3D (13)C-(13)C-(13)C correlation NMR for de novo distance determination of solid proteins and application to a human alpha-defensin

Abstract

The de novo structure of an antimicrobial protein, human alpha-defensin 1 (HNP-1), is determined by combining a 3D (13)C-(13)C-(13)C (CCC) magic-angle spinning (MAS) correlation experiment with standard resonance assignment experiments. Using a short spin diffusion mixing time to assign intra-residue cross peaks and a long mixing time to detect inter-residue correlation peaks, we show that the 3D CCC experiment not only reduces the ambiguity of resonance assignment, but more importantly yields two orders of magnitude more long-range distances without recourse to existing crystal structures. Most of these distance constraints could not be obtained in a de novo fashion from 2D correlation spectra due to significant resonance overlap. Combining the distance constraints from the 3D CCC experiment and the chemical-shift-derived torsion angles, we obtained a de novo high-resolution NMR structure of HNP-1, with a heavy-atom RMSD of 3.4A from the crystal structure of the analogous HNP-3. The average energy of the minimum-energy ensemble is less than of 40kcal/mol. Thus, the 3D CCC experiment provides a reliable means of restraining the three-dimensional structure of insoluble proteins with unknown conformations.

Fig. 1

Pulse sequence for the 3D CCC MAS correlation experiment. Phase cycles are: ϕ₁ = 0 2, ϕ₂ = 1, ϕ₃= 0 0 1 1 2 2 3 3, ϕ₄ = 1 1 2 2 3 3 0 0, ϕ₅ = 3 3 0 0 1 1 2 2, ϕ₆ = 1 1 2 2 3 3 0 0, 3 3 0 0 1 1 2 2, ϕ₇ = 0 1 2 3 2 3 0 1, and receiver = 1 0 3 2 3 2 1 0, 3 2 1 0 1 0 3 2. Here 0 = +x, 1 = +y, 2 = −x, and 3 = −y.

Fig. 2

2D ¹³C-¹³C correlation spectrum of microcrystalline HNP-1 at a DARR mixing time of 100 ms. The spectrum was measured under 12 kHz MAS at 253 K. Inter-residue cross peaks are annotated in blue, and some intra-residue peaks are also assigned, in black.

Fig. 3

F2–F3 planes of the 3D CCC spectrum at various F1 frequencies. (a) F1=54.0 ppm, (b) F1=49.0 ppm, (c) F1=33.8 ppm, (d) F1=27.1 ppm. The amino acid spin systems are connected by dashed lines. All inter-residue cross peaks are assigned. The non-sequential inter-residue cross peaks are shown in brown while the sequential cross peaks are in black.

Fig. 4

Number of assigned peaks and their degeneracies from 2D and 3D ¹³C NMR. (a) Statistics of the 100 ms 2D CC spectrum. (b) Statistics of the 3D CCC spectrum, measured with mixing times of 20 ms and 100 ms. The 3D spectrum contains two orders of magnitude more resolved peaks than the 2D spectrum, with lower assignment degeneracies.

Fig. 5

2D contour map of the number of inter-residue cross peaks in HNP-1. A red diagonal line (red) indicating auto-correlation is drawn to distinguish from sequential correlation peaks.

Fig. 6

Three-dimensional structures of HNP-1 from SSNMR a n d HNP-3 from crystallography. (a) Average minimum-energy SSNMR structure obtained from long-distance constraints detected in the 2D CC spectrum. (b) Ensemble of 10 minimum-energy SSNMR structures obtained from long-distance constraints detected in the 3D CCC spectrum. (c) Average SSNMR structure of (b). All NMR structures shared the same torsion angle constraints obtained from chemical shifts. (d) Crystal structure of HNP-3. Yellow, purple, and red ribbons represent the three β-strands β1, β2, and β3. Black indicates loops. The three disulfide bonds are shown in orange.

Fig. 7

Effects of linewidths on the number of resolved cross peaks from 3D CCC and 2D DARR spectra. (a) F1=54.0 ppm plane of the 3D CCC spectrum, processed with Gaussian line broadening values of 0.5 ppm (left), 1.0 ppm (middle), and 2.0 ppm (right). The numbers of cross peaks are 47, 33, and 35, respectively. (b) 2D 100 ms DARR spectrum, processed with Gaussian line broadening values of 0.5 ppm, 1.0 ppm, and 2.0 ppm, from the left to the right. The numbers of cross peaks are 194, 126, and 84, respectively.