Force-dependent isomerization kinetics of a highly conserved proline switch modulates the mechanosensing region of filamin

Abstract

Proline switches, controlled by cis-trans isomerization, have emerged as a particularly effective regulatory mechanism in a wide range of biological processes. In this study, we use single-molecule mechanical measurements to develop a full kinetic and energetic description of a highly conserved proline switch in the force-sensing domain 20 of human filamin and how prolyl isomerization modulates the force-sensing mechanism. Proline isomerization toggles domain 20 between two conformations. A stable cis conformation with slow unfolding, favoring the autoinhibited closed conformation of filamin's force-sensing domain pair 20-21, and a less stable, uninhibited conformation promoted by the trans form. The data provide detailed insight into the folding mechanisms that underpin the functionality of this binary switch and elucidate its remarkable efficiency in modulating force-sensing, thus combining two previously unconnected regulatory mechanisms, proline switches and mechanosensing. O wing to its ring structure (Fig. 1A), the peptidyl-prolyl bond is special among the peptide bonds in that it can populate slowly exchanging cis and trans conformations with similar probability (1). Therefore, proline residues can drastically affect the kinetics of folding (2, 3) as well as conformational changes of proteins (4, 5). Although most native protein conformations require trans prolines, there are a number of examples where native cis prolines have been reported (1,(6)(7)(8). In a few cases, both cis and trans conformations can be accommodated in the folded state thus leading to two distinct native states (9-11). Beyond its importance for protein folding, peptidyl-prolyl isomerization has also been discussed as a molecular timer switch regulating different functional conformations of proteins (5, 12).Filamins are essential actin cross-linkers as well as mechanosignaling hubs in the cytoskeleton. Recently, a special conformation in the rod 2 region of filamin has been shown to act as a force-sensing region mediating the interaction to transmembrane receptors like integrins or the von Willebrand receptor glycoprotein Ib (GPIb) (13-15) (Fig. 1B). Lad et al. (13) have shown that the first β-strand of domain 20 (FLNa20) of human filamin A binds to the subsequent domain 21 (FLNa21) thus covering and autoinhibiting its transmembrane receptor-binding site forming the domain pair shown in Fig. 1B. This domain pair has a mechanosensor function (15, 16). Mechanical force transmitted through the cytoskeleton leads to the opening of the domain pair and an increase in receptor-binding affinity of up to 17-fold (15). The crystal structure of FLNa20 exhibits a native cis proline just in front of the last β-strand (Fig. 1B, Inset). Interestingly, a multiple-sequence alignment shows that this cis proline residue is strictly conserved in all available 3D structures of filamin Ig-like domains (Fig. S1).Although proline isomerization has been investigated in numerous bulk studies, the structural heterogeneity caused by a native-state isomerization...

Prune belly syndrome in surviving males can be caused by Hemizygous missense mutations in the X-linked Filamin A gene

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Optimizing the calculation of energy landscape parameters from single-molecule protein unfolding experiments

“…A recent review provides details of studies that have employed the Zhurkov-Bell model to extract parameters of the unfolding energy landscape of a protein from experimental data on polyproteins [43]. In addition, since that review, 12 additional studies have used the Zhurkov-Bell model to extract information from force spectroscopy experiments [15,[44][45][46][47][48][49][50][51][52][53][54]. Given the prevalence of the Zhurkov-Bell model in force spectroscopy experiments, it is interesting to examine its application and accuracy in more detail.…”

Unforgettable force – crosstalk and memory of mechanosensitive structures

“…How do other types of cell adhesion complexes such as tight junctions, desmosomes or hemidesmosomes process mechanical information, and what are relevant length and time scales that characterize the underlying molecular processes? The development of highly sensitive technologies such as single-molecule force spectroscopy (Rognoni et al, 2014;Grison et al, 2017), atomic force microscopy (Kong et al, 2009;Kong et al, 2013;Strohmeyer et al, 2017), and molecular tension sensors (Austen et al, 2015;Ringer et al, 2017a) will certainly be helpful to develop a quantitative understanding of molecular force transduction in cells. It will be crucial, however, to improve the applicability of these techniques to investigate processes of molecular force transduction in complex but physiologically more relevant settings, specifically mammalian tissues.…”