Main Interaction Site Research Articles

Effects of collagen hydrolysate on the stability of anthocyanins: Degradation kinetics, conformational change and interactional characteristics

Anthocyanins are desirable compounds in the food industry owing to their attractive color and high biological activity; however, their poor stability remains a substantial challenge. Here, we show that low-concentration (15 mg/mL) collagen hydrolysate (CH) exhibits a potent stabilization effect on red cabbage anthocyanins (RCAs). CH extended the half-life of RCA by 6.2-fold from 40.7 to 251.1 h. Dynamic light scattering and transmission electron microscopy confirmed the formation of CH–RCA complexes, which exhibited stronger antioxidant activity than RCA alone. Ultraviolet-vis and infrared spectra demonstrated that RCA binding resulted in a more open and disordered CH structure. Centaureidin-3-O-glucoside (C3G) exhibited high affinity for CH, with a binding ratio close to 1.5:1. 1H nuclear magnetic resonance confirmed that the main interaction sites with CH were at the C3G A- and C-rings. This study clarifies how protein hydrolysates protect against anthocyanin degradation from experimental and theoretical aspects.

High internal phase emulsions stabilized by fluorescent phycocyanin for improved stability and bioaccessibility of β-carotene.

High internal phase emulsions (HIPE) are distinguished from ordinary emulsions by higher oil-phase percentage and better storage stability. Recently, HIPE stabilized with protein-based particles has received more attention. However, organic precipitation, chemical cross-linking and thermal denaturation are often needed to stabilize emulsions with natural proteins, and there is an urgent need to reduce the pollution of organic reagents. HIPE loaded with β-carotene stabilized by phycocyanin was prepared under mild conditions. It demonstrated strong stability in terms of temperature and storage, as evidenced by its 94.17% retention rate and 81.06% bioavailability. This stability was ascribed to the efficient defense against heat and UV rays, which was probably associated with the oil-droplet environment and interfacial protection of phycocyanin. It is speculated that the possible main interaction site between phycocyaninand sorbitol exists near amino acids 110 to 120 of the B chain. The hydrogen bond and hydrophobic interaction between them make the phycocyanin fully adsorbed on the oil-water interface when sorbitol is stable, forming a strong oil-water structure, which increases the stability of the emulsion. The outstanding fluorescence characteristics provide a feasible alternative for fluorescent emulsions to distribute and trace active compounds in vitro. HIPE loaded with β-carotene might have potential as a 3D printing material for edible functional foods. © 2024 Society of Chemical Industry.

TripHLApan: predicting HLA molecules binding peptides based on triple coding matrix and transfer learning.

Human leukocyte antigen (HLA) recognizes foreign threats and triggers immune responses by presenting peptides to T cells. Computationally modeling the binding patterns between peptide and HLA is very important for the development of tumor vaccines. However, it is still a big challenge to accurately predict HLA molecules binding peptides. In this paper, we develop a new model TripHLApan for predicting HLA molecules binding peptides by integrating triple coding matrix, BiGRU + Attention models, and transfer learning strategy. We have found the main interaction site regions between HLA molecules and peptides, as well as the correlation between HLA encoding and binding motifs. Based on the discovery, we make the preprocessing and coding closer to the natural biological process. Besides, due to the input being based on multiple types of features and the attention module focused on the BiGRU hidden layer, TripHLApan has learned more sequence level binding information. The application of transfer learning strategies ensures the accuracy of prediction results under special lengths (peptides in length 8) and model scalability with the data explosion. Compared with the current optimal models, TripHLApan exhibits strong predictive performance in various prediction environments with different positive and negative sample ratios. In addition, we validate the superiority and scalability of TripHLApan's predictive performance using additional latest data sets, ablation experiments and binding reconstitution ability in the samples of a melanoma patient. The results show that TripHLApan is a powerful tool for predicting the binding of HLA-I and HLA-II molecular peptides for the synthesis of tumor vaccines. TripHLApan is publicly available at https://github.com/CSUBioGroup/TripHLApan.git.

Interaction of Glycated Albumin with Receptor for Glycation End Products According to Molecular Modeling Data

In diabetes mellitus (DM) patients, the accumulation of advanced glycation end products (AGE) leads to inflammation and oxidative stress through the activation of specific receptors for AGE (RAGE). Glycated albumin (gHSA) makes a significant contribution to the overall level of AGE in human body and, as a result, to the pathogenesis of DM and concomitant diseases. The mechanism of interaction of gHSA with RAGE is practically not studied. The purpose of the present paper is to study the binding of gHSA to RAGE using molecular modeling methods, to find the main sites of interaction and structural features of glycation sites that determine the efficiency of complex formation with RAGE. Ten gHSA models were constructed using molecular docking and molecular dynamics (MD) methods; each model corresponded to one modified lysine residue (carboxymethyl-lysine): Lys64, Lys73, Lys137, Lys233, Lys262, Lys317, Lys378, Lys525, Lys573, Lys574. Complexes of gHSA with the V-domain of RAGE were constructed using the macromolecular docking method, and their stability was studied using MD simulation. In the constructed gHSA models, the carboxyl groups of glycated Lys317 and Lys525 form intramolecular salt bridges with surrounding amino acids; in other cases, the carboxyl groups of the modified lysines are free to interact with positively charged amino acid residues on the RAGE surface. According to the macromolecular docking data and subsequent MD simulation, the complex of RAGE with gHSA glycated at Lys233 is most effective in terms of strength and specificity. Specific RAGE complexes with gHSA glycated at Lys317 and Lys574 are not formed. The obtained data on the interaction of gHSA with RAGE will help to understand the role of albumin in the pathophysiology of DM and advance towards the prevention and development of effective therapy for this disease.

Supercritical CO2/Deep Eutectic Solvent Biphasic System as a New Green and Sustainable Solvent System for Different Applications: Insights from Molecular Dynamics Simulations.

Deep eutectic solvents (DESs) are one of the most interesting research subjects in green chemistry nowadays. Due to their low toxicity, simple synthesis, and lower prices, they have gradually taken the place of other green solvents such as ionic liquids (ILs) in sustainable processes. However, problems such as high viscosity and high polarity limit the applications of DESs in areas such as extraction, catalysis, and biocatalysis. In this work, we introduce and evaluate the potential application of scCO2/DES for the first time. Molecular dynamics simulations were used to examine the phase behavior, polarity, molecular mobilities, and microstructure of this system. Results show that CO2 molecules can significantly diffuse to the DES phase, while DES components do not appear in the scCO2 phase. The diffused CO2 molecules significantly enhanced the molecular mobility of the DES components. The presence of CO2 molecules changes the DES polarity so that hexane can be solubilized and dispersed in the DES phase. Radial distribution functions show that the solubilized CO2 molecules have negligible effects on the microstructure of DES. It was shown that chloride and urea are the main interaction sites of CO2 in DES. The results of this study show that scCO2/DES as a new class of green and versatile solvents can open a new promising window for research in sustainable chemistry and engineering.

Effects of coenzyme Q10 in a propofol infusion syndrome model of rabbits.

Coenzyme Q (CoQ) might be the main site of interaction with propofol on the mitochondrial respiratory chain in the propofol infusion syndrome (PRIS) because of the structural similarity between coenzyme Q10 (CoQ10) and propofol. To investigate the effects of CoQ10 on survival and organ injury in a PRIS model in rabbits. Sixteen male New Zealand white rabbits were divided into 4 groups: (1) propofol infusion group, (2) propofol infusion and CoQ10, 100 mg/kg was administered intravenously, (3) sevoflurane inhalation was administered, and (4) sevoflurane inhalation and CoQ10, 100 mg/kg intravenously, was administered. Arterial blood gas and biochemical analyses were repeated every 2 h and every 12 h, respectively. Animals that were alive on the 24th hour after anesthesia induction were euthanized. The organ damages were investigated under light and transmission electron microscopy (TEM). The propofol infusion group had the highest troponin T levels when compared with the other three groups at the 12th hour. The propofol + CoQ10 group had lower troponin T levels when compared with the propofol and sevoflurane groups (P < 0.05). Administration of CoQ10 decreased total liver injury scores and total organ injury scores both in the propofol and sevoflurane groups. The propofol and sevoflurane organ toxicities were attenuated with CoQ10 in liver, gallbladder, urinary bladder, and spleen. The addition of CoQ10 to propofol and sevoflurane anesthesia prevented the propofol-associated increase in troponin T levels at the 12th hour of infusion and decreased anesthetic-induced total liver and organ injury scores.

Investigation of bovine β-lactoglobulin-procyanidin complexes interactions and its utilization in O/W emulsion

Procyanidins are bioactive polyphenols that have a strong affinity to proteins. Beta-lactoglobulin (BLG) is widely used as an emulsifier in the food and other industries. This study evaluated the interaction between BLG and A-type procyanidin dimer (PA2) using the spectroscopic, thermodynamic, and molecular simulation. PA2 decreased the transmissivity and quenched the intrinsic fluorescence of BLG, suggesting that the two molecules formed a complex. The binding of PA2 reduced the surface hydrophobicity and altered the conformation of BLG with increasing the random coil regions. Thermodynamic and isothermal titration calorimetry analyses suggested that the main driving force of PA2-BLG interaction was hydrophobic attraction. Molecular docking simulations were used to identify the main interaction sites and forces in the BLG-PA2 complexes, which again indicated that hydrophobic interactions dominated. In addition, the influence of PA2 on the ability of BLG to form and stabilize O/W emulsions was analyzed. Emulsions formulated using BLG-PA2 complexes contained relatively small droplets (D4,3 ≈ 0.7 μm) and high surface potentials (absolute value >50 mV). Compared to BLG alone, BLG-PA2 complexes improved the storage stability of the emulsions. This study provides valuable new insights into the formation, properties, and application of protein-polyphenol complexes as functional ingredients in foods.

Study on the structural characteristics and interaction mechanisms of ionic liquid mixtures with a common imidazolium cation

Recently, ionic liquid mixtures have been widely studied and successfully applied in many fields. However, the basic knowledge of their molecular interactions is not yet fully understood. In this work, the structural characteristics and interaction mechanisms of 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (EMIMTFSI) and 1-ethyl-3-methylimidazolium thiocyanate (EMIMSCN) mixtures in different proportions were researched by combining theoretical and experimental methods. The conclusions drawn are summarized as follows: (1) The main interaction sites for [EMIM]+, [TFSI]− and [SCN]− are C2−H, O and N atoms, respectively. (2) With the increase in x(EMIMTFSI), the hydrogen bonding interactions in the EMIMTFSI−EMIMSCN system were weakened, and the tendency to aggregate between two anions ([TFSI]−, [SCN]−) and [EMIM]+ was enhanced. (3) The interaction between [EMIM]+ and [SCN]− is stronger than that between [EMIM]+ and [TFSI]−. The order of diffusion speed is [EMIM]+ > [TFSI]− > [SCN]−. (4) New forming EMIMTFSI and EMIMSCN interacting complexes were assigned by excess spectra.

Mixing behavior of 1-Ethyl-3-methylimidazolium Bis(trifluoromethylsulfonyl)imide and 1-Ethyl-3-methylimidazolium tetrafluoroborate binary ionic liquids mixtures

Mixing two (or more) ionic liquids (IL) to form IL mixtures has become a new research hotspot for the design of ILs to achieve the desired properties. However, fundamental studies on interactions and mixing behaviors of the IL mixtures were reported less. In this work, the two ILs containing the same cation [EMIM]+ (1-ethyl-3-methylimidazolium) were selected to prepare ILs mixtures. The mixing behavior of the two IL mixtures were investigated by experimental method and quantum chemical calculations. The main conclusions were shown as follows: (1) DMSO form stronger hydrogen bonds with EMIMTFSI than EMIMBF4. (2) With addition of EMIMBF4, the hydrogen bonding interactions in EMIMTFSI−EMIMBF4 system were weakened, while the coulombic interactions were strengthened. (3) With the dilution, the C2−H has the smallest changes. A possible reason is the C2−Hs were still the main interaction site. (4) New formation complexes between EMIMBF4 and EMIMTFSI were assigned by excess infrared spectra.

Molecular Insights on the Adsorption of Polycyclic Aromatic Hydrocarbons on Soil Clay Minerals

The role of soil clay minerals is often ignored in studying the polycyclic aromatic hydrocarbons (PAHs) adsorption in soil. In this work, Monte Carlo and molecular dynamics simulations are used to study the adsorption of PAHs on several typical soil clay mineral surfaces, such as (0 0 1) and (0 0 − 1) of kaolinite, pyrophyllite, mica, and montmorillonite. Results show that van der Waals and cation-π interactions dominate the interactions between PAHs and mineral surfaces. Bridging oxygen atom on the mineral surface structure is the main site of hydrophobic interaction with PAHs. The cation-π effect can significantly enhance the PAHs adsorption energy. In addition, the adsorption on clay minerals is enhanced with the increase in the number of PAHs aromatic rings. In the presence of water, different minerals exhibit different adsorption properties. A layer of water molecules is formed close to the kaolinite (0 0 1) surface, reducing PAHs adsorption. The self-diffusion coefficient (D) of PAHs in Ca2+-montmorillonite is more significant than that in K+-mica, due to the different hydration properties of mineral cations. These findings reveal the adsorption mechanism of PAHs on the surfaces of typical clay minerals at a molecular scale and provide a new perspective for soil adsorption of PAHs.

Claudin-10b cation channels in tight junction strands: Octameric-interlocked pore barrels constitute paracellular channels with low water permeability

Claudin proteins constitute the backbone of tight junctions (TJs) regulating paracellular permeability for solutes and water. The molecular mechanism of claudin polymerization and paracellular channel formation is unclear. However, a joined double-rows architecture of claudin strands has been supported by experimental and modeling data. Here, we compared two variants of this architectural model for the related but functionally distinct cation channel-forming claudin-10b and claudin-15: tetrameric-locked-barrel vs octameric-interlocked-barrels model. Homology modeling and molecular dynamics simulations of double-membrane embedded dodecamers indicate that claudin-10b and claudin-15 share the same joined double-rows architecture of TJ-strands. For both, the results indicate octameric-interlocked-barrels: Sidewise unsealed tetrameric pore scaffolds interlocked with adjacent pores via the β1β2 loop of the extracellular segment (ECS) 1. This loop mediates hydrophobic clustering and, together with ECS2, cis- and trans-interaction between claudins of the adjacent tetrameric pore scaffolds. In addition, the β1β2 loop contributes to lining of the ion conduction pathway. The charge-distribution along the pore differs between claudin-10b and claudin-15 and is suggested to be a key determinant for the cation- and water permeabilities that differ between the two claudins. In the claudin-10b simulations, similar as for claudin-15, the conserved D56 in the pore center is the main cation interaction site. In contrast to claudin-15 channels, the claudin-10b-specific D36, K64 and E153 are suggested to cause jamming of cations that prevents efficient water passage. In sum, we provide novel mechanistic information about polymerization of classic claudins, formation of embedded channels and thus regulation of paracellular transport across epithelia.

Structural microheterogeneity and hydrogen bonding properties in the mixtures of two ionic liquids with a common imidazolium cation

A recent focus in the ionic liquid field is to mix two (or more) ionic liquids to expand the available properties. In this work, the interactions and structural properties of 1-ethyl-3-methylimidazolium bis(trifluoromethyl)sulfonylimide (EMIMTFSI) and 1-ethyl-3-methylimidazolium dicyanamide (EMIMDCA) mixtures were investigated by employing multiple experimental methods and DFT calculations. Microheterogeneity of the liquid mixtures was characterized in detail and four complexes or clusters were identified, i.e. 2EMIMTFSI, 2EMIMDCA, 2EMIMTFSI-[DCA]− and 2EMIMDCA-[TFSI]−. Hydrogen bonding interactions between cations and anions were found to be ubiquitous but all in the category of weak interactions. The main interaction sites for [EMIM]+, [TFSI]−, and [DCA]− were found to be C2−H, O/N atoms, and N (in C≡N). This work adds value to an in-depth understanding of the microstructures and molecular interactions in the specified ionic liquid mixtures and would shed light to the general understanding of the ionic liquid systems as a whole.

The First Class of Small Molecules Potently Disrupting the YAP-TEAD Interaction by Direct Competition.

Inhibition of the YAP-TEAD protein-protein interaction is an attractive therapeutic concept under intense investigation with the objective to treat cancers associated with a dysregulation of the Hippo pathway. However, owing to the very extended surface of interaction of the two proteins, the identification of small drug-like molecules able to efficiently prevent YAP from binding to TEAD by direct competition has been elusive so far. We disclose here the discovery of the first class of small molecules potently inhibiting the YAP-TEAD interaction by binding at one of the main interaction sites of YAP at the surface of TEAD. These inhibitors, providing a path forward to pharmacological intervention in the Hippo pathway, evolved from a weakly active virtual screening hit advanced to high potency by structure-based design.

Microglial control of neuronal development via somatic purinergic junctions.

SummaryMicroglia, the resident immune cells of the brain, play important roles during development. Although bi-directional communication between microglia and neuronal progenitors or immature neurons has been demonstrated, the main sites of interaction and the underlying mechanisms remain elusive. By using advanced methods, here we provide evidence that microglial processes form specialized contacts with the cell bodies of developing neurons throughout embryonic, early postnatal, and adult neurogenesis. These early developmental contacts are highly reminiscent of somatic purinergic junctions that are instrumental for microglia-neuron communication in the adult brain. The formation and maintenance of these junctions is regulated by functional microglial P2Y12 receptors, and deletion of P2Y12Rs disturbs proliferation of neuronal precursors and leads to aberrant cortical cytoarchitecture during development and in adulthood. We propose that early developmental formation of somatic purinergic junctions represents an important interface for microglia to monitor the status of immature neurons and control neurodevelopment.

Omicron Binding Mode: Contact Analysis and Dynamics of the Omicron Receptor-Binding Domain in Complex with ACE2.

On 26 November 2021, the WHO classified the Omicron variant of the SARS-CoV-2 virus (B.1.1.529 lineage) as a variant of concern (VOC) (COVID-19 Variant Data, Department of Health, 2022). The Omicron variant contains as many as 26 unique mutations of effects not yet determined (Venkatakrishnan, A., Open Science Framework, 2021). Out of its total of 34 Spike protein mutations, 15 are located on the receptor-binding domain (S-RBD) (Stanford Coronavirus Antiviral & Resistance Database, 2022) that directly contacts the angiotensin-converting enzyme 2 (ACE2) host receptor and is also a primary target for antibodies. Here, we studied the binding mode of the S-RBD domain of the Spike protein carrying the Omicron mutations and the globular domain of human ACE2 using molecular dynamics (MD) simulations. We identified new and key Omicron-specific interactions such as R493 (of mutation Q493R), which forms salt bridges both with E35 and D38 of ACE2, Y501 (N501Y), which forms an edge-to-face aromatic interaction with Y41, and Y505 (Y505H), which makes an H-bond with E37 and K353. The glycan chains of ACE2 also bind differently in the WT and Omicron variants in response to different charge distributions on the surface of Spike proteins. However, while the Omicron mutations considerably improve the overall electrostatic fit of the two interfaces, the total number of specific and favorable interactions between the two does not increase. The dynamics of the complexes are highly affected too, making the Omicron S-RBD:ACE2 complex more rigid; the two main interaction sites, Patches I and II, isolated in the WT complex, become connected in the Omicron complex through the alternating interaction of R493 and R498 with E35 and D38.

ERp57 chaperon protein protects neuronal cells from Aβ-induced toxicity.

Alzheimer's disease (AD) is a neurodegenerative disorder whose main pathological hallmark is the accumulation of Amyloid‐β peptide (Aβ) in the form of senile plaques. Aβ can cause neurodegeneration and disrupt cognitive functions by several mechanisms, including oxidative stress. ERp57 is a protein disulfide isomerase involved in the cellular stress response and known to be present in the cerebrospinal fluid of normal individuals as a complex with Aβ peptides, suggesting that it may be a carrier protein which prevents aggregation of Aβ. Although several studies show ERp57 involvement in neurodegenerative diseases, no clear mechanism of action has been identified thus far. In this work, we gain insights into the interaction of Aβ with ERp57, with a special focus on the contribution of ERp57 to the defense system of the cell. Here, we show that recombinant ERp57 directly interacts with the Aβ25−35 fragment in vitro with high affinity via two in silico‐predicted main sites of interaction. Furthermore, we used human neuroblastoma cells to show that short‐term Aβ25−35 treatment induces ERp57 decrease in intracellular protein levels, different intracellular localization, and ERp57 secretion in the cultured medium. Finally, we demonstrate that recombinant ERp57 counteracts the toxic effects of Aβ25−35 and restores cellular viability, by preventing Aβ25−35 aggregation. Overall, the present study shows that extracellular ERp57 can exert a protective effect from Aβ toxicity and highlights it as a possible therapeutic tool in the treatment of AD.

Polylactide nanoparticle impregnation with carbamazepine in supercritical media and its subsequent release in liquid solvents: insights from molecular simulation

A full-atomic classical molecular dynamics simulation of polylactide nanoparticle impregnation with carbamazepine in supercritical carbon dioxide was performed. The effect of temperature (333 K, 373 K), pressure (20 MPa, 40 MPa) and ethanol addition (1.7 mol.%) on the impregnation process was studied. Based on the solvent accessible surface area values it was concluded that a pressure increase and cosolvent addition have a positive effect on polymer swelling, and a temperature increase at a similar pressure value has a negative effect. It was demonstrated that carbamazepine gets adsorbed on the polymer nanoparticle surface and penetrates inside the polymer matrix. The main interaction sites were determined. The average number and lifetime of hydrogen bonds, the solute mobility before and after the impregnation process were estimated. The possibility of carbamazepine release from the polylactide nanoparticle in water, ethanol and dichloromethane was studied at 310 K and 0.1 MPa. Since the drug and polymer affinities to the solvents are not equal, their release behavior is completely different.

Chimeric Investigations into the Diamide Binding Site on the Lepidopteran Ryanodine Receptor.

Alterations to amino acid residues G4946 and I4790, associated with resistance to diamide insecticides, suggests a location of diamide interaction within the pVSD voltage sensor-like domain of the insect ryanodine receptor (RyR). To further delineate the interaction site(s), targeted alterations were made within the same pVSD region on the diamondback moth (Plutella xylostella) RyR channel. The editing of five amino acid positions to match those found in the diamide insensitive skeletal RyR1 of humans (hRyR1) in order to generate a human–Plutella chimeric construct showed that these alterations strongly reduce diamide efficacy when introduced in combination but cause only minor reductions when introduced individually. It is concluded that the sites of diamide interaction on insect RyRs lie proximal to the voltage sensor-like domain of the RyR and that the main site of interaction is at residues K4700, Y4701, I4790 and S4919 in the S1 to S4 transmembrane domains.

A barbed end interference mechanism reveals how capping protein promotes nucleation in branched actin networks

Heterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP potentiates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we combine structural biology with in vitro reconstitution to demonstrate that CP not only terminates filament elongation, but indirectly stimulates the activity of Arp2/3 activating nucleation promoting factors (NPFs) by preventing their association to filament barbed ends. Key to this function is one of CP’s C-terminal “tentacle” extensions, which sterically masks the main interaction site of the terminal actin protomer. Deletion of the β tentacle only modestly impairs capping. However, in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes—capping and nucleation—in branched actin network assembly.

PIP2-induced membrane binding of the vinculin tail competes with its other binding partners

Vinculin plays a key role during the first phase of focal adhesion formation and interacts with the plasma membrane through specific binding of its tail domain to the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Our understanding of the PIP2-vinculin interaction has been hampered by contradictory biochemical and structural data. Here, we used a multiscale molecular dynamics simulation approach, in which unbiased coarse-grained molecular dynamics were used to generate starting structures for subsequent microsecond-long all-atom simulations. This allowed us to map the interaction of the vinculin tail with PIP2-enriched membranes in atomistic detail. In agreement with experimental data, we have shown that membrane binding is sterically incompatible with the intramolecular interaction between vinculin’s head and tail domain. Our simulations further confirmed biochemical and structural results, which identified two positively charged surfaces, the basic collar and the basic ladder, as the main PIP2 interaction sites. By introducing a valency-disaggregated binding network analysis, we were able to map the protein-lipid interactions in unprecedented detail. In contrast to the basic collar, in which PIP2 is specifically recognized by an up to hexavalent binding pocket, the basic ladder forms a series of low-valency binding sites. Importantly, many of these PIP2 binding residues are also involved in maintaining vinculin in a closed, autoinhibited conformation. These findings led us to propose a molecular mechanism for the coupling between vinculin activation and membrane binding. Finally, our refined binding site suggests an allosteric relationship between PIP2 and F-actin binding that disfavors simultaneous interaction with both ligands, despite nonoverlapping binding sites.