Comparison of the early stages of forced unfolding for fibronectin type III modules

Abstract

The structural changes accompanying stretch-induced early unfolding events were investigated for the four type III fibronectin (FN-III) modules, FN-III 7, FN-III8, FN-III9, and FN-III10 by using steered molecular dynamics. Simulations revealed that two main energy barriers, I and II, have to be overcome to initiate unraveling of FN-III's tertiary structure. In crossing the first barrier, the two opposing ␤-sheets of FN-III are rotated against each other such that the ␤-strands of both ␤-sheets align parallel to the force vector (aligned state). All further events in the unfolding pathway proceed from this intermediate state. A second energy barrier has to be overcome to break the first major cluster of hydrogen bonds between adjacent ␤-strands. Simulations revealed that the height of barrier I varied significantly among the four modules studied, being largest for FN-III 7 and lowest for FN-III10, whereas the height of barrier II showed little variation. Key residues affecting the mechanical stability of FN-III modules were identified. These results suggest that FN-III modules can be prestretched into an intermediate state with only minor changes to their tertiary structures. T he mechanical properties of fibronectin (FN) and its extracellular matrix fibers have drawn considerable attention recently. FN is a 450-to 500-kDa dimeric protein composed of more than 20 modules per monomer. FN primarily consists of three structurally homologous modules, FN-I, FN-II, and FN-III, presented schematically in Fig. 1A. Cells assemble FN into fibrillar networks that provide mechanical stability to the extracellular matrix and connective tissue. Furthermore, cells have been found to mechanically stretch FN fibers by up to four times their normal length (1). This wide extension range is attributed to the unfolding of individual FN modules under tension, which are presumed to refold when tension is released (2, 3). The FN-III modules are of particular interest because they contain several cell recognition sites, including the synergy site on FN-III 9 and an RGD loop on FN-III 10 (4). Beyond its role in FN, the FN-III motif is structurally ubiquitous and found in 2% of all animal proteins (5). The FN-III motif is a Greek key ␤-sandwich (Fig. 1B) with four ␤-strands in the upper sheet and three in the lower sheet (6).Mechanical forces, in addition to chemical cues, have been implicated in playing a critical role in regulating the functional states of FN. Binding sites buried within the protein in its native state, also referred to as cryptic sites, can be exposed by denaturation, or potentially through mechanical stretching (7,8). Forced unfolding of the FN-III 10 module also has been suggested to modify exposure of the RGD loop, thus influencing FNЈs accessibility to integrins (9). Mechanical forces, furthermore, are essential in initiating FN fibril assembly (10), potentially through exposure of cryptic sites, and͞or by swapping complementary ␤-strands of partially unfolded FN-III modules during refolding (11,12). The functional ...

Cell mechanics and mechanotransduction: pathways, probes, and physiology

“…As discussed above, cryptic binding sites on fibronectin can be exposed by force-induced lengthening of the molecule, leading to the formation of fibrils. This process has been extensively studied by experiment and molecular dynamic simulation (18,29), and the results of these studies show that forces of 3-5 pN are sufficient to unfold individual fibronectin modules and that a force of 5 pN is sufficient to stretch the molecule to five times its initial length (29,87). These levels of force are comparable to those estimated above for the conformational changes that could lead to intracellular mechanotransduction.…”

Phage-based molecular probes that discriminate force-induced structural states of fibronectin in vivo

“…In particular, forces emanating from contractile cells such as pathogenic myofibroblasts have been hypothesized to partially unfold ECM proteins like Fn, thus engaging/disengaging theorized integrin switches (25). Despite considerable in silico and in vitro evidence for the extensibility of Fn within fibers and Fn type III domain unfolding (26)(27)(28), there is still no direct evidence that such molecular events occur in vivo, a fact that perpetuates the debate regarding the validity of such observations. To fill this void, we combined controlled Fn fiber straining with random peptide phage display to isolate peptide-based molecular probes capable of discriminating Fn fibers under relaxed and strained conditions.…”

Fibronectin extension and unfolding within cell matrix fibrils controlled by cytoskeletal tension

“…The increase in energy transfer after treatment with cytoD suggests that FnIII modules refold when tension is released. Atomic force microscopy and steered molecular dynamics simulations have shown that forced unfolding of a single FnIII module results in an extended peptide about 10 times the length of the native module (13,28). FnIII modules lack internal disulfide bonds, making them mechanically less stable than FnI or FnII modules.…”