset in the data. The total numbers of scans used to obtain each spectrum in panels a, b, c, d, e, f, g, and h are 10240, 10240, 10240, 8192, 77056, 80736, 102432, and 152064, respectively.

Figure 7

Dependence of fpCTDQBU of GFF Gly-1 13CO on 13C π pulse rf field: (a) (Δ_S_/S_0)i cor and (Δ_S/S_0)i sim vs. dephasing time; and (b) signal-to-noise ratio of (Δ_S/S_0)i cor and (Δ_S/S_0)i sim vs. dephasing time. The MAS frequency = 8000 Hz, δ_13C transmitter = 175.7 ppm, M = 336, and the constant-time = 84 ms. The symbol legend is: squares, cor, 10 kHz 13C π pulse rf field; crosses, sim, 10 kHz field; diamonds, cor, 43 kHz field; circles, sim, 43 kHz field. Uncertainties are displayed for the cor points in plot a and lines are drawn between like symbols in plot b. Each (Δ_S_/S_0)i cor was based on a (Δ_S/S_0)i exp determined from spectral integrations over a 0.5 ppm region centered at the Gly-1 13CO peak chemical shift. Each (Δ_S/_S_0)i cor value was determined using intensities from 4096 total (_S_0 + S_1) scans. The S_0_i sim and S_1_i sim were calculated with dCC = 42 Hz which yielded the best overall agreement between (Δ_S/S_0)i cor and (Δ_S/_S_0)i sim values. The value of k (Eq. 5) was set to give the best agreement at 10 kHz field between the sim and cor signal-to-noise ratios.

Figure 8

(a) fpCTDQBU (Δ_S_/S_0)i cor (open squares with error bars) and (Δ_S/S_0)i sim (crosses) vs. dephasing time for GFF Gly-1 13CO. The MAS frequency = 8000 Hz, the 13C π pulse rf field = 10 kHz, δ_13C transmitter = 175.7 ppm, M = 336, and the constant-time = 84 ms. Each (Δ_S_/S_0)i cor was based on a (Δ_S/S_0)i exp determined from spectral integrations over a 0.5 ppm region centered at the Gly-1 13CO peak chemical shift. Each (Δ_S/_S_0)i cor value for τ i = 16, 24, 32, 40, 48, 56, and 64 ms was determined using intensities from 20480 total (_S_0 + S_1) scans and the (Δ_S/S_0)i cor value for τ_i = 72 ms was determined using intensities from 16384 total scans. (b) A plot of χ_2 vs. dCC yields dCC = 42.3 ± 0.5 Hz which corresponds to rCC = 5.67 ± 0.02 Å for the Gly-1/Phe-3 labeled pair. The (Δ_S/S_0)i sim values in panel a were calculated with d = 42.3 Hz. (c) Plot of (Δ_S/S_0)i cor(open squares with error bars) and (Δ_S/S_0)i sim (crosses) calculated with dCC = 49 Hz which corresponds to rCC = 5.40 Å, the Gly-1 CO/Phe-3 CO distance in the X-ray structure. The (Δ_S/S_0)i cor were fitted to (Δ_S/S_0)i sim as a function of the D-GFF:unlabeled GFF ratio and the displayed (Δ_S/_S_0)i cor were calculated with the best-fit value of the ratio (1:71) for which _χ_2 = 7.

Figure 9

fpCTDQBU (Δ_S_/S_0)i cor and (Δ_S/S_0)i sim vs. dephasing time for the HFPtr-L7CF11N/LM3e sample. The MAS frequency = 8000 Hz, the 13C π pulse rf field = 10 kHz, δ13C transmitter = 178.4 ppm, M = 336, and the constant-time = 84 ms. The (Δ_S/S_0)i cor values are open squares with error bars. Each (Δ_S/S_0)i cor was based on a (Δ_S/_S_0)i exp determined from spectral integrations over a 1 ppm region centered at 173.4 ppm, the peak shift in the _S_0 spectra. The total (_S_0 + S_1) numbers of scans used to obtain the (Δ_S/S_0)i cor values for τi = 32, 48, and 64 ms were 154112, 161472, and 204864, respectively. The (Δ_S/_S_0)i sim values were calculated with two-spin and three-spin models in plots a and b, respectively. In plot a, the up triangles, crosses, and down triangles correspond to dCC = 10, 15 and 20 Hz, respectively and in plot b, they correspond to dCC = 8, 13, and 18 Hz, respectively. The best-fit values of dCC are ∼15 Hz and ∼13 Hz for the two-spin and three-spin models, respectively, and correspond to interstrand Leu-7 13CO-13CO distances of 8.0 Å and 8.4 Å. Reasonable upper limits on dCC in the two-spin and three-spin models are ∼20 Hz and ∼18 Hz respectively, and correspond to distances of 7.3 Å and 7.5 Å.

Figure 10

Structural models for strand arrangements of LM3e-associated HFPtr: (a) parallel in-register; (b) parallel with adjacent strands two-residues out-of-register; and (c) antiparallel with adjacent strand crossing between Phe-8 and Leu-9. The first sixteen residues of each strand are displayed, the 13CO labeled Leu-7 are colored red, and the 15N labeled Phe-11 are colored green.

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Conformational flexibility and strand arrangements of the membrane-associated HIV fusion peptide trimer probed by solid-state NMR spectroscopy - PubMed (original) (raw)