Dissociation of Nicotine from Acetylcholine-Binding Protein under Terahertz Waves Radiation.

The binding of nicotine (NCT) to acetylcholine-binding protein (AChBP) plays an important role in synaptic transmission and neurotransmitter regulation. However, effectively regulating their binding or dissociation processes remains a challenging problem. In this study, we employed all-atom molecular dynamics (MD) simulations to systematically investigate the impact of external terahertz (THz) waves on the binding kinetics between AChBP and NCT. We first identified the key residues (i.e., W143) and the key interactions (i.e., hydrogen bonding and cation-π interaction) in AChBP-NCT binding without THz waves. We then investigated the binding and dissociation of charged NCT with AChBP at three different frequencies (i.e., 13.02, 21.44, 42.55 THz). Importantly, the predominant vibrational modes at 13.02 THz can drive the rotation of the pentagonal ring on NCT. This leads to the disruption of hydrogen bonds between NCT and W143 and a reduced likelihood of forming cation-π interactions, resulting in the dissociation of NCT from AChBP. Additionally, we further investigated the influence of electric field intensities on the dissociation kinetics and found that when the electric field intensity exceeds a critical value (∼0.60 V/nm), the probability of ligand dissociation gradually rises as the intensity increases. In general, this study contributes to a better understanding of the effects of THz waves on protein-ligand interactions, which might also shed some light on potential applications in nicotine addiction treatment and therapeutic strategies for neurodegenerative diseases.

The conformational plasticity of structurally unrelated lipid transport proteins correlates with their mode of action.

Lipid transfer proteins (LTPs) are key players in cellular homeostasis and regulation, as they coordinate the exchange of lipids between different cellular organelles. Despite their importance, our mechanistic understanding of how LTPs function at the molecular level is still in its infancy, mostly due to the large number of existing LTPs and to the low degree of conservation at the sequence and structural level. In this work, we use molecular simulations to characterize a representative dataset of lipid transport domains (LTDs) of 12 LTPs that belong to 8 distinct families. We find that despite no sequence homology nor structural conservation, the conformational landscape of LTDs displays common features, characterized by the presence of at least 2 main conformations whose populations are modulated by the presence of the bound lipid. These conformational properties correlate with their mechanistic mode of action, allowing for the interpretation and design of experimental strategies to further dissect their mechanism. Our findings indicate the existence of a conserved, fold-independent mechanism of lipid transfer across LTPs of various families and offer a general framework for understanding their functional mechanism.

Unbiased MD simulations identify lipid binding sites in lipid transfer proteins.

The characterization of lipid binding to lipid transfer proteins (LTPs) is fundamental to understand their molecular mechanism. However, several structures of LTPs, and notably those proposed to act as bridges between membranes, do not provide the precise location of their endogenous lipid ligands. To address this limitation, computational approaches are a powerful alternative methodology, but they are often limited by the high flexibility of lipid substrates. Here, we develop a protocol based on unbiased coarse-grain molecular dynamics simulations in which lipids placed away from the protein can spontaneously bind to LTPs. This approach accurately determines binding pockets in LTPs and provides a working hypothesis for the lipid entry pathway. We apply this approach to characterize lipid binding to bridge LTPs of the Vps13-Atg2 family, for which the lipid localization inside the protein is currently unknown. Overall, our work paves the way to determine binding pockets and entry pathways for several LTPs in an inexpensive, fast, and accurate manner.

Structure of the human TIP60 complex.

Mammalian TIP60 is a multi-functional enzyme with histone acetylation and histone dimer exchange activities. It plays roles in diverse cellular processes including transcription, DNA repair, cell cycle control, and embryonic development. Here we report the cryo-electron microscopy structures of the human TIP60 complex with the core subcomplex and TRRAP module refined to 3.2-Å resolution. The structures show that EP400 acts as a backbone integrating the motor module, the ARP module, and the TRRAP module. The RUVBL1-RUVBL2 hexamer serves as a rigid core for the assembly of EP400 ATPase and YL1 in the motor module. In the ARP module, an ACTL6A-ACTB heterodimer and an extra ACTL6A make hydrophobic contacts with EP400 HSA helix, buttressed by network interactions among DMAP1, EPC1, and EP400. The ARP module stably associates with the motor module but is flexibly tethered to the TRRAP module, exhibiting a unique feature of human TIP60. The architecture of the nucleosome-bound human TIP60 reveals an unengaged nucleosome that is located between the core subcomplex and the TRRAP module. Our work illustrates the molecular architecture of human TIP60 and provides architectural insights into how this complex is bound by the nucleosome.

Long-chain fatty acids block allergic reaction against lipid transfer protein Sola l 7 from tomato seeds.

Due to the benefits of tomato as an antioxidant and vitamin source, allergy to this vegetable food is a clinically concerning problem. Sola l 7, a class I lipid transfer protein found in tomato seeds, has been identified as an allergen linked to severe anaphylaxis. However, the role of lipid binding in Sola l 7-induced allergy remains unclear. Here, the three-dimensional structure of recombinant Sola l 7 (rSola l 7) has been elucidated using nuclear magnetic resonance spectroscopy (NMR). Its interaction with free fatty acids has been deeply studied; fluorescence emission spectroscopy revealed that different long-chain fatty acids interact with the protein, affecting the only tyrosine residue present in Sola l 7. On the contrary, no changes in the overall secondary structure were observed after the analysis of the circular dichroism spectra in the presence of fatty acids. Unsaturated oleic and linoleic fatty acids presented higher affinity and promoted more significant changes than saturated or short-chain fatty acids. 1H-15N HSQC NMR spectra allowed to determine the regions of the protein that were modified when rSola l 7 interacts with the fatty acids, suggesting epitope modification after the interaction. For corroboration, IgG and IgE binding to rSola l 7 were assessed in the presence of free fatty acids, revealing that both IgE and IgG binding were significantly lower than in their absence, suggesting a potential protective role of unsaturated fatty acids in tomato allergy.

Model Mechanism for Lipid Uptake by the Human STARD2/PC-TP Phosphatidylcholine Transfer Protein.

The human StAR-related lipid transfer domain protein 2 (STARD2), also known as phosphatidylcholine (PC) transfer protein, is a single-domain lipid transfer protein thought to transfer PC lipids between intracellular membranes. We performed extensive µs-long molecular dynamics simulations of STARD2 of its apo and holo forms in the presence or absence of complex lipid bilayers. The simulations in water reveal ligand-dependent conformational changes. In the 2 µs-long simulations of apo STARD2 in the presence of a lipid bilayer, we observed spontaneous reproducible PC lipid uptake into the protein hydrophobic cavity. We propose that the lipid extraction mechanism involves one to two metastable states stabilized by choline-tyrosine or choline-tryptophane cation-π interactions. Using free energy perturbation, we evaluate that PC-tyrosine cation-π interactions contribute 1.8 and 2.5 kcal/mol to the affinity of a PC-STARD2 metastable state, thus potentially providing a significant decrease of the energy barrier required for lipid desorption.

An integrated structural model of the DNA damage-responsive H3K4me3 binding WDR76:SPIN1 complex with the nucleosome.

Serial capture affinity purification (SCAP) is a powerful method to isolate a specific protein complex. When combined with cross-linking mass spectrometry and computational approaches, one can build an integrated structural model of the isolated complex. Here, we applied SCAP to dissect a subpopulation of WDR76 in complex with SPIN1, a histone reader that recognizes trimethylated histone H3 lysine4 (H3K4me3). In contrast to a previous SCAP analysis of the SPIN1:SPINDOC complex, histones and the H3K4me3 mark were enriched with the WDR76:SPIN1 complex. Next, interaction network analysis of copurifying proteins and microscopy analysis revealed a potential role of the WDR76:SPIN1 complex in the DNA damage response. Since we detected 149 pairs of cross-links between WDR76, SPIN1, and histones, we then built an integrated structural model of the complex where SPIN1 recognized the H3K4me3 epigenetic mark while interacting with WDR76. Finally, we used the powerful Bayesian Integrative Modeling approach as implemented in the Integrative Modeling Platform to build a model of WDR76 and SPIN1 bound to the nucleosome.

Structural and Functional Characterization of New Lipid Transfer Proteins with Chitin-Binding Properties: Insights from Protein Structure Prediction, Molecular Docking, and Antifungal Activity.

Faced with the emergence of multiresistant microorganisms that affect human health, microbial agents have become a serious global threat, affecting human health and plant crops. Antimicrobial peptides have attracted significant attention in research for the development of new microbial control agents. This work's goal was the structural characterization and analysis of antifungal activity of chitin-binding peptides from Capsicum baccatum and Capsicum frutescens seeds on the growth of Candida and Fusarium species. Proteins were initially submitted to extraction in phosphate buffer pH 5.4 and subjected to chitin column chromatography. Posteriorly, two fractions were obtained for each species, Cb-F1 and Cf-F1 and Cb-F2 and Cf-F2, respectively. The Cb-F1 (C. baccatum) and Cf-F1 (C. frutescens) fractions did not bind to the chitin column. The electrophoresis results obtained after chromatography showed two major protein bands between 3.4 and 14.2 kDa for Cb-F2. For Cf-F2, three major bands were identified between 6.5 and 14.2 kDa. One band from each species was subjected to mass spectrometry, and both bands showed similarity to nonspecific lipid transfer protein. Candida albicans and Candida tropicalis had their growth inhibited by Cb-F2. Cf-F2 inhibited the development of C. albicans but did not inhibit the growth of C. tropicalis. Both fractions were unable to inhibit the growth of Fusarium species. The toxicity of the fractions was tested in vivo on Galleria mellonella larvae, and both showed a low toxicity rate at high concentrations. As a result, the fractions have enormous promise for the creation of novel antifungal compounds.

Role of AcrAB-TolC and Its Components in Influx-Efflux Dynamics of QAC Drugs in Escherichia coli Revealed Using SHG Spectroscopy.

Multidrug efflux pumps, especially those belonging to the class of resistance-nodulation-division (RND), are the key contributors to the rapidly growing multidrug resistance in Gram-negative bacteria. Understanding the role of efflux pumps in real-time drug transport dynamics across the complex dual-cell membrane envelope of Gram-negative bacteria is thus crucial for developing efficient antibiotics against them. Here, we employ second harmonic generation-based nonlinear spectroscopy to study the role of the tripartite efflux pump and its individual components. We systematically investigate the effect of periplasmic adaptor protein AcrA, inner membrane transporter protein AcrB, and outer membrane channel TolC on the overall drug transport in live Acr-type Escherichia coli and its mutant strain cells. Our results reveal that when one of its components is missing, the tripartite AcrAB-TolC efflux pump machinery in Escherichia coli can effectively function as a bipartite system, a fact that has never been demonstrated in live Gram-negative bacteria.

Investigating Lipid Transporter Protein and Lipid Interactions Using Variable Temperature Electrospray Ionization, Ultraviolet Photodissociation Mass Spectrometry, and Collision Cross Section Analysis.

Gram-negative bacteria develop and exhibit resistance to antibiotics, owing to their highly asymmetric outer membrane maintained by a group of six proteins comprising the Mla (maintenance of lipid asymmetry) pathway. Here, we investigate the lipid binding preferences of one Mla protein, MlaC, which transports lipids through the periplasm. We used ultraviolet photodissociation (UVPD) to identify and characterize modifications of lipids endogenously bound to MlaC expressed in three different bacteria strains. UVPD was also used to localize lipid binding to MlaC residues 130-140, consistent with the crystal structure reported for lipid-bound MlaC. The impact of removing the bound lipid from MlaC on its structure was monitored based on collision cross section measurements, revealing that the protein unfolded prior to release of the lipid. The lipid selectivity of MlaC was evaluated based on titrimetric experiments, indicating that MlaC-bound lipids in various classes (sphingolipids, glycerophospholipids, and fatty acids) as long as they possessed no more than two acyl chains.

Solid Phase Peptide Carrier Conjugation.

Conjugation to carrier proteins is necessary for peptides to be able to induce antibody formation when injected into animals together with a suitable adjuvant. This is usually performed by conjugation in solution followed by mixing with the adjuvant. Alternatively, the carrier may be adsorbed onto a solid support followed by activation and conjugation with the peptide by solid-phase chemistry. Different reagents can be used for conjugation through peptide functional groups (-SH, -NH2, -COOH), and various carrier proteins may be used depending on the peptides and the intended use of the antibodies. The solid phase may be an ion exchange matrix, from which the conjugate can subsequently be eluted and mixed with adjuvant. Alternatively, the adjuvant aluminum hydroxide may be used as the solid-phase matrix, whereupon the carrier is immobilized and conjugated with peptide. The resulting adjuvant-carrier-peptide complexes may then be used directly for immunization.

Using lipid binding proteins and advanced microscopy to study lipid domains.

Sphingomyelin is postulated to form clusters with glycosphingolipids, cholesterol and other sphingomyelin molecules in biomembranes through hydrophobic interaction and hydrogen bonds. These clusters form submicron size lipid domains. Proteins that selectively binds sphingomyelin and/or cholesterol are useful to visualize the lipid domains. Due to their small size, visualization of lipid domains requires advanced microscopy techniques in addition to lipid binding proteins. This Chapter describes the method to characterize plasma membrane sphingomyelin-rich and cholesterol-rich lipid domains by quantitative microscopy. This Chapter also compares different permeabilization methods to visualize intracellular lipid domains.

Structures and Efflux Mechanisms of the AcrAB-TolC Pump.

The global emergence of multidrug resistance (MDR) in gram-negative bacteria has become a matter of worldwide concern. MDR in these pathogens is closely linked to the overexpression of certain efflux pumps, particularly the resistance-nodulation-cell division (RND) efflux pumps. Inhibition of these pumps presents an attractive and promising strategy to combat antibiotic resistance, as the efflux pump inhibitors can effectively restore the potency of existing antibiotics. AcrAB-TolC is one well-studied RND efflux pump, which transports a variety of substrates, therefore providing resistance to a broad spectrum of antibiotics. To develop effective pump inhibitors, a comprehensive understanding of the structural aspect of the AcrAB-TolC efflux pump is imperative. Previous studies on this pump's structure have been limited to individual components or in vitro determination of fully assembled pumps. Recent advancements in cellular cryo-electron tomography (cryo-ET) have provided novel insights into this pump's assembly and functional mechanism within its native cell membrane environment. Here, we present a summary of the structural data regarding the AcrAB-TolC efflux pump, shedding light on its assembly pathway and operational mechanism.

Conservation of C4BP-binding sequence patterns in Streptococcus pyogenes M and Enn proteins.

Antigenically sequence variable M proteins of the major bacterial pathogen Streptococcus pyogenes (Strep A) are responsible for recruiting human C4b-binding protein (C4BP) to the bacterial surface, which enables Strep A to evade destruction by the immune system. The most sequence divergent portion of M proteins, the hypervariable region (HVR), is responsible for binding C4BP. Structural evidence points to the conservation of two C4BP-binding sequence patterns (M2 and M22) in the HVR of numerous M proteins, with this conservation applicable to vaccine immunogen design. These two patterns, however, only partially explain C4BP binding by Strep A. Here, we identified several M proteins that lack these patterns but still bind C4BP and determined the structures of two, M68 and M87 HVRs, in complex with a C4BP fragment. Mutagenesis of these M proteins led to the identification of amino acids that are crucial for C4BP binding, enabling formulation of new C4BP-binding patterns. Mutagenesis was also carried out on M2 and M22 proteins to refine or generate experimentally grounded C4BP-binding patterns. The M22 pattern was the most prevalent among M proteins, followed by the M87 and M2 patterns, while the M68 pattern was rare. These patterns, except for M68, were also evident in numerous M-like Enn proteins. Binding of C4BP via these patterns to Enn proteins was verified. We conclude that C4BP-binding patterns occur frequently in Strep A strains of differing M types, being present in their M or Enn proteins, or frequently both, providing further impetus for their use as vaccine immunogens.

Deciphering the drug delivery potential of Type1 lipid transfer protein from Citrus sinensis for enhancing the therapeutic efficacy of drugs.

Type1 Non-specific Lipid Transfer Protein (CsLTP1) from Citrus sinensis is a small cationic protein possessing a long tunnel-like hydrophobic cavity. CsLTP1 performing membrane trafficking of lipids is a promising candidate for developing a potent drug delivery system. The present work includes in-silico studies and the evaluation of drugs binding to CsLTP1 using biophysical techniques along with the investigation of CsLTP1's ability to enhance the efficacy of drugs employing cell-based bioassays. The in-silico investigations identified Panobinostat, Vorinostat, Cetylpyridinium Chloride, and Fulvestrant with higher affinities and stability of binding to the hydrophobic pocket of CsLTP1. SPR studies revealed strong binding affinities of anticancer drugs, Panobinostat (KD = 1.40 µM) and Vorinostat (KD = 2.17 µM) to CsLTP1 along with the binding and release kinetics. CD and fluorescent spectroscopy revealed drug-induced conformational changes in CsLTP1. CsLTP1-associated drug forms showed remarkably enhanced efficacy in MCF-7 cells, representing increased cell cytotoxicity, intracellular ROS, reduced mitochondrial membrane potential, and up-regulation of proapoptotic markers than the free drugs employing qRT-PCR and western blot analysis. The findings demonstrate that CsLTP1 binds strongly to hydrophobic drugs to facilitate their transport, hence improving their therapeutic efficacy revealed by the in-vitro investigations. This study establishes an excellent foundation for developing CsLTP1-based efficient drug delivery system.

Influence of the interaction between p53 and ZNF568 on mitochondrial oxidative phosphorylation.

The tumor suppressor p53 plays important roles in suppressing the development and progression of cancer by responding to various stress signals. In addition, p53 can regulate the metabolic pathways of cancer cells by regulating energy metabolism and oxidative phosphorylation. Here, we present a mechanism for the interaction between p53 and ZNF568. Initially, we used X-ray crystallography to determine the irregular loop structure of the ZNF568 KRAB domain; this loop plays an important role in the interaction between p53 and ZNF568. In addition, Cryo-EM was used to examine how the p53 DBD and ZNF568 KRAB domains bind together. The function of ZNF568 on p53-mediated mitochondrial respiration was confirmed by measuring glucose consumption and lactate production. These findings show that ZNF568 can reduce p53-mediated mitochondrial respiratory activity by binding to p53 and inhibiting the transcription of SCO2. SIGNIFICANCE: ZNF568 can directly bind to the p53 DBD and transcriptionally regulate the SCO2 gene. SCO2 transcriptional regulation by interaction between ZNF568 and p53 may regulate the balance between mitochondrial respiration and glycolysis.

Navigating the complexities of docking tools with nicotinic receptors and acetylcholine binding proteins in the realm of neonicotinoids.

Molecular docking, pivotal in predicting small-molecule ligand binding modes, struggles with accurately identifying binding conformations and affinities. This is particularly true for neonicotinoids, insecticides whose impacts on ecosystems require precise molecular interaction modeling. This study scrutinizes the effectiveness of prominent docking software (Ledock, ADFR, Autodock Vina, CDOCKER) in simulating interactions of environmental chemicals, especially neonicotinoid-like molecules with nicotinic acetylcholine receptors (nAChRs) and acetylcholine binding proteins (AChBPs). We aimed to assess the accuracy and reliability of these tools in reproducing crystallographic data, focusing on semi-flexible and flexible docking approaches. Our analysis identified Ledock as the most accurate in semi-flexible docking, while Autodock Vina with Vinardo scoring function proved most reliable. However, no software consistently excelled in both accuracy and reliability. Additionally, our evaluation revealed that none of the tools could establish a clear correlation between docking scores and experimental dissociation constants (Kd) for neonicotinoid-like compounds. In contrast, a strong correlation was found with drug-like compounds, bringing to light a bias in considered software towards pharmaceuticals, thus limiting their applicability to environmental chemicals. The comparison between semi-flexible and flexible docking revealed that the increased computational complexity of the latter did not result in enhanced accuracy. In fact, the higher computational cost of flexible docking with its lack of enhanced predictive accuracy, rendered this approach useless for this class of compounds. Conclusively, our findings emphasize the need for continued development of docking methodologies, particularly for environmental chemicals. This study not only illuminates current software capabilities but also underscores the urgency for advancements in computational molecular docking as it is a relevant tool to environmental sciences.

Fluorometric Analysis of Carrier-Protein-Dependent Biosynthesis through a Conformationally Sensitive Solvatochromic Pantetheinamide Probe.

Carrier proteins (CPs) play a fundamental role in the biosynthesis of fatty acids, polyketides, and non-ribosomal peptides, encompassing many medicinally and pharmacologically relevant compounds. Current approaches to analyze novel carrier-protein-dependent synthetic pathways are hampered by a lack of activity-based assays for natural product biosynthesis. To fill this gap, we turned to 3-methoxychromones, highly solvatochromic fluorescent molecules whose emission intensity and wavelength are heavily dependent on their immediate molecular environment. We have developed a solvatochromic carrier-protein-targeting probe which is able to selectively fluoresce when bound to a target carrier protein. Additionally, the probe displays distinct responses upon CP binding in carrier-protein-dependent synthases. This discerning approach demonstrates the design of solvatochromic fluorophores with the ability to identify biosynthetically active CP-enzyme interactions.

Cyanovirin-N Binding to N-Acetyl-d-glucosamine Requires Carbohydrate-Binding Sites on Two Different Protomers.

Cyanovirin-N (CV-N) binds high-mannose oligosaccharides on enveloped viruses with two carbohydrate-binding sites, one bearing high affinity and one low affinity to Manα(1-2)Man moieties. A tandem repeat of two CV-N molecules (CVN2) was tested for antiviral activity against human immunodeficiency virus type I (HIV-1) by using a domain-swapped dimer. CV-N was shown to bind N-acetylmannosamine (ManNAc) and N-acetyl-d-glucosamine (GlcNAc) when the carbohydrate-binding sites in CV-N were free to interact with these monosaccharides independently. CVN2 recognized ManNAc at a Kd of 1.4 µM and bound this sugar in solution, regardless of the lectin making amino acid side chain contacts on the targeted viral glycoproteins. An interdomain cross-contacting residue Glu41, which has been shown to be hydrogen bonding with dimannose, was substituted in the monomeric CV-N. The amide derivative of glucose, GlcNAc, achieved similar high affinity to the new variant CVN-E41T as high-mannose N-glycans, but binding to CVN2 in the nanomolar range with four binding sites involved or binding to the monomeric CVN-E41A. A stable dimer was engineered and expressed from the alanine-to-threonine-substituted monomer to confirm binding to GlcNAc. In summary, low-affinity binding was achieved by CVN2 to dimannosylated peptide or GlcNAc with two carbohydrate-binding sites of differing affinities, mimicking biological interactions with the respective N-linked glycans of interest and cross-linking of carbohydrates on human T cells for lymphocyte activation.

αMI-domain of integrin Mac-1 binds the cytokine pleiotrophin using multiple mechanisms.

The integrin Mac-1 (αMß2, CD11b/CD18, CR3) is an adhesion receptor expressed on macrophages and neutrophils. Mac-1 is also a promiscuous integrin that binds a diverse set of ligands through its αMI-domain. However, the binding mechanism of most ligands remains unclear. We have characterized the interaction of αMI-domain with the cytokine pleiotrophin (PTN), a protein known to bind αMI-domain and induce Mac-1-mediated cell adhesion and migration. Our data show that PTN's N-terminal domain binds a unique site near the N- and C-termini of the αMI-domain using a metal-independent mechanism. However, a stronger interaction is achieved when an acidic amino acid in a zwitterionic motif in PTN's C-terminal domain chelates the divalent cation in the metal ion-dependent adhesion site of active αMI-domain. These results indicate that αMI-domain can bind ligands using multiple mechanisms and that the active αMI-domain has a preference for motifs containing both positively and negatively charged amino acids.