Resonance assignment and three-dimensional structure determination of a human alpha-defensin, HNP-1, by solid-state NMR

Abstract

Human alpha-defensins [human neutrophil peptides (HNPs)] are immune defense mini-proteins that act by disrupting microbial cell membranes. Elucidating the three-dimensional (3D) structures of HNPs in lipid membranes is important for understanding their mechanisms of action. Using solid-state NMR (SSNMR), we have determined the 3D structure of HNP-1 in a microcrystalline state outside the lipid membrane, which provides benchmarks for structure determination and comparison with the membrane-bound state. From a suite of two-dimensional and 3D magic-angle spinning experiments, (13)C and (15)N chemical shifts that yielded torsion angle constraints were obtained, while inter-residue distances were obtained to restrain the 3D fold. Together, these constraints led to the first high-resolution SSNMR structure of a human defensin. The SSNMR structure has close similarity to the crystal structures of the HNP family, with the exception of the loop region between the first and second beta-strands. The difference, which is partially validated by direct torsion angle measurements of selected loop residues, suggests possible conformational variation and flexibility of this segment of the protein, which may regulate HNP interaction with the phospholipid membrane of microbial cells.

Figure 1

Amino acid sequence and disulfide bond connectivities of HNP-1.

Figure 2

An image of microcrystalline HNP-1. The average size of the crystals is ~20 μm.

Figure 3

1D CP-MAS spectra of microcrystalline U-¹³C, ¹⁵N-labeled HNP-1. (a) ¹³C spectrum, measured at 258 K at a ¹³C Larmor frequency of 225 MHz. (b) ¹⁵N spectrum, measured at 268 K at a ¹⁵N Larmor frequency of 60 MHz.

Figure 4

Representative regions of 2D ¹³C-¹³C correlation spectra of HNP-1. (a) Ala Cα-Cβ region from a 40 ms DARR spectrum. (b) Cys Cα-Cβ region from a 40 ms DARR spectrum. (c) Cys Cα-Cβ region from a 0.8 ms CM₅RR spectrum. Blue: positive intensities. Red: negative intensities. (d-f) CO-Cα/Cβ region. (d) 40 ms DARR spectrum, (e) 0.8 ms CM₅RR spectrum. (f) 1.5 ms CM₅RR. The sign of the cross peak intensities distinguishes the one-bond Gly CO-Cα peaks (red, indicating negative) from the two-bond Cys CO-Cβ peaks (blue, indicating positive).

Figure 5

F2-F3 strips of 3D NCACX (blue) and NCOCX (red) spectra for residues G18 to Q23, illustrating sequential resonance assignment. The strip width for the F2 dimension is 2.3 ppm. The F1 ¹⁵N chemical shifts for the strips are indicated on the right end of each strip.

Figure 6

HNP-1 (φ, ψ) torsion angles determined from solid-state NMR ¹³C and ¹⁵N chemical shifts (filled squares). The torsion angles of HNP-3 are shown for comparison (open squares and ribbons) (PDB accession code: 1DFN). Residues with significantly different torsion angles from the crystal structure values of HNP-3 are shaded.

Figure 7

Direct measurement of φ torsion angles by HNCH. (a) Aliphatic region of the 1D ¹³C spectrum. (b) Cα region (44-64 ppm) of the ¹³C spectrum, indicating the assignment of the resolved peaks and the φ_H torsion angles (φ_H+60°=φ) of each residue. For most residues the φ_H angles agree between TALOS and the crystal structure within ±10°. But for C10 and Y22, significant deviations exist. (c) Measured HNCH spectra with dipolar evolution time of 0 (S₀) and half a rotor period (S, shown in blue). The measured S.S₀ values are indicated along with calculated values. The 50.8-ppm peak has an S/S₀ value that is consistent with the chemical shift derived φ angles and not the HNP-3 value. (d) Calculated HNCH curves for the 50.8 ppm C10/A28 Cα peak. The A28 φ_H angle is fixed while the C10 φ_H angle is either 173° or 132°. The measured HNCH intensity at the middle of the rotor period is consistent with the TALOS φ_H angle but not the crystal structure value.

Figure 8

2D ¹³C-¹³C DARR spectra of HNP-1 with mixing times (a) 40 ms, (b) 100 ms, and (c) 200 ms. In (a), all peaks are assigned. In (b) and (c), only inter-residue and intra-residue multiple-bond correlations not observed in (a) are assigned. Red: non-sequential inter-residue correlations; blue: sequential correlations; black: intra-residue multiple-bond correlations.

Figure 9

¹⁵N-¹⁵N 2D PDSD spectrum with a mixing time of 3 s. Black and red assignments indicate sequential and non-sequential correlations, respectively.

Figure 10

SSNMR structures of HNP-1 compared with the X-ray crystal structure of HNP-3. (a) Eleven minimum energy SSNMR structures of HNP-1. (b) Average structure of (a). (c) Crystal structure of HNP-3. The disulfide bonds are shown in orange. (d) Comparison of the β1-β2 loop conformation between the crystal structure (red), the current solid-state NMR structure (blue), and the NMR structure with the additional distance restraints from the 3D CCC experiment (green).

Figure 11

2D ¹³C-¹H LG-CP spectrum to determine the mobility of microcrystalline HNP-1. (a) 2D spectrum, measured under 11 kHz MAS and 293 K. (b-k) 1D dipolar cross sections at selected chemical shifts. Most Cα and Cβ cross sections show rigid-limit values (after taking into account the LG scaling factor of 0.577), except for the 56.2-ppm cross section (c), which has a second coupling that is smaller than the rest.