The actin cytoskeleton is involved in numerous cellular functions, such as cell shape maintenance, proliferation, and migration. Studies using α-actin, the main actin isoform present in skeletal muscle tissue as a model of choice, revealed that actin undergoes large conformational changes as it executes its function. Thus, it is well established that actin monomers assemble to nucleate and elongate filaments, which form the basis of the cytoskeleton. Structurally, addition of actin monomers at filament ends induces a transition from the globular actin monomer (G-actin) to a flattened F-actin structure. In the filament, the actin structural plasticity is also important for its ATPase activity. Actin molecules achieve this plasticity through movement of its four distinct subdomains (SD1-4) that change relative orientations in response to nucleotide binding, ATP hydrolysis, and inorganic phosphate (Pi) release . These events, in turn, modulate the mechanical properties of the filament and interactions with many actin binding proteins.
In addition to being modulated by ATP and ADP binding, and ATP hydrolysis, actin plasticity also depends on other environmental factors, such as the surrounding ions. Furthermore, recent work has highlighted the importance of the methylation of histidine 73 (H73) in affecting filament formation and the rate of ATP hydrolysis in the monomeric form. Although first reported nearly six decades ago, H73 methylation has remained a biochemical oddity and continues to be somewhat of a functional mystery.
Here, we address some of these mechanistic questions using β-actin as a model system. We chose to focus on β-actin because it remains far less studied actin isoform, despite functional relevance. Firstly, unlike α-actin, which is only expressed in skeletal muscles, β-actin is ubiquitously expressed in all cells. Furthermore, β-actin plays a key role in early embryonic development, cell migration and growth . More recently, β-actin has been shown to participate in chromatin remodeling, and there is accumulating evidence that β-actin is important for epithelial-to-mesenchymal transition and of interest in cancer research. However, β-actin remains less studied than the α isoform, especially in terms of its biochemical mechanism and dynamics.
Over the last two decades, molecular dynamics simulations have become an increasingly popular strategy for studying biological systems at different scales. Notably, this methodology has been extensively employed to delineate various properties of actin, such as structural differences between nucleotide states, the localization of water molecules in the actin binding site and their impact on protein plasticity and enzymatic properties , interactions with small molecules and the dynamic behavior of filaments in diverse environments . However, these computational studies almost exclusively focused on α-actin, while the β-actin isoform has remained understudied, limiting our understanding of its dynamics and functional implications.
Even if MD simulations have been instrumental in the understanding of how ion and water molecules interact with proteins the precise parametrization of these components is still challenging and depends on the system studied. Recent methodological developments on polarizable force-fields have drastically increased the accuracy of interactions between these molecules and proteins. In addition, the use of enhanced sampling methods allows exploring diverse unknown states hard to reach through the use of classical molecular dynamics simulations. Therefore, here, we employed large scale molecular dynamics simulations that overcome the limitations of classical approaches by combining adaptive sampling with the AMOEBA polarizable force field to more accurately model interatomic interactions via the high-performance Tinker-HP software. We used this strategy to provide insights into β-actin biochemistry and on how H73 methylation together with key environmental factors (ions and nucleotides), affect its dynamics and plasticity both in its core and extremities.
As this work sheds light onto the plasticity of the β-actin isoform, where subtle structural changes can impact actin function, from the monomer to the filament, our results highlighted how the post translational modification of histidine 73 can drastically change the dynamic properties of actin in different forms, from the monomer to the filament. Our work reveals how subtle structural changes, such as a single methylation buried within the core of the protein, could have numerous consequences that affect protein plasticity at different scales. Importantly, although understudied, histidine methylation has now been reported in other proteins, such as S100A9, myosin, skeletal muscle myosin light chain kinase (MLCK 2), and ribosomal protein Rpl3, with a recent analysis identifying about 300 histidine methylation sites in the proteome of HeLa cells. Similar to what we observed in β-actin, we expect that this PTM plays a major regulatory role in many of these systems. Thus, our study highlights the value of using MD simulations combined with polarizable force fields as a method for understanding the effects of this PTM in actin and beyond.