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

Molecular Dynamics of Mechanosensing and Mechanotransduction at the Focal Adhesions

Focal adhesions are critical to cellular processes such as cell migration and cell-substrate adhesion. Focal adhesion formation can be mechanically regulated: forces either from outside the cell or from contracting actin filaments can induce rapid growth and maturation of the focal adhesions. One hypothesis explored here contends that force-induced focal adhesion formation results from mechanosensing by individual protein components. Molecular dynamics computational simulations are developed to evaluate mechanosensing by talin and vinculin. In Part 1 of this dissertation, two force-induced conformational changes are suggested for talin's activation: (i) the cryptic vinculin-binding sites (VBS) can be activated by stretch of an individual talin rod domain, and (ii) talin can adopt multiple dimer orientations in response to forces applied from outside the cell. The mechanisms of vinculin activation are then explored in Part 2. The trajectory of a vinculin conformational changes that would render it activated is predicted along with the structure of an activated vinculin. Domain 1 (D1) is predicted to separate from the vinculin tail (Vt) during activation. In this context, the PIP2 from the cell membrane is shown to preferentially bind basic residues on the vinculin surface and recruit vinculin to membrane proximal regions, potentially allowing for vinculin phosphorylation. The impact of phosphorylation on the vinculin structure is simulated and it is suggested that phosphorylation could prime vinculin for activation by reducing the strength of inter-domain interactions stabilizing the auto-inhibited vinculin conformation. In Part 3 of this dissertation, the interaction of activated vinculin with its binding partners is simulated. It is demonstrated that a talin VBS can only link vinculin prior to activation but can completely bind vinculin following activation. Vinculin activation by movement at D1 is shown to be necessary and sufficient for linking vinculin to actin filaments. Furthermore, simulation of F-actin caping by vinculin suggests that a second vinculin conformational change, releasing Vt from all vinculin head domains, facilitates effective capping of the actin filament. Three vinculinbinding sites on F-actin are predicted. These simulations demonstrate that indeed both talin and vinculin can exhibit molecular mechanosensing

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-actinin's 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