Ultrahigh resolution protein structures using NMR chemical shift tensors

Abstract

NMR chemical shift tensors (CSTs) in proteins, as well as their orientations, represent an important new restraint class for protein structure refinement and determination. Here, we present the first determination of both CST magnitudes and orientations for (13)Cα and (15)N (peptide backbone) groups in a protein, the β1 IgG binding domain of protein G from Streptococcus spp., GB1. Site-specific (13)Cα and (15)N CSTs were measured using synchronously evolved recoupling experiments in which (13)C and (15)N tensors were projected onto the (1)H-(13)C and (1)H-(15)N vectors, respectively, and onto the (15)N-(13)C vector in the case of (13)Cα. The orientations of the (13)Cα CSTs to the (1)H-(13)C and (13)C-(15)N vectors agreed well with the results of ab initio calculations, with an rmsd of approximately 8°. In addition, the measured (15)N tensors exhibited larger reduced anisotropies in α-helical versus β-sheet regions, with very limited variation (18 ± 4°) in the orientation of the z-axis of the (15)N CST with respect to the (1)H-(15)N vector. Incorporation of the (13)Cα CST restraints into structure calculations, in combination with isotropic chemical shifts, transferred echo double resonance (13)C-(15)N distances and vector angle restraints, improved the backbone rmsd to 0.16 Å (PDB ID code 2LGI) and is consistent with existing X-ray structures (0.51 Å agreement with PDB ID code 2QMT). These results demonstrate that chemical shift tensors have considerable utility in protein structure refinement, with the best structures comparable to 1.0-Å crystal structures, based upon empirical metrics such as Ramachandran geometries and χ(1)/χ(2) distributions, providing solid-state NMR with a powerful tool for de novo structure determination.

Fig. 1.

Dipolar:CST correlation spectra for both ¹³Cα and ¹⁵N sites. Experimental spectrum is presented in black, with simulations in red. Ratios provided are the ratio of dipolar to CST evolution. Row two of A indicates ratio of ¹⁵N-¹³Cα dipolar:CST evolution. (A) Fit lineshapes for [¹H-¹³C]∶[¹³C CST] correlation spectra for lysines with different secondary structures are presented. K4 is located in a β-sheet, K28 in the α-helix, and K50 in a β-turn with an unusual positive value of ϕ. (b) Fit ensemble of [¹H-¹⁵N]∶[¹⁵N CST] correlation spectra. Fit is representative of limited variations of ¹⁵N tensors throughout GB1.

Fig. 2.

Analysis of [¹H-¹³C] dipolar:¹³C CST correlation spectra. (A) Fit α angles, defining orientation of each tensor element to the HC dipole, as a function of residue number. All angles over 180° were converted to their < 90° complement for clarity. Clear trends are observed where δ₁₁ is oriented within 20° of the dipole in β-strands but moves within 30° of the bond normal in the α-helix. δ₂₂ and δ₃₃ are near perpendicular to the HC bond in the β-sheet, while δ₁₁ and δ₂₂ reorient up to 80° in the α-helix. (B) Fit β angles defining the orientation of each tensor element to the NC bond vector. While overall variation of orientation is not as pronounced, there is a strong shift in the β₂ angle between helical and sheet conformations with a concerted, smaller adjustment of β₁ and β₃.

Fig. 3.

Agreement of fit ¹³Cα CST orientations with ab initio predictions and reconstructed Δσ^∗ values as a function of residue number. For most sites, fits are within 30° of the predicted orientation, within the maximum experimental error. (A) Experimental ¹³C CST orientations α₁, α₂, and α₃ (black dots) and (B) experimental orientations β₁, β₂, and β₃ (black dots) plotted against theoretical angles predicted by ab initio surfaces of Sun et al. assuming 2QMT crystal structure geometries. Blue lines indicate a deviation of ± 30°. (c) Reconstructed Δσ^∗ magnitudes. β-sheets sites range from 20–33 ppm and α-helical from -6–8 ppm, largely consistent with values reported by Tjandra and Bax.

Fig. 4.

GB1 structure calculated using all CST information, vector angles, TALOS dihedrals, and all distances. (A) The 10 lowest energy structures (out of 200) are presented in blue with 2QMT crystal structure represented in red; bbrmsd = 0.16 Å and agreement with 2QMT is 0.51 Å. (B) The lowest 10 energy structures are presented in CPK to illustrate the overall heavy atom order. The all heavy atom rmsd is 0.72 Å.

Fig. 5.

Amide chemical shift tensor analysis for protein GB1. (A) Principal elements of ¹⁵N tensor in the traceless representation compared to previously published slow-MAS data. Principal elements are presented in black with error bars corresponding to one standard deviation. Tensor values from previous work are presented in red. The rmsd between δ₁₁ elements from both datasets is 1.6 ppm, and 6 ppm for δ₂₂ and δ₃₃ values corresponding to a deviation of η of 0.13. (B) Chemical shift tensor elements plotted against isotropic chemical shift. The correlation reveals that the changes in the isotropic chemical shift result from largely correlated shifts of all three principal elements. R² for each element (δ₁₁, δ₂₂, δ₃₃) are 0.82, 0.75, and 0.62, respectively. (C) The angle β (a1) as a function of residue number. The angle β defines the orientation of the δ₁₁/δ_zz tensor element to the HN bond dipole.