Javad Golji

Molecular Mechanics of the α-Actinin Rod Domain: Bending, Torsional, and Extensional Behavior

α-Actinin is an actin crosslinking molecule that can serve as a scaffold and maintain dynamic actin filament networks. As a crosslinker in the stressed cytoskeleton, α-actinin can retain conformation, function, and strength. α-Actinin has an actin binding domain and a calmodulin homology domain separated by a long rod domain. Using molecular dynamics and normal mode analysis, we suggest that the α-actinin rod domain has flexible terminal regions which can twist and extend under mechanical stress, yet has a highly rigid interior region stabilized by aromatic packing within each spectrin repeat, by electrostatic interactions between the spectrin repeats, and by strong salt bridges between its two anti-parallel monomers. By exploring the natural vibrations of the α-actinin rod domain and by conducting bending molecular dynamics simulations we also predict that bending of the rod domain is possible with minimal force. We introduce computational methods for analyzing the torsional strain of molecules using rotating constraints. Molecular dynamics extension of the α-actinin rod is also performed, demonstrating transduction of the unfolding forces across salt bridges to the associated monomer of the α-actinin rod domain.

A Molecular Dynamics Investigation of Vinculin Activation

Vinculin activation plays a critical role in focal adhesion initiation and formation. In its native state, vinculin is in an autoinhibitory conformation in which domain 1 prevents interaction of the vinculin tail domain with actin by steric hindrance. Once activated, vinculin is able to interact with both actin and talin. Several hypotheses have been put forth addressing the mechanisms of vinculin activation. One set of studies suggests that vinculin interaction with talin is sufficient to cause activation, whereas another set of studies suggests that a simultaneous interaction with several binding partners is necessary to achieve vinculin activation. Using molecular-dynamics (MD) simulations, we investigate the mechanisms of vinculin activation and suggest both a trajectory of conformational changes leading to vinculin activation, and key structural features that are likely involved in stabilizing the autoinhibited conformation. Assuming that the simultaneous interaction of vinculin with both actin and talin causes a stretching force on vinculin, and that vinculin activation results from a removal of steric hindrance blocking the actin-binding sites, we simulate with MD the stretching and activation of vinculin. The MD simulations are further confirmed by normal-mode analysis and simulation after residue modification. Taken together, the results of these simulations suggest that bending of the vinculin-binding-site region in vinculin away from the vinculin tail is the likely trajectory of vinculin activation.

Vinculin Activation Is Necessary for Complete Talin Binding

Focal adhesions are critical to a number of cellular processes that involve mechanotransduction and mechanical interaction with the cellular environment. The growth and strengthening of these focal adhesions is dependent on the interaction between talin and vinculin. This study investigates said interaction and how vinculin activation influences it. Using molecular dynamics, the interaction between talins vinculin binding site (VBS) and vinculins domain 1 (D1) is simulated both before and after vinculin activation. The simulations of VBS binding to vinculin before activation suggest the proximity of the vinculin tail to D1 prevents helical movement in D1 and thus prevents binding of VBS. In contrast, interaction of VBS with activated vinculin shows the possibility of complete VBS insertion into D1. In the simulations of both activated and autoinhibited vinculin where VBS fails to fully bind, VBS demonstrates significant hydrophobic interaction with surface residues in D1. These interactions link VBS to D1 even without its proper insertion into the hydrophobic core. Together these simulations suggest VBS binds to vinculin with the following mechanism: 1), VBS links to D1 via surface hydrophobic interactions; 2), vinculin undergoes activation and D1 is moved away from the vinculin tail; 3), helices in D1 undergo conformational change to allow VBS binding; and 4), VBS inserts itself into the hydrophobic core of D1.

Phosphorylation Primes Vinculin for Activation

Vinculin phosphorylation has been implicated as a potential mechanism for focal adhesion growth and maturation. Four vinculin residues-Y100, S1033, S1045, and Y1065-are phosphorylated by kinases during focal adhesion maturation. In this study, phosphorylation at each of these residues is simulated using molecular dynamics models. The simulations demonstrate that once each phosphorylated vinculin structure is at equilibrium, significant local conformational changes result that may impact either vinculin activation or vinculin binding to actin and PIP2. Simulation of vinculin activation after phosphorylation shows that the added phosphoryl groups can prime vinculin for activation. It remains to be seen if vinculin can be phosphorylated at S1033 in vivo, but these simulations highlight that in the event of a S1033 phophorylation vinculin will likely be primed for activation.

A Molecular Trajectory of α-Actinin Activation

The mechanisms by which living cells respond to mechanical stimuli are not yet fully understood. It has been suggested that mechanosensing proteins play an important role in mechanotransduction because their binding affinities are directly affected by the external stress. α-Actinin is an actin cross-linker and may act as a mechanosensor in adhesion sites. Its interaction with vinculin is suggested to be mechanically regulated. In this study, the free energy of activation is explored using the umbrella sampling method. An activation trajectory is generated in which α-actinins vinculin-binding site swings out of the rod domain, leading to approximately an 8 kcal/mol free energy release. The activation trajectory reveals several local and global conformational changes along the activation pathway accompanied by the breakage of a number of key interactions stabilizing the inhibited structure. These results may shed light on the role of α-actinin in cellular mechanotransduction and focal adhesion formation.

The interaction of vinculin with actin.

Vinculin can interact with F-actin both in recruitment of actin filaments to the growing focal adhesions and also in capping of actin filaments to regulate actin dynamics. Using molecular dynamics, both interactions are simulated using different vinculin conformations. Vinculin is simulated either with only its vinculin tail domain (Vt), with all residues in its closed conformation, with all residues in an open I conformation, and with all residues in an open II conformation. The open I conformation results from movement of domain 1 away from Vt; the open II conformation results from complete dissociation of Vt from the vinculin head domains. Simulation of vinculin binding along the actin filament showed that Vt alone can bind along the actin filaments, that vinculin in its closed conformation cannot bind along the actin filaments, and that vinculin in its open I conformation can bind along the actin filaments. The simulations confirm that movement of domain 1 away from Vt in formation of vinculin 1 is sufficient for allowing Vt to bind along the actin filament. Simulation of Vt capping actin filaments probe six possible bound structures and suggest that vinculin would cap actin filaments by interacting with both S1 and S3 of the barbed-end, using the surface of Vt normally occluded by D4 and nearby vinculin head domain residues. Simulation of D4 separation from Vt after D1 separation formed the open II conformation. Binding of open II vinculin to the barbed-end suggests this conformation allows for vinculin capping. Three binding sites on F-actin are suggested as regions that could link to vinculin. Vinculin is suggested to function as a variable switch at the focal adhesions. The conformation of vinculin and the precise F-actin binding conformation is dependent on the level of mechanical load on the focal adhesion.

The Talin Dimer Structure Orientation Is Mechanically Regulated

Formation of a stable cell-substrate contact can be regulated by mechanical force, especially at the focal adhesion. Individual proteins that make up the focal adhesions, such as talin, can exhibit mechanosensing. We previously described one mode of talin mechanosensing in which the vinculin-binding site of talin is exposed after force-induced stretch of a single talin rod domain. Here, we describe a second mode of talin mechanosensing in which the talin dimer itself can adopt different orientations in response to mechanical stimulation. Using molecular dynamics models, we demonstrate that the C-terminus region of the talin dimer is flexible mainly at the linker between the dimerization helices and the nearby actin-binding helical bundle. Our molecular dynamics simulations reveal two possible orientations of the talin dimer at its C-terminus. The extracellular matrix (ECM)-bound integrins cross-linked by talin can be forced apart leading to an elongated orientation of the talin dimer, and the ECM-bound integrins can be forced together by the ECM producing a collapsed orientation of the talin dimer. Formation of the elongated orientation is shown to be more favorable. Switching between the two talin dimer orientations constitutes a mode of mechanosensing.

Dynamic Regulation of α-Actinin’s Calponin Homology Domains on F-Actin

α-Actinin is an essential actin cross-linker involved in cytoskeletal organization and dynamics. The molecular conformation of α-actinins actin-binding domain (ABD) regulates its association with actin and thus mutations in this domain can lead to severe pathogenic conditions. A point mutation at lysine 255 in human α-actinin-4 to glutamate increases the binding affinity resulting in stiffer cytoskeletal structures. The role of different ABD conformations and the effect of K255E mutation on ABD conformations remain elusive. To evaluate the impact of K255E mutation on ABD binding to actin we use all-atom molecular dynamics and free energy calculation methods and study the molecular mechanism of actin association in both wild-type α-actinin and in the K225E mutant. Our models illustrate that the strength of actin association is indeed sensitive to the ABD conformation, predict the effect of K255E mutation--based on simulations with the K237E mutant chicken α-actinin--and evaluate the mechanism of α-actinin binding to actin. Furthermore, our simulations showed that the calmodulin domain binding to the linker region was important for regulating the distance between actin and ABD. Our results provide valuable insights into the molecular details of this critical cellular phenomenon and further contribute to an understanding of cytoskeletal dynamics in health and disease.

Focal Adhesion Mechanotransduction: Molecular Events Leading to Vinculin Activation

Focal adhesions are formed as a molecular glue linking cytoskeletal actin filaments to the extracellular matrix (ECM). They are formed at the site of mechanical stimulation (1) and involve and initial recruitment of talin and vinculin to ECM bound integrin molecules at the site of external stimulation. Talin recruitment and its force-induced activation and subsequent interaction with vinculin have been extensively studied (2–4). Vinculin is natively in an auto-inhibited conformation and its activation involves removal of a steric hindrance preventing binding of Vt with actin (5) (Figure 1). Several hypotheses have been put forth regarding vinculin activation and its subsequent interaction with actin: 1) vinculin activation requires only interaction with talin at domain 1 (D1) (6), 2) a simultaneous interaction with both actin and talin is necessary to achieve vinculin activation (7), 3) once activated vinculin interacts with actin via an electrostatic interaction between Vt and two regions on F-actin (5). Each of these hypotheses is evaluated through molecular dynamics simulation and analysis.Copyright