Phosphatidylserine clustering by the Ebola virus matrix protein is a critical step in viral budding.

Phosphatidylserine (PS) is a critical lipid factor in the assembly and spread of numerous lipid-enveloped viruses. Here, we describe the ability of the Ebola virus (EBOV) matrix protein eVP40 to induce clustering of PS and promote viral budding in vitro, as well as the ability of an FDA-approved drug, fendiline, to reduce PS clustering and subsequent virus budding and entry. To gain mechanistic insight into fendiline inhibition of EBOV replication, multiple in vitro assays were run including imaging, viral budding and viral entry assays. Fendiline lowers PS content in mammalian cells and PS in the plasma membrane, where the ability of VP40 to form new virus particles is greatly lower. Further, particles that form from fendiline-treated cells have altered particle morphology and cannot significantly infect/enter cells. These complementary studies reveal the mechanism by which EBOV matrix protein clusters PS to enhance viral assembly, budding, and spread from the host cell while also laying the groundwork for fundamental drug targeting strategies.

Biophysical and computational studies of membrane penetration by the GRP1 pleckstrin homology domain.

The pleckstrin homology (PH) domain of the general receptor for phosphoinositides 1 (GRP1) exhibits specific, high-affinity, reversible binding to phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P(3)) at the plasma membrane, but the nature and extent of the interaction between this bound complex and the surrounding membrane environment remains unclear. Combining equilibrium and nonequilibrium molecular dynamics (MD) simulations, NMR spectroscopy, and monolayer penetration experiments, we characterize the membrane-associated state of GRP1-PH. MD simulations show loops flanking the binding site supplement the interaction with PI(3,4,5)P(3) through multiple contacts with the lipid bilayer. NMR data show large perturbations in chemical shift for these loop regions on binding to PI(3,4,5)P(3)-containing DPC micelles. Monolayer penetration experiments and further MD simulations demonstrate that mutating hydrophobic residues to polar residues in the flanking loops reduces membrane penetration. This supports a "dual-recognition" model of binding, with specific GRP1-PH-PI(3,4,5)P(3) interactions supplemented by interactions of loop regions with the lipid bilayer.

The origin of C1A-C2 interdomain interactions in protein kinase Calpha.

The regulatory domain of protein kinase Calpha (PKCalpha) contains three membrane-targeting modules, two C1 domains (C1A and C1B) that bind diacylglycerol and phorbol ester, and the C2 domain that is responsible for the Ca2+-dependent membrane binding. Accumulating evidence suggests that C1A and C2 domains of PKCalpha are tethered in the resting state and that the tethering is released upon binding to the membrane containing phosphatidylserine. The homology modeling and the docking analysis of C1A and C2 domains of PKCalpha revealed a highly complementary interface that comprises Asp55-Arg252 and Arg42-Glu282 ion pairs and a Phe72-Phe255 aromatic pair. Mutations of these residues in the predicted C1A-C2 interface showed large effects on in vitro membrane binding, enzyme activity, phosphatidylserine selectivity, and cellular membrane translocation of PKCalpha, supporting their involvement in interdomain interactions. In particular, D55A (or D55K) and R252A (or R252E) mutants showed much higher basal membrane affinity and enzyme activity and faster subcellular translocation than wild type, whereas a double charge-reversal mutant (D55K/R252E) behaved analogously to wild type, indicating that a direct electrostatic interaction between the two residues is essential for the C1A-C2 tethering. Collectively, these studies provide new structural insight into PKCalpha C1A-C2 interdomain interactions and the mechanism of lipid-mediated PKCalpha activation.