An official website of the United States government
Cell signaling is an important process involving complex interactions between lipids and proteins. The myristoylated alanine-rich C-kinase substrate (MARCKS) has been established as a key signaling regulator, serving a range of biological roles. Its effector domain (ED), which anchors the protein to the plasma membrane, induces domain formation in membranes containing phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylserine (PS). The mechanisms governing the MARCKS-ED binding to membranes remain elusive. Here, we investigate the composition-dependent affinity and MARCKS-ED-binding-induced changes in interfacial environments using two-dimensional infrared spectroscopy and fluorescence anisotropy. Both negatively charged lipids facilitate the MARCKS-ED binding to lipid vesicles. Although the hydrogen-bonding structure at the lipid-water interface remains comparable across vesicles with varied lipid compositions, the dynamics of interfacial water show divergent patterns due to specific interactions between lipids and peptides. Our findings also reveal that PIP2 becomes sequestered by bound peptides, while the distribution of PS exhibits no discernible change upon peptide binding. Interestingly, PIP2 and PS become colocalized into domains both in the presence and absence of MARCKS-ED. More broadly, this work offers molecular insights into the effects of membrane composition on binding.
more »
« less
This content will become publicly available on April 1, 2026
Cooperativity of PIP2 and PS lipids modulates PH domain binding
Phosphatidylinositides constitute only 1%–3% of plasma membranes but play vital roles in cellular signaling. In particular, phosphatidylinositol 4,5-bisphosphate (PIP2) is involved in processes such as cytoskeleton organization and ion channel regulation. Pleckstrin homology (PH) domains are modular domains found in many proteins and are known for their strong affinity for PIP2 headgroups. The role of lipid composition in PH domain binding to PIP2, particularly the inclusion of phos phatidylserine (PS), is not well understood. This study explores the mechanisms of PH domain binding to PIP2 using fluores cence spectroscopy, Fourier transform infrared spectroscopy, two-dimensional infrared spectroscopy, and molecular dynamics simulations. We find that anionic PIP2 and PS alter the interfacial environment compared to phosphatidylcholines. Additionally, the PH domain promotes the localization of anionic lipid domains upon binding. Our results highlight the role of PSinlipid domain formation within membranes and its potential influence on protein binding affinities and lipid geometries. Spe cifically, we discovered a strong interaction between PIP2 and PS whereby hydrogen bonding within these anionic lipids drives localization in the membrane. This interaction also regulates protein binding at the membrane interface. Our findings suggest that cooperativity between PIP2 and PS is key to the formation of localized lipid domains and the recruitment of proteins such as the PH domain of phospholipase C-d1
more »
« less
- Award ID(s):
- 2129209
- PAR ID:
- 10645460
- Publisher / Repository:
- Cell Press
- Date Published:
- Journal Name:
- Biophysical Journal
- Volume:
- 124
- Issue:
- 7
- ISSN:
- 0006-3495
- Page Range / eLocation ID:
- 1146 to 1157
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Electrostatic interactions drive molecular assembly and organization in the plasma membrane. Specific protein-lipid interactions, however, are difficult to resolve. Here we report on a unique approach to investigate these interactions with time-resolved fluorescence spectroscopy. The experiments were performed on a model membrane system consisting of a supported lipid bilayer with an asymmetric distribution of PIP2 in the upper leaflet of the bilayer. The bilayer also contained nickel-chelating lipids that bind to a histidine-tagged peptide of interest. Both the peptide and the lipid were labeled with orthogonal fluorescent probes, so that diffusion and binding could be measured with two-color, pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). Our PIE-FCCS data showed significant lipid-peptide cross-correlation between PIP2 lipids and membrane-bound cationic peptides. Cross-correlation is a direct indication of lipid-peptide binding and complexation. Together with mobility data, we quantified the degree of binding, which offers new insight into this class of lipid-peptide interactions. Overall, this is the first report of lipid-peptide cross-correlation by FCCS, and provides a new route to quantifying the interactions between proteins and lipid membranes, a key interface in cell signaling.more » « less
-
Understanding the mechanisms of nanoparticle interaction with cell membranes is essential for designing materials for applications such as bioimaging and drug delivery, as well as for assessing engineered nanomaterial safety. Much attention has focused on nanoparticles that bind strongly to biological membranes or induce membrane damage, leading to adverse impacts on cells. More subtle effects on membrane function mediated via changes in biophysical properties of the phospholipid bilayer have received little study. Here, we combine electrophysiology measurements, infrared spectroscopy, and molecular dynamics simulations to obtain insight into a mode of nanoparticle-mediated modulation of membrane protein function that was previously only hinted at in prior work. Electrophysiology measurements on gramicidin A (gA) ion channels embedded in planar suspended lipid bilayers demonstrate that anionic gold nanoparticles (AuNPs) reduce channel activity and extend channel lifetimes without disrupting membrane integrity, in a manner consistent with changes in membrane mechanical properties. Vibrational spectroscopy indicates that AuNP interaction with the bilayer does not perturb the conformation of membrane-embedded gA. Molecular dynamics simulations reinforce the experimental findings, showing that anionic AuNPs do not directly interact with embedded gA channels but perturb the local properties of lipid bilayers. Our results are most consistent with a mechanism in which anionic AuNPs disrupt ion channel function in an indirect manner by altering the mechanical properties of the surrounding bilayer. Alteration of membrane mechanical properties represents a potentially important mechanism by which nanoparticles induce biological effects, as the function of many embedded membrane proteins depends on phospholipid bilayer biophysical properties.more » « less
-
null (Ed.)Intrinsic apoptosis is orchestrated by a group of proteins that mediate the coordinated disruption of mitochondrial membranes. Bax is a multi-domain protein that, upon activation, disrupts the integrity of the mitochondrial outer membrane by forming pores. We strategically introduced glutamic acids into a short sequence of the Bax protein that constitutively creates membrane pores. The resulting BaxE5 peptide efficiently permeabilizes membranes at acidic pH, showing low permeabilization at neutral pH. Atomic force microscopy (AFM) imaging showed that at acidic pH BaxE5 established several membrane remodeling modalities that progressively disturbed the integrity of the lipid bilayer. The AFM data offers vistas on the membrane disruption process, which starts with pore formation and progresses through localized exposure of membrane monolayers leading to stable and thin (16 Å) lipid-peptide complexes. The different types of membrane morphology observed in the presence of BaxE5 suggest that the peptide can establish different types of membrane interaction. BaxE5 adopts a rare unstructured conformation when bound to membranes, which might facilitate the dynamic transition between those different states, and then promote membrane digestion.more » « less
-
Members of the saposin-fold protein family and related proteins sharing a similar fold (saposin-like proteins; SAPLIP) are peripheral-membrane binding proteins that perform essential cellular functions. Saposins and SAPLIPs are abundant in both plant and animal kingdoms, and peripherally bind to lipid membranes to play important roles in lipid transfer and hydrolysis, defense mechanisms, surfactant stabilization, and cell proliferation. However, quantitative studies on the interaction between proteins and membranes are challenging due to the different nature of the two components in relation to size, structure, chemical composition, and polarity. Using liposomes and the saposin-fold member saposin C (sapC) as model systems, we describe here a method to apply solution NMR and dynamic light scattering to study the interaction between SAPLIPs and synthetic membranes at the quantitative level. Specifically, we prove with NMR that sapC binds reversibly to the synthetic membrane in a pH-controlled manner and show the dynamic nature of its fusogenic properties with dynamic light scattering. The method can be used to infer the optimal pH for membrane binding and to determine an apparent dissociation constant (KDapp) for protein-liposome interaction. We propose that these experiments can be applied to other proteins sharing the saposin fold.more » « less
