Characterization of the Sec14 Domain Membrane Binding Interface

Abstract

Phosphoinositides, the phosphorylated derivatives of phosphatidylinositol, are critical lipid signaling molecules that regulate a variety of intracellular processes. Sec14 and Sec14 domain proteins employ a heterotypic lipid exchange reaction that stimulates PtdIns kinases, making them critical regulators of many cellular signaling events. The location of ligand binding sites and critical conformational elements of these proteins are well-established, but the dynamics that drive ligand exchange and kinase activation at the membrane interface are not understood.

The overarching aim of this research is to characterize the interface of Sec14 at the membrane. This work includes 1) a mutagenesis approach guided by molecular dynamics simulations to identify the residues that make up the binding interface of Sec14 and its homologs (Chapter 2); 2) an assessment of Sec14 homolog 1 (Sfh1) as a candidate for 5-fluorotryptophan substitution and Nuclear Magnetic Resonance (NMR) experiments (Chapter 3); and 3) a characterization of the oligomerization states of Sfh1, several Sfh1 mutants, and Sec14, as well as a simple protocol for generating a monodisperse population of Sec14 monomers enable cleaner biophysical experimentation (Chapter 3). Several other proof-of-concept studies are also examined (Appendix). This research lays a foundation for future endeavors to characterize the Sec14 membrane binding interface and understand the dynamics that drive PtdIns- kinase stimulation.