Integrated description of protein dynamics from room-temperature X-ray crystallography and NMR

Abstract

Detailed descriptions of atomic coordinates and motions are required for an understanding of protein dynamics and their relation to molecular recognition, catalytic function, and allostery. Historically, NMR relaxation measurements have played a dominant role in the determination of the amplitudes and timescales (picosecond-nanosecond) of bond vector fluctuations, whereas high-resolution X-ray diffraction experiments can reveal the presence of and provide atomic coordinates for multiple, weakly populated substates in the protein conformational ensemble. Here we report a hybrid NMR and X-ray crystallography analysis that provides a more complete dynamic picture and a more quantitative description of the timescale and amplitude of fluctuations in atomic coordinates than is obtainable from the individual methods alone. Order parameters (S(2)) were calculated from single-conformer and multiconformer models fitted to room temperature and cryogenic X-ray diffraction data for dihydrofolate reductase. Backbone and side-chain order parameters derived from NMR relaxation experiments are in excellent agreement with those calculated from the room-temperature single-conformer and multiconformer models, showing that the picosecond timescale motions observed in solution occur also in the crystalline state. These motions are quenched in the crystal at cryogenic temperatures. The combination of NMR and X-ray crystallography in iterative refinement promises to provide an atomic resolution description of the alternate conformational substates that are sampled through picosecond to nanosecond timescale fluctuations of the protein structure. The method also provides insights into the structural heterogeneity of nonmethyl side chains, aromatic residues, and ligands, which are less commonly analyzed by NMR relaxation measurements.

Keywords: B factor; Lipari-Szabo; atomic displacement parameters; nuclear magnetic resonance.

Fig. 1.

Motions reported by the order parameter are represented in the qFit ensembles as the product of two components for the pair of atoms u and v. The angular component (blue) that is defined by θ_ij is sensitive to multiple states within the structural model, and the orthogonal component (red) accounts for the motion within each state. Note that the orthogonal component of the order parameter can differ between states when the atoms involved have different B factors in each state.

Fig. 2.

Correlation plots of experimental for methyl groups with values calculated from a single structure (A) and a multistate qFit model (B) at room temperature. Residues in green improve their fit to the experimental data in the qFit model, whereas residues in orange appear as outliers in both single and qFit models. Dashed lines indicate ±0.2 from ideal agreement. For comparison, C and D show order parameters for a crystal at cryogenic temperature, single and multistate qFit models, respectively. Residues in blue improve agreement in the qFit model, whereas residues in red appear to have the same order parameters at both temperatures.

Fig. 3.

(A) Conformational states observed for representative methyl-containing residues. The backbone is shown as black, side-chain bonds in blue, and bonds to the methyls are identified as green and red. (B) Anharmonic motions of the peptide planes in the central β-sheet of the room-temperature qFit multiconformer model.

Fig. 4.

Correlation plots of experimental NH S² with values calculated from a single structure (A) and a multistate qFit model (B) at room temperature. The residue in green improves in fit whereas orange points appear as outliers in both single and qFit models. Dashed lines indicate ±0.2 from ideal agreement. For comparison, C and D show order parameters for a crystal at cryogenic temperature, single and multistate qFit models, respectively. Residues in red appear to have the similar or increased order parameters at the cryogenic temperature.

Fig. 5.

Correlation plots of room-temperature and cryogenic and components of the order parameters for the methyl (A and B) and NH (C and D) order parameters. The line of best fit for the temperature dependence of (green) is shown in B and D.

Fig. 6.

Experimental and calculated S² values from the room-temperature qFit ensemble (bars). Order parameters from all side-chain methyls (red) and Trp indoles (blue) (A) and NH groups (B) in the protein. Residues that do not contain methyl or amide groups are indicated with gray shading, and residues whose calculated ¹⁵N order parameters are affected by lattice contacts are indicated in green.

Fig. 7.

Alternative models for Leu-8, 2F_oF_c electron density around L8 is contoured at 0.3σ (blue) and the difference density is contoured at 3σ (red). qFit model conformational ensemble in the on-rotamer g−g+ (χ₁, χ₂) conformation for all states (A). One state built in the on-rotamer g−t (B). One rotamer built in an off-rotamer g−t conformation (C). The backbone is shown as black, side-chain bonds in blue, and bonds to the methyls are identified as green and red.