34 research outputs found
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Steric regulation of tandem calponin homology domain actin-binding affinity.
Tandem calponin homology (CH1-CH2) domains are common actin-binding domains in proteins that interact with and organize the actin cytoskeleton. Despite regions of high sequence similarity, CH1-CH2 domains can have remarkably different actin-binding properties, with disease-associated point mutants known to increase as well as decrease affinity for F-actin. To investigate features that affect CH1-CH2 affinity for F-actin in cells and in vitro, we perturbed the utrophin actin-binding domain by making point mutations at the CH1-CH2 interface, replacing the linker domain, and adding a polyethylene glycol (PEG) polymer to CH2. Consistent with a previous model describing CH2 as a steric negative regulator of actin binding, we find that utrophin CH1-CH2 affinity is both increased and decreased by modifications that change the effective "openness" of CH1 and CH2 in solution. We also identified interface mutations that caused a large increase in affinity without changing solution "openness," suggesting additional influences on affinity. Interestingly, we also observe nonuniform subcellular localization of utrophin CH1-CH2 that depends on the N-terminal flanking region but not on bulk affinity. These observations provide new insights into how small sequence changes, such as those found in diseases, can affect CH1-CH2 binding properties
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Molecular Mechanisms of Mechanosensitivity in Focal Adhesions
Physical environment guides tissue regeneration and morphology in both health and disease. In the past three decades, several experiments illustrated that mechanical cues are captured and transduced to biochemical signals in the cellular level (mechanotransduction) mediated by cell adhesion. Cells adhere to their microenvironment through large protein assemblies known as focal adhesions that directly couple intra- and extra-cellular matrices and play a critical role in many vital cell functions including proliferation, differentiation and cell fate. It is inherently difficult to investigate the molecular basis of focal adhesion formation and growth using current experimental methodologies due to the fine time- and length-scales of protein-protein interactions. Here, I used molecular dynamics simulations to investigate the underlying molecular mechanisms of focal adhesion formation and maturation with atomic resolution. Integrins are key focal adhesion receptors that reside on the cell membrane and mediate bi-directional signaling between cell cytoskeleton and ECM. Focal adhesions are a mixture of integrin-associated protein complexes known as integrin modules that forms the basic adhesion units. Integrin module formation is initiated by talin binding to the integrin tail, which is shown to be sufficient for integrin activation. A few other focal adhesion proteins can also directly engage with integrin’s cytoplasmic tail and link it to the actin cytoskeleton. It is not yet clear how simultaneous (cooperative) versus sequential (competitive) binding of focal adhesion proteins to integrin with respect to talin result in different functionalities of integrin modules. In the first part of this study, competitive versus cooperative integrin binding between two important focal adhesion proteins –filamin and α-actinin– with talin were studied. The purpose of this aim was to gain insight on integrin module formation that eventually determines its functional properties. Maturation of focal adhesions follows an increase in local forces. A well-established hypothesis on force transmission across focal adhesion complexes is the presence of mechanosensitive elements that change their conformations in response to force. In the second part of this study, we investigated and argued force-induced conformational changes of two important focal adhesion proteins –vinculin and α-actinin– in order to shed light on their role in transmission of forces across focal adhesions leading to adhesion maturation and growth. In conclusion, this study unravels the structural basis of mechanosensitivity of key focal adhesions. Furthermore, important molecular interactions that give rise to mechanosensitivite characteristics of focal adhesions were studies. Important impacts of the current study include but are not limited to the following: 1) our results was used to complement previous experimental studies and also construct new hypotheses for future experiments; 2) Understanding regulatory mechanisms of focal adhesions is critical for developing novel therapeutics for many diseases involving cell adhesion including cancer as it enhances target recognition and the accuracy of drug delivery systems. 3) In addition, performing extensive simulations on protein complexes will contribute to improving the accuracy of various aspects of computational methods including empirical force fields that is indicative of our understanding of fundamental physical and chemical principles governing protein-protein interactions. 4) And most importantly, this work provides a fundamental insight into the relation between structure and function of mechanosensitive proteins in focal adhesions
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α-Actinin Induces a Kink in the Transmembrane Domain of β 3-Integrin and Impairs Activation via Talin
Integrin-mediated signaling is crucial for cell-substrate adhesion and can be triggered from both intra- and extracellular interactions. Although talin binding is sufficient for inside-out activation of integrin, other cytoplasmic proteins such as α-actinin and filamin can directly interfere with talin-mediated integrin activation. Specifically, α-actinin plays distinct roles in regulating αIIbβ3 versus α5β1 integrin. It has been shown that α-actinin competes with talin for binding to the cytoplasmic tail of β3-integrin, whereas it cooperates with talin for activating integrin α5β1. In this study, molecular dynamics simulations were employed to compare and contrast molecular mechanisms of αIIbβ3 and α5β1 activation in the presence and absence of α-actinin. Our results suggest that α-actinin impairs integrin signaling by both undermining talin binding to the β3-integrin cytoplasmic tail and inducing a kink in the transmembrane domain of β3-integrin. Furthermore, we showed that α-actinin promote talin association with β1-integrin by restricting the motion of the cytoplasmic tail and reducing the entropic barrier for talin binding. Taken together, our results showed that the interplay between talin and α-actinin regulates signal transmission via controlling the conformation of the transmembrane domain and altering natural response modes of integrins in a type-specific manner