A multiscale biophysical model for the recruitment of actin nucleating proteins at the membrane interface.

The dynamics and organization of the actin cytoskeleton are crucial to many cellular events such as motility, polarization, cell shaping, and cell division. The intracellular and extracellular signaling associated with this cytoskeletal network is communicated through cell membranes. Hence the organization of membrane macromolecules and actin filament assembly are highly interdependent. Although the actin-membrane linkage is known to happen through many routes, the major class of interactions is through the direct interaction of actin-binding proteins with the lipid class containing poly-phosphatidylinositols (PPIs). Among the PPIs, phosphatidylinositol bisphosphate (PI(4,5)P2) acts as a significant factor controlling actin polymerization in the proximity of the membrane by binding to actin-associated proteins. The molecular interactions between these actin-binding proteins and the membrane lipids remain elusive. Here, using molecular modeling, analytical theory, and experimental methods, we investigate the binding of three different actin-binding proteins, mDia2, NWASP, and gelsolin, to membranes containing PI(4,5)P2 lipids. We perform molecular dynamics simulations on the protein-bilayer system and analyze the membrane binding in the form of hydrogen bonds and salt bridges at various PI(4,5)P2 and cholesterol concentrations. Our experimental study with PI(4,5)P2-containing large unilamellar vesicles mimics the computational experiments. Using the multivalencies of the proteins obtained in molecular simulations and the cooperative binding mechanisms of the proteins, we also propose a multivalent binding model that predicts the actin filament distributions at various PI(4,5)P2 and protein concentrations.

Lateral distribution of phosphatidylinositol 4,5-bisphosphate in membranes regulates formin- and ARP2/3-mediated actin nucleation.

Spatial and temporal control of actin polymerization is fundamental for many cellular processes, including cell migration, division, vesicle trafficking, and response to agonists. Many actin-regulatory proteins interact with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and are either activated or inactivated by local PI(4,5)P2 concentrations that form transiently at the cytoplasmic face of cell membranes. The molecular mechanisms of these interactions and how the dozens of PI(4,5)P2-sensitive actin-binding proteins are selectively recruited to membrane PI(4,5)P2 pools remains undefined. Using a combination of biochemical, imaging, and cell biologic studies, combined with molecular dynamics and analytical theory, we test the hypothesis that the lateral distribution of PI(4,5)P2 within lipid membranes and native plasma membranes alters the capacity of PI(4,5)P2 to nucleate actin assembly in brain and neutrophil extracts and show that activities of formins and the Arp2/3 complex respond to PI(4,5)P2 lateral distribution. Simulations and analytical theory show that cholesterol promotes the cooperative interaction of formins with multiple PI(4,5)P2 headgroups in the membrane to initiate actin nucleation. Masking PI(4,5)P2 with neomycin or disrupting PI(4,5)P2 domains in the plasma membrane by removing cholesterol decreases the ability of these membranes to nucleate actin assembly in cytoplasmic extracts.

Interactions of Haptoglobin with Monomeric Globin Species: Insights from Molecular Modeling and Native Electrospray Ionization Mass Spectrometry.

Haptoglobin (Hp) binds free hemoglobin (Hb) dimers to prevent negative consequences of Hb circulation in the extracellular environment. Although both monomeric Hb and myoglobin (Mb) species also present potential risks, their interactions with Hp have not been extensively studied. Mb is homologous to both the α- and ß-chains of Hb and shares many conserved Hb/Hp interface residues, yet whether Hp binds Mb remains unclear. To address this, computational biology tools were used to predict the interactions required for Hp to bind monomeric globins, and the predicted association was tested using native electrospray ionization mass spectrometry (ESI-MS). The Hb/Hp crystal structure was used as the template to create molecular models of two Mb molecules bound to an Hp heterodimer (Mb2/Hp). Molecular modeling suggests that Mb can bind at the Hp α-chain binding site, where 73% of the globin/Hp interactions are conserved. By contrast, several ionic ß-chain residues involved in complementary electrostatic interactions with Hp correspond to residues with the opposite charge in Mb, suggesting unfavorable electrostatic Hp/Mb interactions at the ß-chain binding site. As shown by native ESI-MS, isolated monomeric Hbα subunits can form 2:1 complexes with Hp heterotetramers in the absence of Hb ß-chains. Native ESI-MS also confirmed that Mb can bind to Hp heterotetramers in solution with stoichiometries of 1:1 and 2:1 at physiological pH and ionic strength. The affinity of Hp for Mb appears to be diminished relative to that of Hb α-chains. Our in silico experiments rationalize this change and demonstrate that molecular modeling of protein/protein interactions is a valuable aid for MS experiments.