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[Image: see text] The host defense peptide LL-37 is the only human cathelicidin, characterized by pleiotropic activity ranging from immunological to anti-neoplastic functions. However, its overexpression has been associated with harmful inflammatory responses and apoptosis. Thus, for the latter cases, the development of strategies aiming to reduce LL-37 toxicity is highly desired as these have the potential to provide a viable solution. Here, we demonstrate that the reduction of LL-37 toxicity might be achieved by the impairment of its cell surface binding through interaction with small organic compounds that are able to alter the peptide conformation and minimize its cell penetration ability. In this regard, the performed cell viability and internalization studies showed a remarkable attenuation of LL-37 cytotoxicity toward colon and monocytic cells in the presence of the polysulfonated drug suramin. The mechanistic examinations of the molecular details indicated that this effect was coupled with the ability of suramin to alter LL-37 secondary structure via the formation of peptide–drug complexes. Moreover, a comparison with other therapeutic agents having common features unveiled the peculiar ability of suramin to optimize the binding to the peptide sequence. The newly discovered suramin action is hoped to inspire the elaboration of novel repurposing strategies aimed to reduce LL-37 cytotoxicity under pathological conditions.

The intracellular pathogen Mycobacterium tuberculosis can survive and replicate within host macrophages. Among various immunomodulatory substances, macrophages also produce α 1 -acid glycoprotein (AAG) which is secreted into the extracellular matrix of tuberculosis granulomas that represents a specific binding environment. Employing circular dichroism (CD) and UV/VIS absorption spectroscopic methods, we demonstrated and evaluated the AAG binding properties of novel antitubercular drug candidates developed against sensitive and multidrug-resistant strains of M. tuberculosis. As inferred from the CD spectroscopic data, these chemically diverse organic molecules are engulfed within the β -barrel of the protein either in a monomeric or dimeric form. Molecular docking simulations suggested the importance of H-bonds and ligand-aromatic residue π - π stacking interactions in stabilizing the drug molecules at the protein binding site. Based on the estimated Kd values (7–20 μM), AAG could be considered as the significant binding partner of the antitubercular agents studied herein. As such, it may affect the drug distribution and bioavailability not only in serum but also in macrophages and in the extracellular matrix of tuberculosis granulomas.

Host defense antimicrobial peptides (HDPs) constitute an integral component of the innate immune system having nonspecific activity against a broad spectrum of microorganisms. They also have diverse biological functions in wound healing, angiogenesis, and immunomodulation, where it has also been demonstrated that they have a high affinity to interact with human lipid signaling molecules. Within bacterial biofilms, quorum sensing (QS), the vital bacterial cell-to-cell communication system, is maintained by similar diffusible small molecules which control phenotypic traits, virulence factors, biofilm formation, and dispersion. Efficient eradication of bacterial biofilms is of particular importance as these colonies greatly help individual cells to tolerate antibiotics and develop antimicrobial resistance. Regarding the antibacterial function, for several HDPs, including the human cathelicidin LL-37, affinity to eradicate biofilms can exceed their activity to kill individual bacteria. However, related underlying molecular mechanisms have not been explored yet. Here, we employed circular dichroism (CD) and UV/VIS spectroscopic analysis, which revealed that LL-37 exhibits QS signal affinity. This archetypal representative of HDPs interacts with the Pseudomonas quinolone signal (PQS) molecules, producing co-assemblies with peculiar optical activity. The binding of PQS onto the asymmetric peptide chains results in chiral supramolecular architectures consisting of helically disposed, J-aggregated molecules. Besides the well-known bacterial membrane disruption activity, our data propose a novel action mechanism of LL-37. As a specific case of the so-called quorum quenching, QS signal molecules captured by the peptide are sequestered inside co-assemblies, which may interfere with the microbial QS network helping to prevent and eradicate bacterial infections.

In the emerging era of antimicrobial resistance, the susceptibility to co-infections of patients suffering from either acquired or inherited hemolytic disorders can lead to dramatic increase in mortality rates. Closely related, heme liberated during hemolysis is one of the major sources of iron, which is vital for both host and invading microorganisms. While recent intensive research in the field has demonstrated that heme exerts diverse local effects including impairment of immune cells functions, it is almost completely unknown how it may compromise key molecules of our innate immune system, such as antimicrobial host defense peptides (HDPs). Since HDPs hold great promise as natural therapeutic agents against antibiotic-resistant microbes, understanding the effects that may modulate their action in microbial infection is crucial. Here we explore how hemin can interact directly with selected HDPs and influence their structure and membrane activity. It is revealed that induced helical folding, large assembly formation, and altered membrane activity is promoted by hemin. However, these effects showed variations depending mainly on peptide selectivity toward charged lipids, and the affinity of the peptide and hemin to lipid bilayers. Hemin-peptide complexes are sought to form semi-folded co-assemblies, which are present even with model membranes resembling mammalian or bacterial lipid compositions. In vitro cell-based toxicity assays supported that toxic effects of HDPs could be attenuated due to their assembly formation. These results are in line with our previous findings on peptide-lipid-small molecule systems suggesting that small molecules present in the complex in vivo milieu can regulate HDP function. Inversely, diverse effects of endogenous compounds could also be manipulated by HDPs.

New methods for quantifying extracellular vesicles (EVs) in complex biofluids are critically needed. We report the development of a new technology combining size exclusion chromatography (SEC), a commonly used EV purification technique, with fluorescence detection of specifically labelled EVs. The resulting platform, Flu-SEC, demonstrates a linear response to concentration of specific EVs and could form the basis of a system with phenotyping capability. Flu-SEC was validated using red blood cell derived EVs (REVs), which provide an ideal EV model with monodisperse size distribution and high EV concentration. Microfluidic Resistive Pulse Sensing (MRPS) was used to accurately determine the size distribution and concentration of REVs. Anti-CD235a antibody, specific to glycophorin A, and the more general wheat germ agglutinin (WGA), were selected to label REVs. The results show the quantitative power of Flu-SEC: a highly linear fluorescence response over a wide range of concentrations. Moreover, the Flu-SEC technique reports the ratio of EV-bound and free-antibody molecules, an important metric for determining optimal labelling conditions for other applications. Flu-SEC represents an orthogonal tool to single-particle fluorescent methods such as flow cytometry and fluorescent NTA, for the quantification and phenotyping of EVs.

Antimicrobial peptides (AMPs) kill bacteria by targeting their membranes through various mechanisms involving peptide assembly, often coupled with disorder‐to‐order structural transition. However, for several AMPs, similar conformational changes in cases in which small organic compounds of both endogenous and exogenous origin have induced folded peptide conformations have recently been reported. Thus, the function of AMPs and of natural host defence peptides can be significantly affected by the local complex molecular environment in vivo; nonetheless, this area is hardly explored. To address the relevance of such interactions with regard to structure and function, we have tested the effects of the therapeutic drug suramin on the membrane activity and antibacterial efficiency of CM15, a potent hybrid AMP. The results provided insight into a dynamic system in which peptide interaction with lipid bilayers is interfered with by the competitive binding of CM15 to suramin, resulting in an equilibrium dependent on peptide‐to‐drug ratio and vesicle surface charge. In vitro bacterial tests showed that when CM15⋅suramin complex formation dominates over membrane binding, antimicrobial activity is abolished. On the basis of this case study, it is proposed that small‐molecule secondary structure regulators can modify AMP function and that this should be considered and could potentially be exploited in future development of AMP‐based antimicrobial agents.

Owing to their potential applicability against multidrug-resistant bacteria, antimicrobial peptides (AMPs) or host defense peptides (HDPs) gain increased attention. Besides diverse immunomodulatory roles, their classical mechanism of action mostly involves membrane disruption of microbes. Notably, their unbalanced overexpression has also been associated with host cell cytotoxicity in various diseases. Relatedly, AMPs can be subject to aggregate formation, either via self-assembly or together with other compounds, which has demonstrated a modulation effect on their biological functions, thus highly relevant both for drug targeting projects and understanding their in vivo actions. However, the molecular aspects of the related assembly formation are not understood. Here, we focused in detail on an experimentally studied AMP-drug system, i.e., CM15-suramin, and performed all-atom and coarse-grain (CG) simulations. Results obtained for all systems were in close line with experimental observations and indicate that the CM15-suramin aggregation is an energetically favorable and dynamic process. In the presence of bilayers, the peptide-drug assembly formation was highly dependent on lipid composition, and peptide aggregates themselves were also capable of binding to the membranes. Interestingly, longer CG simulations with zwitterionic membranes indicated an intermediate state in the presence of both AMP-drug assemblies and monomeric peptides located on the membrane surface. In sharp contrast, larger AMP-drug aggregates could not be detected with a negatively charged membrane, rather the AMPs penetrated its surface in a monomeric form, in line with previous in vitro observations. Considering experimental and theoretical results, it is promoted that in biological systems, cationic AMPs may often form associates with anionic compounds in a reversible manner, resulting in lower bioactivity. This is only mildly affected by zwitterionic membranes; however, membranes with a negative charge strongly alter the energetic preference of AMP assemblies, resulting in the dissolution of the complexes into the membrane. The phenomenon observed here at a molecular level can be followed in several experimental systems studied recently, where peptides interact with food colors, drug molecules, or endogenous compounds, which strongly indicates that reversible associate formation is a general phenomenon for these complexes. These results are hoped to be exploited in novel therapeutic strategies aiming to use peptides as drug targets and control AMP bioactivity by directed assembly formation.

Besides the outstanding potential in biomedical applications, extracellular vesicles (EVs) are also promising candidates to expand our knowledge on interactions between vesicular surface proteins and small-molecules which exert biomembrane-related functions. Here we provide mechanistic details on interactions between membrane active peptides with antimicrobial effect (MAPs) and red blood cell derived EVs (REVs) and we demonstrate that they have the capacity to remove members of the protein corona from REVs even at lower than 5 μM concentrations. In case of REVs, the Soret-band arising from the membrane associated hemoglobins allowed to follow the detachment process by flow-Linear Dichroism (flow-LD). Further on, the significant change on the vesicle surfaces was confirmed by transmission electron microscopy (TEM). Since membrane active peptides, such as melittin have the affinity to disrupt vesicles, a combination of techniques, fluorescent antibody labeling, microfluidic resistive pulse sensing, and flow-LD were employed to distinguish between membrane destruction and surface protein detachment. The removal of protein corona members is a newly identified role for the investigated peptides, which indicates complexity of their in vivo function, but may also be exploited in synthetic and natural nanoparticle engineering. Furthermore, results also promote that EVs can be used as improved model systems for biophysical studies providing insight to areas with so far limited knowledge.

Body fluids flow all over the body and affect the biological processes at biointerfaces. To simulate such a case, sum frequency generation (SFG) vibrational spectroscopy and a self-designed microfluidic chip were combined together to investigate the interaction between a pH-responsive polymeric drug, poly(α-propylacrylic acid) (PPAAc), and the model cell membranes in different liquid environments. By examining the SFG spectra under the static and flowing conditions, the drug-membrane interaction was revealed comprehensively. The interfacial water layer was screened as the key factor affecting the drug-membrane interaction. The interfacial water layer can prevent the side propyl groups on PPAAc from inserting into the model cell membrane but would be disrupted by numerous ions in buffer solutions. Without flowing, at pH 6.6, the interaction between PPAAc and the model cell membrane was strongest; with flowing, at pH 5.8, the interaction was strongest. Flowing was proven to substantially affect the interaction between PPAAc and the model cell membranes, suggesting that the fluid environment was of key significance for biointerfaces. This work demonstrated that, by combining SFG and microfluidics, new information about the molecular-level interaction between macromolecules and the model cell membranes can be acquired, which cannot be obtained by collecting the normal static SFG spectra.

Dynamic increase of resistant bacterial infectious diseases continuously requires development of novel compounds against them. The molecular level understanding of the mechanism and interactions of natural host-defense peptides or antimicrobial peptides (AMPs) is an important step towards rational design and development of compounds inspired by their function. A particular set of these peptides have disordered structure, the ordering of which may modify their antimicrobial properties. Recent experiments demonstrate that such conformational transitions of AMPs could be mediated by the presence of small organic compounds, such as approved drug molecules. However, the molecular mechanisms underlying these structural changes are unclear. In this study, we apply molecular docking and molecular dynamics-based approaches to rigorously analyze the interactions between the drug suramin and the AMP CM15, a synthetic unstructured hybrid peptide. We characterize the energetic properties of putative CM15-suramin complexes revealing particular impacts of CM15 residues as well as the parts of suramin on these interactions. We find that α-helical content of the peptide is increased in the presence of suramin, which is in agreement with the experimental data. Kinetics analysis from canonical molecular dynamics and metadynamics simulations suggest that the effect of suramin does not promote the formation of α-helix but rather results from its ability to stabilize the α-helical population in the conformational pool of the peptide. Potentially, understanding the physico-chemical basis underlying the interactions between drug molecules and disordered AMPs will prove useful in strategies for antimicrobial compound development. Further on, the given computational protocol for the analysis of such flexible systems provide a basis for future theoretical investigation of similar biomolecular complexes.

A peculiar polygonal protein scaffolding that resembles to spectrin-based skeleton of red blood cells can be reconstructed on the outer surface of vesicle-like nanoerythrosomes. The approximately 130 nm sized nanoerythrosomes are produced from red blood cell ghosts by addition of phospholipids (dipalmitoylphosphatidylcholine, DPPC). The scaffolding, constructed from the structural proteins of the cell membrane skeleton, covers the whole object resulting an enhanced stiffness. The protein pattern of the scaffolding is thermosensitive, reversible transformable in the biologically relevant temperature range. When the lipid additive is changed from DPPC to lysophospholipid (LPC), the protein network/scaffolding ceases to exist. By the variation of lipid type and ratio, a tailoring of the nanoerythrosomes can be achieved. During the tailoring process nanoerythrosomes or micelles, in a wide size range from 200 to 30 nm, are produced.

The cell membrane is an ordered environment, which anisotropically affects the structure and interactions of all of its molecules. Monitoring membrane orientation at a local level is rather challenging but could reward crucial information on protein conformation and interactions in the lipid bilayer. We monitored local lipid ordering changes upon varying the cholesterol concentration using polarized light spectroscopy and pyrene as a membrane probe. Pyrene, with a shape intermediate between a disc and a rod, can detect microscopic orientation variations at the level of its size. The global membrane orientation was determined using curcumin, a probe with nonoverlapping absorption relative to that of pyrene. While the macroscopic orientation of a liquid-phase bilayer decreases with increasing cholesterol concentration, the local orientation is improved. Pyrene is found to be sensitive to the local effects induced by cholesterol and temperature on the bilayer. Disentangling local and global orientation effects in membranes could provide new insights into functionally significant interactions of membrane proteins.

Membrane-active, basic amphipathic peptides represent a class of biomolecules with diverse functions. Sequentially close protein segments also show similar behaviour in several ways. Here we investigated the effect of the lipid mediator lysophosphatidic acid (LPA) on the conformation of structurally disordered peptides including extracellular antimicrobial peptides (AMPs), and calmodulin-binding motifs derived from cytosolic and membrane target proteins. The interaction with associated LPA resulted in gain of ordered secondary structure elements, which for most cases were previously uncharacteristic of the particular peptide. Results revealed mechanism of the LPA-peptide interactions with regulation of the lipid on peptide conformation and oligomerization in a concentration-dependent manner involving (1) relocation of tryptophan residues into the lipid cluster, (2) multiple contacts between the binding partners dictated by complex driving forces, (3) multiple peptide binding to LPA associates with an affinity in the low micromolar range, and (4) selectivity for LPA compared with structurally related lipids. In line with recent findings showing endogenous molecules inducing structural changes in AMPs, we propose that accumulation of LPA in signalling or pathological processes might modulate host-defense activity or trigger certain processes by direct interaction with cationic amphipathic peptide sequences.

This study is aimed to assess the binding interaction between the antiparasitic cationic drug imidocarb (IMD) and sulfated glycosaminoglycans (GAGs), the ubiquitious nonprotein macromolecules of living organisms. These complex, heterogeneous polyanions are the integral constituents of cell membranes and the extracellular matrix and display affinity toward basic compounds, the binding of which may affect their biological functions. Exciton-type circular dichroism (CD) spectroscopic features measured at low salt concentration verify the heparin and heparan sulfate binding of IMD, which occurs in a cooperative manner by association of several drug molecules to a disaccharide unit. Isothermal titration calorimetry (ITC) measurements reassured the heparin interaction, resulting in a Kd value in the low micromolar range. In contrast, when considering high molar excess of the heparin-binding sites, closer resembling in vivo conditions, an entirely different CD signature was induced, suggesting a shift from the o...

Human serum albumin (HSA) binding of anticancer plant alkaloid ellipticine has been studied in conjunction with assessing its association to serum α1-acid-glycoprotein (AAG). Taking advantage of the ability of chiral protein environments to induce optical activity, circular dichroism (CD) spectroscopy was employed to characterize the alkaloid-protein non-covalent adducts. CD competition experiments revealed the simultaneous accommodation of two ellipticine molecules within the large crevice of HSA located in subdomain IB that induces an exciton coupling signature in the CD spectrum. Note that subdomain IIA was also found to host an ellipticine molecule. Supporting molecular docking simulations were also performed, which showed a hydrophobicity-driven stabilization of two ellipticine molecules within subdomain IB in a left-handed helical orientation. Similar to HSA, a pair of alkaloid ligands are inserted into the β-barrel cavity of AAG producing a polyphasic CD curve. The ‘F1/S’ and ‘A’ genetic variants of the protein bind ellipticine with comparable affinity albeit the distinct topography of the pockets provokes large intensity differences in the CD signals.

We present a computational model of the Vascular Endothelial Growth Factor (VEGF), an important regulator of blood vessels formation, which function is affected by its heparin interactions. Although structures of a receptor binding (RBD) and a heparin binding domain (HBD) of VEGF are known, there are structural data neither on the 12 amino acids interdomain linker nor on its complexes with heparin. We apply molecular docking and molecular dynamics techniques combined with circular dichroism spectroscopy to model the full structure of the dimeric VEGF and to propose putative molecular mechanisms underlying the function of VEGF/VEGF receptors/heparin system. We show that both the conformational flexibility of the linker and the formation of HBD-heparin-HBD sandwich-like structures regulate the mutual disposition of HBDs and so affect the VEGF-mediated signalling.

DJ-1 (PARK7) is a multifunctional protein linked to the onset and progression of a number of diseases, most of which are associated with high oxidative stress. The Cys106 of DJ-1 is unusually reactive and thus sensitive to oxidation, and due to high oxidative stress it was observed to be in various oxidized states in disease condition. The oxidation state of Cys106 of DJ-1 is believed to determine the specific functions of the protein in normal and disease conditions. Here we report molecular dynamics simulation and biophysical experimental studies on DJ-1 in reduced (Cys106, S-), oxidized (Cys106, SO2-), and over-oxidized (Cys106, SO3-) states. To simulate the different oxidation states of Cys106 in DJ-1, AMBER related force field parameters were developed and reported for 3-sulfinoalanine and cysteine sulfonic acid. Our studies found that the overall structure of DJ-1 in different oxidation states was similar globally, while it differed locally significantly, which have implications on its stability, function and its link to disease on-set. Importantly, the results suggest that over-oxidation may trigger loss of functions due to local structural modification in the Cys106 containing pocket of DJ-1 and structurally destabilize the dimeric state of DJ-1, which is believed to be its bioactive conformation. Such loss of functions would result in reduced ability of DJ-1 to protect from oxidative stress insults and may lead to increased progression of disease.