Modeling helical proteins using residual dipolar couplings, sparse long-range distance constraints and a simple residue-based force field

Abstract

We present a fast and simple protocol to obtain moderate-resolution backbone structures of helical proteins. This approach utilizes a combination of sparse backbone NMR data (residual dipolar couplings and paramagnetic relaxation enhancements) or EPR data with a residue-based force field and Monte Carlo/simulated annealing protocol to explore the folding energy landscape of helical proteins. By using only backbone NMR data, which are relatively easy to collect and analyze, and strategically placed spin relaxation probes, we show that it is possible to obtain protein structures with correct helical topology and backbone RMS deviations well below 4 Å. This approach offers promising alternatives for the structural determination of proteins in which nuclear Overha-user effect data are difficult or impossible to assign and produces initial models that will speed up the high-resolution structure determination by NMR spectroscopy.

Keywords: Dipolar waves; Helical proteins; NMR; Paramagnetic relaxation enhancements; Residual dipolar couplings; Simulated annealing; Structural determination.

Fig. 1

Angle definitions. The position of a helix is described by three angles as depicted in the left and center figures. The angles are the result of a “ZYZ” Euler angle convention treating counterclockwise rotations as positive. θ is the angle between the central axis of the helix and the z-axis of the reference frame starting from a zero point position where the central axis and z-axis are collinear. ϕ is the angle between the projection of the central axis and the positive x-axis of the reference frame. ρ is defined as the rotation about the central axis referenced to a zero point that occurs when the central axis is collinear with the z-axis, as shown in the center figure, and the first N–H bond vector in the helix lies along the positive x-axis. The figure at the right shows the angles defining the position of an N–H bond vector with respect to the central axis of the helix. β is the polar angle from the positive z’-axis of the helix axis frame, and α is the azimuthal angle in the x’y’-plane measured from the positive x’-axis

Fig. 2

a Kyte–Doolittle hydropathy plot for 1I6Z that was used to determine solvent-accessible sites for the positioning of site-directed spin labels. b Ribbon structure of 1I6Z showing the location of MTSSL spin labels along the protein backbone

Fig. 3

Comparing energy and RMSD for RDC, PRE and EPR constraint methods. a Force field plus a single set of RDCs (method 2); b force field plus five sets of RDCs (method 4); c force field plus PRE distance constraints (method 5); d force field plus EPR distance constraints (method 6); e force field plus PRE constraints and a single set of RDCs (method 7); f force field plus EPR constraints and a single set of RDCs (method 8). In each case, the six proteins are represented by circles (1I6Z), squares (1G2H), diamonds (1DV5), up-triangles (1J7O), left-triangles (1A6S) and down-triangles (2ABD). For each protein, data points represent an independent starting configuration

Fig. 4

Average RMSD (black bars) and δ_IA (gray bars) for each simulation type. Averages are taken over the six proteins studied. a Structures with the lowest RMSD; b structures giving the lowest energy for each protein. The simulations are as follows: the force field alone (1), the force field plus a single set of RDC constraints (2), the force field plus two sets of RDC constraints (3), the force field plus five sets of RDC constraints (4), the force field plus PRE distance constraints (5), the force field plus EPR constraints (6), the force field plus RDC/PRE constraints (7), the force field plus RDC/EPR constraints (8)

Fig. 5

Percentage of total structures with δ_IA (a) and RMSD (b) values above the following cutoffs: 25° or 6 Å (black bars), 10° or 4 Å (light gray bars), and 5° or 3 Å (dark gray bars). The methods are the same as described in the caption of Fig. 4

Fig. 6

Ribbon diagrams of the lowest energy structure found for each protein in the best three methods. Diagrams are colored, so the first helix is red, the second helix is blue, the third helix is green, and the fourth helix is yellow. Unstructured loop regions are shown in gray only for the target structure