Conformational dynamics and ligand binding in the multi-domain protein PDC109

Abstract

PDC109 is a modular multi-domain protein with two fibronectin type II (Fn2) repeats joined by a linker. It plays a major role in bull sperm binding to the oviductal epithelium through its interactions with phosphorylcholines (PhCs), a head group of sperm cell membrane lipids. The crystal structure of the PDC109-PhC complex shows that each PhC binds to the corresponding Fn2 domain, while the two domains are on the same face of the protein. Long timescale explicit solvent molecular dynamics (MD) simulations of PDC109, in the presence and absence of PhC, suggest that PhC binding strongly correlates with the relative orientation of choline-phospholipid binding sites of the two Fn2 domains; unless the two domains tightly bind PhCs, they tend to change their relative orientation by deforming the flexible linker. The effective PDC109-PhC association constant of 28 M(-1), estimated from their potential of mean force is consistent with the experimental result. Principal component analysis of the long timescale MD simulations was compared to the significantly less expensive normal mode analysis of minimized structures. The comparison indicates that difference between relative domain motions of PDC109 with bound and unbound PhC is captured by the first principal component in the principal component analysis as well as the three lowest normal modes in the normal mode analysis. The present study illustrates the use of detailed MD simulations to clarify the energetics of specific ligand-domain interactions revealed by a static crystallographic model, as well as their influence on relative domain motions in a multi-domain protein.

Figure 1. X-ray structure of BSP-A1.

(A) Sequence and associated secondary structure organization of PDC109. Cystine bridges are indicated by black lines. (B) Crystal structure of PDC109 . The N-terminal Fn2 domain (PDC109/a, residues 24–61) and the C-terminal Fn2 domain (PDC109/b, residues 69–109) are connected by a linker peptide (residues 62–68) shown in blue. The net charges are 1, 1, and 2 for PDC109/a, linker, and PDC109/b, respectively. Loop 1 (H41-L44) between 2 and 3 strands in PDC109/a and loop 2 (G87-M91) between the 2 and 3 strands in PDC109/b are denoted by green arrows .

Figure 2. Stereoviews of the homodimer crystal structure of PDC109 complexed with PhCs (PDB ID: 1h8p) .

(A) BSP-A1 protomer and (B) BSP-A2 protomer. Bound PhC molecules are shown as vdW spheres, aromatic sidechains at the PhC binding sites are shown in orange, and loops 1 and 2 that neighbor the binding sites and interact with PhC ligand are denoted in green .

Figure 3. Distances between the ligand and protein interaction sites in PDC109 domains.

Time series are shown for distances between the quaternary ammonium nitrogen of PhC and the center of geometry of six carbon atoms in indole rings of W47 (A), W93 (B), W58 (C), W106 (D); and between the average position of anionic PhC phosphoryl oxygens and the hydroxyl oxygens of Y30 (E), Y75 (F), Y54 (G), Y100 (H), Y60 (I), Y108 (J).

Figure 4. Ligand-binding site interactions.

Four snapshots from the MD simulations (AD) are compared with corresponding binding site conformations from the crystallographic protomers A and B (EH). Snapshots showing PhC phosphate moiety interactions with specific protein side-chains are arranged as follows: (A) Y30 and Y54 at 60 ns, (B) Y60 at 100 ns, (C) Y75 and Y100 at 150 ns, and (D) Y108 at 175 ns. Binding site residues Y30, Y54, W47, W58 (from PDC109/a), and Y75, Y100, W93, W106 (from PDC109/b) are shown in orange, residues Y60 and Y108 (from PDC109/b) are shown in purple, while bound PhC molecules are shown as charge-colored spheres. Residue labels are indicated in the crystal structure panels E and G.

Figure 5. Potentials of mean force (PMFs) between PhC and the individual PDC109 domains.

PMFs for PDC109/a (A) and PDC109/b (B) are displayed with standard deviations every 10 points. The insets display histogram of the number of sampling points with respect to the distance coordinate (scaled by 10).

Figure 6. Full MD results for PMF profiles.

PMFs for PDC109/a (A) and PDC109/b (B) are displayed, including those in overlapping regions of windows.

Figure 7. Histograms of number of samples as a function of atom-pair distances in the two minima of PMF.

The distance between W47/W58 of PDC109/a and the N atom of PhC in the first (2.5 Å3.5 Å) (A) and second (5.2 Å6.0 Å) (B) minima were calculated from the trajectory saved from the PMF calculation in the window 2.0 Å6.0 Å. The corresponding results for the separation between Y60 of PDC109/a and PhC phosphate group are shown in (C) and (D).

Figure 8. Snapshots of PDC109 during the MD simulations.

Structures of the PDC109-PhC complex at 200 ns (red) and 350 ns (blue) and of ligand-free PDC109 at 350 ns (green) are compared against the X-ray crystallographic structure (yellow) via a best-fit of PDC109/b .

Figure 9. Relative orientation and distance of the two domains of PDC109.

is the angle between two vectors, V and V in (A) , defined as displacements of C atom between W47 and W58 of PDC109/a and between W93 and W106 of PDC109/b, respectively. Fluctuations of are shown for ligand-free PDC109 in red (B) and for PDC109 complexed with PhC in blue (C). Fluctuations of center-to-center distance between two domains of PDC109 are in red (D) and of PhC-bound PDC109 are in blue (E).

Figure 10. RMSDs for (A) PDC109/a, (B) PDC109/b, (C) linker, and (D) the entire protein.

The time series for ligand-bound and free PDC109 is shown in blue and red, respectively.

Figure 11. Normal mode (NM) spectrum for ligand-free PDC109 ( = NM index).

The inset shows an expansion of the low frequency range.

Figure 12. Comparison of the first three normal modes and principal components.

Overlapping ribbon conformations for the three lowest normal modes are shown at t = 0 (red) and (blue) with the normalized eigenvector vibrational amplitudes scaled by a factor of 200. The normal mode index corresponds to specific vibrational frequencies as follows:  = 1 (hinge-bend), 0.80 cm;  = 2 (twist), 1.72 cm;  = 3 (tilt), 3.08 cm. Overlapping ribbon conformations for the three largest amplitude principal components (p = 1, 2, 3) are shown with the reference structure (red) as displacements scaled by a factor of 200 standard deviations along each principal component (blue) .

Figure 13. Projections of the principal components on the normal modes for PDC109.

(A) p = 1, (B) p = 2, and (C) p = 3. Insets show a magnified view for low frequency modes.

Figure 14. Influence of ligand binding on normal modes and principal components of PDC109.

Involvement coefficients () and thermal involvement coefficients () in the space spanned by normal modes are displayed in (A) and (B), respectively. The corresponding decompositions into the principal components are shown in (C) and (D). Insets show a magnified view for low mode frequencies. (E) Residue-wise comparison of amplitudes of PC1 eigenvectors at 0 and 180 (PC1, dashed line) and conformational difference (DIFF, solid line) between ligand-free and PhC-bound PDC109. At each residue, backbone atom coordinates were averaged out and the normalized difference between opposite components was scaled by a factor of 2.36 to obtain a best-fit conformational difference.