Department of Chemistry
Wichita State University
Regulation of palladin structure and function (2014-2015)
Palladin is one of the latest proteins to join the sizeable group of actin binding proteins that coordinate the remodeling of the actin cytoskeleton. Increased expression of palladin has been linked to metastatic progression, and cells from palladin knockout mice have disorganized actin arrays and reduced motility. While it is clear that palladin is involved in diverse cellular functions and signaling pathways, how signaling and functional specificity are achieved is not understood. Our hypothesis is that conformational changes in palladin, brought about by critical intermolecular interactions, modulate palladin’s role in cell motility. In this proposal two distinct regulatory mechanisms will be investigated: one involving membrane phospholipids, which are capable of anchoring ABPs at the cell membrane, and another linking actin-induced dimerization of palladin with enhanced crosslinking of actin filaments. Specifically, we will use a combination of structural and biochemical techniques to gain valuable insights regarding the biochemical mechanisms underlying the palladin-actin interaction and their relationship to cell motility.
First, we will investigate the interaction between palladin and a variety of membrane lipids. Based on our finding that the actinbinding domain of palladin also binds to phospholipid vesicles, we will now explore the consequences of these two binding events. Quantitative analysis of lipid binding by palladin will be facilitated by Surface Plasmon Resonance
experiments with the assistance of the Protein Production Core Facility. We also will investigate how actin binding regulates the structure and function of palladin. Based on our finding that palladin oligomerizes upon binding actin, we will now use a detailed structure-function analysis to investigate how this conformational change effects actin crosslinking by palladin. In addition to essential data collection in the BioNMR core, our studies will benefit from continued collaborations with the Analytical Proteomics Lab for MS analysis of chemical crosslinking and limited proteolysis data to map the actin-binding site on palladin. Unraveling and illuminating the structural and biochemical determinants that play a critical role in paladin-actin interactions will enable us to define their relationship to the metastatic process. Furthermore achievement of the stated research goals may ultimately impact our ability to control this process by disrupting or controlling this interaction to limit invasive motility in cancer cells.