A Novel Electrostatic Regulatory Mechanism in a Calcium Binding Protein, L-plastin

Abstract

Plastins are a group of highly conserved actin binding proteins. L-plastin is a human isoform of this protein found active in hemopoietic cells. This isoform is also expressed in cancer cells. The N-terminal of this isoform contains a Calcium-binding EF-hand domain that allosterically regulates this protein for binding to its target. It is, however, very difficult to delineate the effects that underlie the allosteric mechanism. To investigate the regulatory mechanisms of the activation of this protein domain, we have used both computational and experimental methods. Working with calcium in MD simulations has previously caused significant problems as classical force fields are not well equipped to deal with calcium. This is because calcium is a divalent ion, which can induce a strong local electrostatic field, and is capable of charge transfer. Although classical force fields are insufficient, a new polarizable force field now known as a Drude force field, provides a more accurate solution to this problem. We describe results from simulations with both Classical and Drude force fields for the calcium-sensitive regions of L-plastin. These ongoing simulations have already provided valuable insight an unexpected and previously unseen electrostatic regulatory mechanism of L-plastin. In addition to computational simulations, to further validate this novel mechanism we obtained promising experimental validation using a number of biophysical methods including isothermal calorimetry (ITC) and differential scanning calorimetry (DSC). Although we are looking into this mechanism in L-plastin, the conserved nature of this protein may indicate that this mechanism is present in a wide range of related proteins, and may help provide us with a deeper understanding of the mechanisms associated with calcium activation and deactivation.