Filamin family members (A, B, and C) are composed of an actin-binding domain and subsequent 24 immunoglobulin(Ig)-like domains. Filamins have been shown to play an important role in maintaining the rheological properties of the actin cytoskeleton.1
Filamin molecules can cross-link actin fibrils into actin-networks. As mechanical proteins, filamins localize to the cortex, stress fibers, and muscle Z-line. Besides their critical roles as mechanical proteins, filamins are also involved in cellular response to stress. Filamin A has been postulated to respond to cues from the stress environment of cells, as reported from its involvement in cell-fate determination,2
activation of platelets,3-5
How could filamin A transduce the stress state of cells? Some studies report that filamin A turnover and ability to bind actin can be regulated through its interactions with calmodulin and calpain, proteins that are activated by changes in cellular calcium level.2,8,9
Calcium levels in turn fluctuate with cellular stress levels.10
Other studies suggest that filamin A’s role as a scaffold protein, binding over 70 proteins,11
is modulated by its quaternary structure, which in turn could be regulated by either physical or thermal stress.12,13
Based on these studies, we hypothesize that stress-induced conformational changes of filamin A play a direct role in signaling either by disrupting existing interactions or by introducing new interactions. Therefore, it is important to investigate possible stress-induced conformational changes that could play a functional role.
Atomic force microscope (AFM) has been commonly used to study a protein’s response to mechanical stress at the single molecule level. Several AFM studies on other proteins with Ig-like fold indicate that Ig domains can have force-induced metastable conformations. D. discoideum
filamin (ddFLN), a six Ig-like domain homolog of mammalian filamin, has been reported to have a metastable conformation in domain 4 (ddFLN4), which lacks β strands A and/or B.14-16
Ig-like domain of titin (TNI27) has been reported to have a force-induced17-19
stable intermediate that lacks β strand A.20
Fibronectin has been reported to have a forced-induced21
conformation in its fribronectin III domain 10 (FN-III10
) that lacks β strand A and B.22
Although AFM by itself has been used to detect the presence of intermediate state in these domains, complementary studies have been used to determine the atomistic conformation of the intermediate states. Mutational studies, such as amino acid inserts and
-value analysis, have been used to obtain structural insights into conformational changes in proteins under stress. These studies have been successful at proposing hypotheses about the conformation of intermediates of ddFLN414
as well as identifying critical residues for the mechanical stability of N-terminal β strands of TNI27.23
However, a limitation of these studies is that mutations can alter the conformational changes under stress. Thus, while existing biochemical and biophysical techniques are effective at generating hypotheses about stress-induced metastable states, they are lack detailed direct structural information about these conformations.
Being able to provide a direct atomistic picture of conformational changes under forces, computational modeling has been used in the study protein conformational changes under mechanical stress. However, the time-scales accessible by traditional molecular dynamic simulations are severely limited (~10-100 nanoseconds) and are not sufficient to study large-scale conformational dynamics of proteins (sub-microsecond to millisecond time-scales). Two major strategies have been employed to overcome this limitation. The first approach is to use simplified proteins models, in which residues are represented as one or several pseudo-atoms having empirical structural-based interactions.24,25
For example, West et al. used a simplified model to study the thermal and forced unfolding of TNI27, and found distinct unfolding pathways.26
The second approach often relies on simulating the system under extreme conditions, such as high temperatures or high forces, to promote rapid protein unfolding events.27
For example, Fowler et al., used traditional MD and a pulling protocol similar to AFM with pulling forces approximately two times the experimentally observed critical unfolding force (>=300 pN) for TNI27.19
Steered molecular dynamics was used by Lu et al. to understand the unfolding pathways of TNI27 with very high forces (~1,000 pN).28
Many other studies have been recently reviewed by Sotomayor et al.29
. One major limitation of these approaches is that unfolding pathways could be altered under extreme conditions.
To avoid the artifacts arising out of using unphysical simulation parameters and coarse-grained models in the study of force-induced filamin A conformational changes, we employ all-atom discrete molecular dynamics (DMD) simulations.30-35
DMD is similar to traditional MD in that it calculates the evolution of the coordinates of the system under study as a function of time and follows the same laws of conservation of energy and momentum as MD, but it affords much more rapid and diverse sampling. DMD solves the ballistic equations of motion rather than Newtonian ones, which reduces the simulation algorithm to an iterative search of the immediate collision events in the system, making it several orders of magnitude faster compared to MD. The reduction in simulation time achieved by DMD gives us the ability to study conformational dynamics over physiologically relevant time-scales. DMD has been shown to have a higher sampling efficiency than traditional molecular dynamics and has been used to study protein folding thermodynamics and protein aggregation.36
All-atom DMD features a transferable force field and has been successfully used to fold several small proteins ab initio
(<60 amino acids)35
and to investigate the conformational dynamics of superoxide dismutase.37
To study force-induced unfolding of filamin A using DMD we need an atomistic structure of filamin A. However, structures of only five of the 24 Ig-like domains of filamin A have been experimentally determined.12,38-40
Hence, in this study we use the all-atom structure of filamin A derived from comparative modeling in an earlier study (Kesner, et al. submitted
). The average sequence similarity of all the Ig-like domains is > 40%. Therefore, we expect the homology-derived structures to be close to their native structures.41
In that study we observed that there are two classes of Ig-like domains in filamin: six-stranded (lacking the N-terminal strand) and canonical seven-stranded. Domains 16, 18, and 20 are six stranded. In a recent crystal structure of domains 19-21 it was observed that the putative first strand of domain 20 interacts with domain 21 instead of forming a β sheet with the second strand. This interaction has been proposed to inhibit domain 21’s interaction with other binding partners.12
Based on this quaternary structure it is possible that β strand A of an Ig-like domain could form complementary strands with adjacent Ig-like domains. Hence, we hypothesize that unraveling of the N-terminal strand of Ig-like domains could initiate inter-domain interactions through that strand. We study the six-stranded Ig-like domains from two perspectives. First, we compare domains 16 and 18 to that of domain 20 (whose structure has been determined)12
to study if the six-stranded domains have similar physical characteristics. Second, we compare these domains to the seven-stranded domains to understand the role of the missing strand.
For each of the 24 Ig-like domains that make up filamin, we perform constant-force simulations by applying a set of physiologically relevant ranges of forces (0-315 pN).42
Based on these systematic DMD simulations, we estimate the critical force of unfolding of each individual domain and characterize the conformational changes under mechanical stress. We find that the critical forces are heterogeneous among the 24 domains due to the sequence differences. We also find that there is no significant difference between the critical unfolding forces of domains with and without N-terminal strands. We find that under small forces up to ~35 pN, which correspond to the physiological forces43
, most Ig-like domains remain in the native states. As the force increases up to the intermediate force levels of ~70 pN, large conformational changes takes place. Interestingly, Ig-like domains feature a common initial conformational change, where the first β strand unfurls. Domains that lack their N-terminal β strands appear similar to one another, maintaining their native-like conformation under low forces. At intermediate forces, they unfold to a heterogeneous population of intermediate states similar to seven-stranded domains.
Additionally, the effect of temperature versus mechanical stress on unfolding pathways has been the subject of much debate.44
The general hypothesis is that thermally induced and force-induced unfolding pathways are independent from one another since the thermal fluctuations are exerted on the protein globally, while the effect of mechanical forces is localized and non-homogeneous. To examine whether the thermally and force-induced unfolding of filamin Ig-like domains are different from each other, we also perform thermal unfolding of three Ig-like domains, 14, 21, and 24. Interestingly, we find that thermally induced unfolding shares the same initial unfolding intermediate state as forced unfolding, where the first strand unfolds. This observation suggests that filamin evolved to feature the intermediate state of unfolded N-terminal strand(s). Given the observation that the putative first strand of domain 20 interacts with domain 21 instead of forming a β sheet with the second strand, we propose that N-terminal strand may act as a conformational switch that unfolds under stressed physiological conditions leading to exposure of cryptic binding sites, removing of native binding sites, and modulating the quaternary structure of domains.