Interplay of mechanical and binding properties of Fibronectin type I

Abstract

Fibronectins (FNs) are a major component of the extracellular matrix (ECM), and provide important binding sites for a variety of ligands outside and on the surface of the cell. Similar to other ECM proteins, FNs are consistently subject to mechanical stress in the ECM. Therefore, it is important to study their structure and binding properties under mechanical stress and understand how their binding and mechanical properties might affect each other. Although certain FN modules have been extensively investigated, no simulation studies have been reported for the FN type I (Fn1) domains, despite their prominent role in binding of various protein modules to FN polymers in the ECM. Using equilibrium and steered molecular dynamics simulations, we have studied mechanical properties of Fn1 modules in the presence or the absence of a specific FN-binding peptide (FnBP). We have also investigated how the binding of the FnBP peptide to Fn1 might be affected by tensile force. Despite the presence of disulfide bonds within individual Fn1 modules that are presumed to prevent their extension, it is found that significant internal structural changes within individual modules are induced by the forces applied in our simulations. These internal structural changes result in significant variations in the accessibility of different residues of the Fn1 modules, which affect their exposure, and, thus, the binding properties of the Fn1 modules. Binding of the FnBP appears to reduce the flexibility of the linker region connecting individual Fn1 modules (exhibited in the form of reduced fluctuation and motion of the linker region), both with regard to bending and stretching motions, and hence stabilizes the inter-domain configuration under force. Under large tensile forces, the FnBP peptide unbinds from Fn1. The results suggest that Fn1 modules in FN polymers do contribute to the overall extension caused by force-induced stretching of the polymer in the ECM, and that binding properties of Fn1 modules can be affected by mechanically induced internal protein conformational changes in spite of the presence of disulfide bonds which were presumed to completely abolish the capacity of Fn1 modules to undergo extension in response to external forces.

Fig. 1

a Major structural features of Fn1 modules. An ¹Fn1 module (purple) with its five β strands, as well as a portion of the FnBP peptide (red) are shown. Two disulfide bonds connect β-strands A and D, and D and E, respectively. b A representative simulation system with ¹Fn1²Fn1 drawn in purple, the FnBP peptide in red and water in transparent surface representation. The binding peptide forms an additional β-strand with both ¹Fn1 and ²Fn1 modules. In pulling simulations, one end of ¹Fn1²Fn1 was fixed, while the other end was coupled to a constraint moving at a constant velocity. The water box is large enough to accommodate the stretching of the protein under tensile loading. Equilibrium simulations used a very similar setup, though with a smaller water box

Fig. 2

Stress-induced extension of ¹Fn1²Fn1. The snapshots are taken at a t = 0 ns, b t = 3 ns, c t = 9 ns, and d t = 11 ns of the pulling simulation. The extensions of the individual modules and that of the linker (the three distinct regions defined in d) are plotted in (e), along with the corresponding applied force, as functions of time. The disulfide bonds and a key salt bridge in the linker region are also shown

Fig. 3

Change in accessibility of residues in ¹Fn1²Fn1 upon tensile loading. The average surface accessibility (c) has been calculated using two complementary methods (resulting in similar conclusions): the number of water molecules within 3.5 Å of individual residues or the solvent accessible surface area (SASA). Both values are averaged over a period of 100 ps, at t = 11 ns and at t = 0 ns of the pulling simulation, and the differences are plotted in (c) and used to color the ¹Fn1²Fn1 molecule in front (a) and back (b) views, with residues that become exposed in blue and those that become buried in red

Fig. 4

Linker rigidification induced by peptide binding. Shown are 100 frames of backbone structures of the ¹Fn1²Fn1 taken from the 10 ns of the equilibrium simulation with the ¹Fn1 modules used to align the frames. The results are shown for the peptide-free system (left), as well as for the peptide-bound system (right). The time interval between the frames is 100 ps, and ¹Fn1²Fn1 is colored according to the time steps, with the first step in red and the last one in blue

Fig. 5

Stress-induced extension of ¹Fn1²Fn1 (purple) in the presence of the FnBP peptide (red). Snapshots of the FnBP-¹Fn1²Fn1 complex are taken at a t = 0 ns, b t = 3 ns, c t = 7 ns, and d t = 10 ns from one of the four pulling simulations; the extensions of the individual modules and the linker (defined in Fig. 2) are shown in e, along with the corresponding applied force, as functions of time

Fig. 6

Force-extension profiles of ¹Fn1²Fn1. Applied forces as functions of the length of ¹Fn1²Fn1 during the pulling of ¹Fn1²Fn1 in the absence (dotted line) and four independent simulations in the presence of the FnBP peptide (solid lines)