The mechano-sensing role of the unique SH3 insertion in plakin domains revealed by Molecular Dynamics simulations

Abstract

The plakin family of proteins, important actors in cross-linking force-bearing structures in the cell, contain a curious SH3 domain insertion in their chain of spectrin repeats (SRs). While SH3 domains are known to mediate protein-protein interactions, here, its canonical binding site is autoinhibited by the preceding SR. Under force, however, this SH3 domain could be released, and possibly launch a signaling cascade. We performed large-scale force-probe molecular dynamics simulations, across two orders of magnitude of loading rates, to test this hypothesis, on two prominent members of the plakin family: desmoplakin and plectin, obligate proteins at desmosomes and hemidesmosomes, respectively. Our simulations show that force unravels the SRs and abolishes the autoinhibition of the SH3 domain, an event well separated from the unfolding of this domain. The SH3 domain is free and fully functional for a significant portion of the unfolding trajectories. The rupture forces required for the two proteins significantly decrease when the SH3 domain is removed, which implies that the SH3 domain also stabilizes this junction. Our results persist across all simulations, and support a force-sensing as well as a stabilizing role of the unique SH3 insertion, putting forward this protein family as a new class of mechano-sensors.

Figure 1

Plakin family proteins contain a curious SH3 domain insertion. (a) The three systems we study using force-probe molecular dynamics: (b) four spectrin repeats and an SH3 domain (only desmoplakin). (c) two spectrin repeats and an SH3 domain (desmoplakin and plectin), and (d) only two spectrin repeats with the SH3 domain removed (desmoplakin and plectin). The blue surface denotes the SH3-SR4 interface for cases. (b,c) and the SR4-SR5 interface for case. (d) shown are snapshots from MD simulations on desmoplakin, starting from PDB code 3R6N. (e) The assignment of helices 4A-C and 5A-C in desmoplakin with the SH3 domain removed, for clarity. The structure is colored by residue index (N-terminal pure red, C-terminal pure green) and the labels of the helices are placed close to the N-terminus of all helices.

Figure 2

Unfolding events observed in all single force-probe simulations. Boxes extend from the start to the end of the loss in contact area, either within a single domain (SR3, SR4, SR5, SR6, and SH3) or at inter-domain interfaces (SR3-SR4, SH3-helix 4C5A, and SR5-SR6), see Methods for details. Each set of horizontally aligned boxes correspond to one FPMD trajectory. The unfolding events are represented as a function of inter-spring distance, which is very close to the net increase of the protein end-to-end distance. Three sets of systems are presented: (a) four spectrin repeats and an SH3 domain (only desmoplakin (20 runs)), (b) two spectrin repeats and an SH3 domain (left: desmoplakin and right: plectin (33 runs each)), and (c) only two spectrin repeats with the SH3 domain removed (left: desmoplakin and right: plectin (33 runs each)). The purple area in subplot a. shows that the domains SR3, SR6 and the associated interfaces are first to unfold (until vt $\approx 60$ nm), while the green areas for subplots a, b show the minimum active area, i.e., the region of the unfolding trajectories after the SH3–4C5A interface is lost and before the beginning of the SH3 domain unfolding.

Figure 3

Unfolding forces for desmoplakin (DESP) and plectin (PLEC). (a) The force profiles (smoothed with a Gaussian of standard deviation of 1 ns) of desmoplakin (red) and plectin (blue) at the pulling velocity of 0.1 nm/ns. The dots show the highest force within the first peak, i.e., the considered rupture force. We mark with green boxes the region of the pulling trajectories in which the proteins are active (the SH3 domain is freed but fully folded). (b) The average rupture forces of desmoplakin and plectin, with and without the SH3 domain, at each pulling velocity. The bars show the standard deviation at each velocity and the line is the Bell model (see Eq. 1) fit of all four considered systems. The distance between the y-ticks is 400 pN.

Figure 4

The two different ways the SH3-SR4 interface can be lost: (a) “shearing” and (b) “tearing” (representative snapshots with red = SR4, green = SR5, blue surface = residues at helix 4 C in contact with the SH3 domain, and orange highlight: the N-terminal domains of helix 5 C, whose unfolding triggers the tilt and therefore the “tearing” topology), and (c) the maximum tilt angle observed for the 33 unfolding trajectories of desmoplakin (left) and plectin (right). Red points refer to force profiles with three distinct peaks and blue points show a lack of a middle peak. See also the ROC curve based on this plot in the Supplementary Information.

Figure 5

The various unfolding mechanisms exhibited by desmoplakin (red numbers) and plectin (blue numbers). The SH3 domain is represented as a gray box, while SR4 and SR5 are shown in red and green, respectively. Solid arrows show force-resistant unfolding events, while dashed arrows force-compliant ones and an orange star represents the rupture event that activates the SH3 domain. Plectin samples a more diverse set of unfolding sequences, including about a quarter of them with an early activation of the SH3 domain followed by a forceless unfolding.