Kinetic frustration by limited bond availability controls the LAT protein condensation phase transition on membranes.

LAT is a membrane-linked scaffold protein that undergoes a phase transition to form a two-dimensional protein condensate on the membrane during T cell activation. Governed by tyrosine phosphorylation, LAT recruits various proteins that ultimately enable condensation through a percolation network of discrete and selective protein-protein interactions. Here, we describe detailed kinetic measurements of the phase transition, along with coarse-grained model simulations, that reveal that LAT condensation is kinetically frustrated by the availability of bonds to form the network. Unlike typical miscibility transitions in which compact domains may coexist at equilibrium, the LAT condensates are dynamically arrested in extended states, kinetically trapped out of equilibrium. Modeling identifies the structural basis for this kinetic arrest as the formation of spindle arrangements, favored by limited multivalent binding interactions along the flexible, intrinsically disordered LAT protein. These results reveal how local factors controlling the kinetics of LAT condensation enable formation of different, stable condensates, which may ultimately coexist within the cell.

Characterization of Protein-Phospholipid/Membrane Interactions Using a "Membrane-on-a-Chip" Microfluidic System.

It is now clear that organelles of a mammalian cell can be distinguished by phospholipid profiles, both as ratios of common phospholipids and by the absence or presence of certain phospholipids. Organelle-specific phospholipids can be used to provide a specific shape and fluidity to the membrane and/or used to recruit and/or traffic proteins to the appropriate subcellular location and to restrict protein function to this location. Studying the interactions of proteins with specific phospholipids using soluble derivatives in isolation does not always provide useful information because the context in which the headgroups are presented almost always matters. Our laboratory has shown this circumstance to be the case for a viral protein binding to phosphoinositides in solution and in membranes. The system we have developed to study protein-phospholipid interactions in the context of a membrane benefits from the creation of tailored membranes in a channel of a microfluidic device, with a fluorescent lipid in the membrane serving as an indirect reporter of protein binding. This system is amenable to the study of myriad interactions occurring at a membrane surface as long as a net change in surface charge occurs in response to the binding event of interest.

Zn2+ Binds to Phosphatidylserine and Induces Membrane Blebbing.

Herein, we show that Zn2+ binds to phosphatidylserine (PS) lipids in supported lipid bilayers (SLBs), forming a PS-Zn2+ complex with an equilibrium dissociation constant of ∼100 µM. Significantly, Zn2+ binding to SLBs containing more than 10 mol % PS induces extensive reordering of the bilayer. This reordering is manifest through bright spots of high fluorescence intensity that can be observed when the bilayer contains a dye-labeled lipid. Measurements using atomic force microscopy (AFM) reveal that these spots represent three-dimensional unilamellar blebs. Bleb formation is ion specific, inducible by exposing the bilayer to µM concentrations of Zn2+ but not Mg2+, Cu2+, Co2+, or Mn2+. Moreover, Ca2+ can induce some blebbing at mM concentrations but not nearly as effectively as Zn2+. The interactions of divalent metal cations with PS lipids were further investigated by a combination of vibrational sum frequency spectroscopy (VSFS) and surface pressure-area isotherm measurements. VSFS revealed that Zn2+ and Ca2+ were bound to the phosphate and carboxylate moieties on PS via contact ion pairing, dehydrating the lipid headgroup, whereas Mg2+ and Cu2+ were bound without perturbing the hydration of these functional groups. Additionally, Zn2+ was found to dramatically reduce the area per lipid in lipid monolayers, while Mg2+ and Cu2+ did not. Ca2+ could also reduce the area per lipid but only when significantly higher surface pressures were applied. These measurements suggest that Zn2+ caused lipid blebbing by decreasing the area per lipid on the side of the bilayer to which the salt was exposed. Such findings have implications for blebbing, fusion, oxidation, and related properties of PS-rich membranes in biological systems where Zn2+ concentrations are asymmetrically distributed.

Immobilization of Phosphatidylinositides Revealed by Bilayer Leaflet Decoupling.

Phosphatidylinositol 4,5-bisphosphate (PIP2) has a significantly lower mobile fraction than most other lipids in supported lipid bilayers (SLBs). Moreover, the fraction of mobile PIP2 continuously decreases with time. To explore this, a bilayer unzipping technique was designed to uncouple the two leaflets of the SLB. The results demonstrate that PIP2 molecules in the top leaflet are fully mobile, while the PIP2 molecules in the lower leaflet are immobilized on the oxide support. Over time, mobile PIP2 species flip from the top leaflet to the bottom leaflet and become trapped. It was found that PIP2 flipped between the leaflets through a defect-mediated process. The flipping could be completely inhibited when holes in the bilayer were backfilled with bovine serum albumin (BSA). Moreover, by switching from H2O to D2O, it was shown that the primary interaction between PIP2 and the underlying substrate was due to hydrogen bond formation, which outcompeted electrostatic repulsion. Using substrates with fewer surface silanol groups, like oxidized polydimethylsiloxane, led to a large increase in the mobile fraction of PIP2. Moreover, PIP2 immobilization also occurred when the bilayer was supported on a protein surface rather than glass. These results may help to explain the behavior of PIP2 on the inner leaflet of the plasma membrane, where it is involved in attaching the membrane to the underlying cytoskeleton.

Multistep Interactions between Ibuprofen and Lipid Membranes.

Ibuprofen (IBU) interacts with phosphatidylcholine membranes in three distinct steps as a function of concentration. In a first step (<10 µM), IBU electrostatically adsorbs to the lipid headgroups and gradually decreases the interfacial potential. This first step helps to facilitate the second step (10-300 µM), in which hydrophobic insertion of the drug occurs. The second step disrupts the packing of the lipid acyl chains and expands the area per lipid. In a final step, above 300 µM IBU, the lipid membrane begins to solubilize, resulting in a detergent-like effect. The results described herein were obtained by a combination of fluorescence binding assays, vibrational sum frequency spectroscopy, and Langmuir monolayer compression experiments. By introducing trimethylammonium-propane, phosphatidylglycerol, and phosphatidylethanolamine lipids as well as cholesterol, we demonstrated that both the chemistry of the lipid headgroups and the packing of lipid acyl chains can substantially influence the interactions between IBU and the membranes. Moreover, different membrane chemistries can alter particular steps in the binding interaction.

The RNA-Binding Site of Poliovirus 3C Protein Doubles as a Phosphoinositide-Binding Domain.

Some viruses use phosphatidylinositol phosphate (PIP) to mark membranes used for genome replication or virion assembly. PIP-binding motifs of cellular proteins do not exist in viral proteins. Molecular-docking simulations revealed a putative site of PIP binding to poliovirus (PV) 3C protein that was validated using nuclear magnetic resonance spectroscopy. The PIP-binding site was located on a highly dynamic α helix, which also functions in RNA binding. Broad PIP-binding activity was observed in solution using a fluorescence polarization assay or in the context of a lipid bilayer using an on-chip, fluorescence assay. All-atom molecular dynamics simulations of the 3C protein-membrane interface revealed PIP clustering and perhaps PIP-dependent conformations. PIP clustering was mediated by interaction with residues that interact with the RNA phosphodiester backbone. We conclude that 3C binding to membranes will be determined by PIP abundance. We suggest that the duality of function observed for 3C may extend to RNA-binding proteins of other viruses.

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions.

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-δ1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 ± 0.05 µM, similar to Kd,app values obtained by others. As previously observed, the PLC-δ1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein's capacity to interact with membranes.

Calcium Directly Regulates Phosphatidylinositol 4,5-Bisphosphate Headgroup Conformation and Recognition.

The orchestrated recognition of phosphoinositides and concomitant intracellular release of Ca2+ is pivotal to almost every aspect of cellular processes, including membrane homeostasis, cell division and growth, vesicle trafficking, as well as secretion. Although Ca2+ is known to directly impact phosphoinositide clustering, little is known about the molecular basis for this or its significance in cellular signaling. Here, we study the direct interaction of Ca2+ with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), the main lipid marker of the plasma membrane. Electrokinetic potential measurements of PI(4,5)P2 containing liposomes reveal that Ca2+ as well as Mg2+ reduce the zeta potential of liposomes to nearly background levels of pure phosphatidylcholine membranes. Strikingly, lipid recognition by the default PI(4,5)P2 lipid sensor, phospholipase C delta 1 pleckstrin homology domain (PLC δ1-PH), is completely inhibited in the presence of Ca2+, while Mg2+ has no effect with 100 nm liposomes and modest effect with giant unilamellar vesicles. Consistent with biochemical data, vibrational sum frequency spectroscopy and atomistic molecular dynamics simulations reveal how Ca2+ binding to the PI(4,5)P2 headgroup and carbonyl regions leads to confined lipid headgroup tilting and conformational rearrangements. We rationalize these findings by the ability of calcium to block a highly specific interaction between PLC δ1-PH and PI(4,5)P2, encoded within the conformational properties of the lipid itself. Our studies demonstrate the possibility that switchable phosphoinositide conformational states can serve as lipid recognition and controlled cell signaling mechanisms.

Polyarginine Interacts More Strongly and Cooperatively than Polylysine with Phospholipid Bilayers.

The interactions of two highly positively charged short peptide sequences with negatively charged lipid bilayers were explored by fluorescence binding assays and all-atom molecular dynamics simulations. The bilayers consisted of mixtures of phosphatidylglycerol (PG) and phosphatidylcholine (PC) lipids as well as a fluorescence probe that was sensitive to the interfacial potential. The first peptide contained nine arginine repeats (Arg9), and the second one had nine lysine repeats (Lys9). The experimentally determined apparent dissociation constants and Hill cooperativity coefficients demonstrated that the Arg9 peptides exhibited weakly anticooperative binding behavior at the bilayer interface at lower PG concentrations, but this anticooperative effect vanished once the bilayers contained at least 20 mol % PG. By contrast, Lys9 peptides showed strongly anticooperative binding behavior at all PG concentrations, and the dissociation constants with Lys9 were approximately 2 orders of magnitude higher than with Arg9. Moreover, only arginine-rich peptides could bind to the phospholipid bilayers containing just PC lipids. These results along with the corresponding molecular dynamics simulations suggested two important distinctions between the behavior of Arg9 and Lys9 that led to these striking differences in binding and cooperativity. First, the interactions of the guanidinium moieties on the Arg side chains with the phospholipid head groups were stronger than for the amino group. This helped facilitate stronger Arg9 binding at all PG concentrations that were tested. However, at PG concentrations of 20 mol % or greater, the Arg9 peptides came into sufficiently close proximity with each other so that favorable like-charge pairing between the guanidinium moieties could just offset the long-range electrostatic repulsions. This led to Arg9 aggregation at the bilayer surface. By contrast, Lys9 molecules experienced electrostatic repulsion from each other at all PG concentrations. These insights may help explain the propensity for cell penetrating peptides containing arginine to more effectively cross cell membranes in comparison with lysine-rich peptides.