Ligand binding of interleukin-8: a comparison of glycosaminoglycans and acidic peptides.

Recognition and binding of regulatory proteins to glycosaminoglycans (GAGs) from the extracellular matrix is a process of high biological importance. The interaction between negatively charged sulfate or carboxyl groups of the GAGs and clusters of basic amino acids on the protein is crucial in this binding process and it is believed that electrostatics represent the key factor for this interaction. However, given the rather undirected nature of electrostatics, it is important to achieve a clear understanding of its role in protein-GAG interactions and how specificity and selectivity in these systems can be achieved, when the classical key-lock binding motif is not applicable. Here, we compare protein binding of a highly charged heparin (HP) hexasaccharide with four de novo designed decapeptides of varying negative net charge. The charge density of these peptides was comparable to typical GAGs of the extracellular matrix. We used the regulatory protein interleukin-8 (IL-8) because its interactions with GAGs are well described. All four peptide ligands bind to the same epitope of IL-8 but show much weaker binding affinity as revealed in 1H-15N HSQC NMR titration experiments. Complementary molecular docking and molecular dynamics simulations revealed further atomistic details of the interaction mode of GAG versus peptide ligands. Overall, similar contributions to the binding energy and hydrogen bond formation are determined for HP and the highly charged peptides, suggesting that the entropic loss of the peptides upon binding likely account for the remarkably different affinity of GAG versus peptide ligands to IL-8.

The Structural Basis of Peptide Binding at Class A G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) represent the largest membrane protein family and a significant target class for therapeutics. Receptors from GPCRs' largest class, class A, influence virtually every aspect of human physiology. About 45% of the members of this family endogenously bind flexible peptides or peptides segments within larger protein ligands. While many of these peptides have been structurally characterized in their solution state, the few studies of peptides in their receptor-bound state suggest that these peptides interact with a shared set of residues and undergo significant conformational changes. For the purpose of understanding binding dynamics and the development of peptidomimetic drug compounds, further studies should investigate the peptide ligands that are complexed to their cognate receptor.

The Conformational Equilibrium of the Neuropeptide Y2 Receptor in Bilayer Membranes.

Dynamic structural transitions within the seven-transmembrane bundle represent the mechanism by which G-protein-coupled receptors convert an extracellular chemical signal into an intracellular biological function. Here, the conformational dynamics of the neuropeptide Y receptor type 2 (Y2R) during activation was investigated. The apo, full agonist-, and arrestin-bound states of Y2R were prepared by cell-free expression, functional refolding, and reconstitution into lipid membranes. To study conformational transitions between these states, all six tryptophans of Y2R were 13 C-labeled. NMR-signal assignment was achieved by dynamic-nuclear-polarization enhancement and the individual functional states of the receptor were characterized by monitoring 13 C NMR chemical shifts. Activation of Y2R is mediated by molecular switches involving the toggle switch residue Trp2816.48 of the highly conserved SWLP motif and Trp3277.55 adjacent to the NPxxY motif. Furthermore, a conformationally preserved "cysteine lock"-Trp11623.50 was identified.

Interaction of the small-molecule kinase inhibitors tofacitinib and lapatinib with membranes.

Lapatinib and tofacitinib are small-molecule kinase inhibitors approved for the treatment of advanced or metastatic breast cancer and rheumatoid arthritis, respectively. So far, the mechanisms which are responsible for their activities are not entirely understood. Here, we focus on the interaction of these drug molecules with phospholipid membranes, which has not yet been investigated before in molecular detail. Owing to their lipophilic characteristics, quantitatively reflected by large differences of the partition equilibrium between water and octanol phases (expressed by logP values), rather drastic differences in the membrane interaction of both molecules have to be expected. Applying experimental (nuclear magnetic resonance, fluorescence and ESR spectroscopy) and theoretical (molecular dynamics simulations) approaches, we found that lapatinib and tofacitinib bind to lipid membranes and insert into the lipid-water interface of the bilayer. For lapatinib, a deeper embedding into the membrane bilayer was observed than for tofacitinib implying different impacts of the molecules on the bilayer structure. While for tofacitinib, no influence to the membrane structure was found, lapatinib causes a membrane disturbance, as concluded from an increased permeability of the membrane for polar molecules. These data may contribute to a better understanding of the cellular uptake mechanism(s) and the side effects of the drugs.

NMR and molecular modeling reveal specificity of the interactions between CXCL14 and glycosaminoglycans.

CXCL14, chemokine (C-X-C motif) ligand 14, is a novel highly conserved chemokine with unique features. Despite exhibiting the typical chemokine fold, it has a very short N-terminus of just two amino acid residues responsible for chemokine receptor activation. CXCL14 actively participates in homeostatic immune surveillance of skin and mucosae, is linked to metabolic disorders and fibrotic lung diseases and possesses strong anti-angiogenic properties in early tumor development. In this work, we investigated the interaction of CXCL14 with various glycosaminoglycans (GAGs) by nuclear magnetic resonance spectroscopy, microscale thermophoresis, analytical heparin (HE) affinity chromatography and in silico approaches to understand the molecular basis of GAG-binding. We observed different GAG-binding modes specific for the GAG type used in the study. In particular, the CXCL14 epitope for HE suggests a binding pose distinguishable from the ones of the other GAGs investigated (hyaluronic acid, chondroitin sulfate-A/C, -D, dermatan sulfate). This observation is also supported by computational methods that included molecular docking, molecular dynamics and free energy calculations. Based on our results, we suggest that distinct GAG sulfation patterns confer specificity beyond simple electrostatic interactions usually considered to represent the driving forces in protein-GAG interactions. The CXCL14-GAG system represents a promising approach to investigate the specificity of GAG-protein interactions, which represents an important topic for developing the rational approaches to novel strategies in regenerative medicine.

Transmembrane Helix Induces Membrane Fusion through Lipid Binding and Splay.

The fusion of biological membranes may require splayed lipids whose tails transiently visit the headgroup region of the bilayer, a scenario suggested by molecular dynamics simulations. Here, we examined the lipid splay hypothesis experimentally by relating liposome fusion and lipid splay induced by model transmembrane domains (TMDs). Our results reveal that a conformationally flexible transmembrane helix promotes outer leaflet mixing and lipid splay more strongly than a conformationally rigid one. The lipid dependence of basal as well as of TMD-driven lipid mixing and splay suggests that the cone-shaped phosphatidylethanolamine stimulates basal fusion via enhancing lipid splay and that the negatively charged phosphatidylserine inhibits fusion via electrostatic repulsion. Phosphatidylserine also strongly differentiates basal and helix-driven fusion, which is related to its preferred interaction with the conformationally more flexible transmembrane helix. Thus, the contribution of a transmembrane helix to membrane fusion appears to depend on lipid binding, which results in lipid splay.

Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor.

Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor superfamily and have important roles in food intake, anxiety and cancer biology 1,2 . The NPY-Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in most mammals, namely the Y1, Y2, Y4 and Y5 receptors, with different affinity and selectivity 3 . NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y1 receptor (Y1R) 4 . A number of peptides and small-molecule compounds have been characterized as Y1R antagonists and have shown clinical potential in the treatment of obesity 4 , tumour 1 and bone loss 5 . However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability 6 . Here we report crystal structures of the human Y1R bound to the two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal the binding modes of Y1R to several structurally diverse antagonists and the determinants of ligand selectivity. The Y1R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance, photo-crosslinking and functional studies, provide insights into the binding behaviour of the agonist and for the first time, to our knowledge, determine the interaction of its N terminus with the receptor. These insights into Y1R can enable structure-based drug discovery that targets NPY receptors.

The interaction of sorafenib and regorafenib with membranes is modulated by their lipid composition.

Sorafenib and regorafenib are small-molecule kinase inhibitors approved for the treatment of locally recurrent or metastatic, progressive, differentiated thyroid carcinoma, renal cell carcinoma, and hepatocellular carcinoma (sorafenib) and of colorectal cancer (regorafenib). As of now, the mechanisms, which are responsible for their antitumor activities, are not completely understood. Given the lipophilic nature of the molecules, it can be hypothesized that the pharmacological impact is mediated by the interaction with cellular membranes as it is true for many pharmacologically active molecules. However, an interaction of sorafenib or regorafenib with lipid membranes has not yet been investigated in detail. Here, we characterized the interaction of both drugs with lipid membranes by applying a variety of biophysical approaches including nuclear magnetic resonance, electron spin resonance, and fluorescence spectroscopy. We found that sorafenib and regorafenib bind to lipid membranes by inserting into the lipid-water interface of the bilayer. This membrane embedding causes a disturbance of bilayer structure leading to an increased permeability of the membrane for polar molecules. One approach shows that the extent of the effects depends on the membrane lipid composition underlining a particular role of phosphatidylcholine and cholesterol. Our data for the first time characterize the impact of sorafenib and regorafenib on the lipid membrane structure and dynamics, which may contribute to a better understanding of their effectiveness in the treatment of malignancies as well as of their side effects.

The adrenal specific toxicant mitotane directly interacts with lipid membranes and alters membrane properties depending on lipid composition.

Mitotane (o,p'.-DDD) is an orphan drug approved for the treatment of adrenocortical carcinoma. The mechanisms, which are responsible for this activity of the drug, are not completely understood. It can be hypothesized that an impact of mitotane is mediated by the interaction with cellular membranes. However, an interaction of mitotane with (lipid) membranes has not yet been investigated in detail. Here, we characterized the interaction of mitotane and its main metabolite o,p'-dichlorodiphenyldichloroacetic acid (o,p'-DDA) with lipid membranes by applying a variety of biophysical approaches of nuclear magnetic resonance, electron spin resonance, and fluorescence spectroscopy. We found that mitotane and o,p'-DDA bind to lipid membranes by inserting into the lipid-water interface of the bilayer. Mitotane but not o,p'-DDA directly causes a disturbance of bilayer structure leading to an increased permeability of the membrane for polar molecules. Mitotane induced alterations of the membrane integrity required the presence of phosphatidylethanolamine and/or cholesterol. Collectively, our data for the first time characterize the impact of mitotane on the lipid membrane structure and dynamics, which may contribute to a better understanding of specific mitotane effects and side effects.

A Selective Mucin/Methylcellulose Hybrid Gel with Tailored Mechanical Properties.

Mucin glycoproteins are key components of native mucus which serves as an initial barrier in the human body against microbial attack. Mucins are able to prevent bacterial adhesion and can trap viruses. However, the weak mechanical properties of mucin solutions have so far prevented their application in a physiological environment. Here, methylcellulose biopolymers are used as mechanical adjuvants to overcome this limitation and generate a thermoresponsive mucin/methylcellulose hybrid system. The hybrid material developed combines the selective permeability properties brought about by mucins with the thermal autogelation properties of methylcellulose. As a consequence, triggered by contact with body-warm surfaces, the hybrid material rapidly forms a gel at physiological conditions, and this external temperature stimulus can also be harnessed to stimulate drug release from incorporated thermosensitive liposomes. Finally, the hybrid gel selectively retards the release of embedded molecules which can be used to further control and prolong drug release from the material.

Scleral cross-linking by riboflavin and blue light application in young rabbits: damage threshold and eye growth inhibition.

BACKGROUND: Scleral cross-linking (SXL) by riboflavin and light application has been introduced as a possible treatment to increase scleral tissue stiffness and to inhibit excessive axial elongation of highly myopic eyes. We evaluated an ocular tissue damage threshold for blue light irradiation, and used SXL treatment to induce eye growth inhibition. METHODS: The sclera of 3-week-old rabbits (39 pigmented and 15 albino rabbits) were treated with different blue light intensities (450 ± 50 nm) and riboflavin. Alterations and a damage threshold were detected in ocular tissues by means of light microscopy and immunohistochemistry. The influence of SXL on the eye growth was examined in 21 young rabbits and was measured by using A-scan ultrasonography, micrometer caliper, and for selected eyes additionally by MR imaging. RESULTS: Light microscopic examinations demonstrated degenerative changes in ocular tissue after irradiation with blue light intensities above 400 mW/cm(2) (with and without riboflavin application). Therefore, that light intensity was defined as the damage threshold. Tissue alteration in retina, choroid, and sclera and activation of retinal microglia cells and Müller cells could be earlier observed at blue light intensities of 150 and 200 mW/cm(2). Albino rabbits were less sensitive to this SXL treatment. A significant reduction of the eye growth could be detected by SXL treatment with the minimal efficient blue light intensity of 15 mW/cm(2) and maintained stable for 24 weeks. CONCLUSIONS: SXL with riboflavin and blue light intensities below a defined damage threshold can induce a long lasting growth inhibitory effect on young rabbit eyes. Therefore, SXL might be a realistic approach to inhibit eye elongation in highly myopic eyes.

Structural Thermodynamics of myr-Src(2-19) Binding to Phospholipid Membranes.

Many proteins are anchored to lipid bilayer membranes through a combination of hydrophobic and electrostatic interactions. In the case of the membrane-bound nonreceptor tyrosine kinase Src from Rous sarcoma virus, these interactions are mediated by an N-terminal myristoyl chain and an adjacent cluster of six basic amino-acid residues, respectively. In contrast with the acyl modifications of other lipid-anchored proteins, the myristoyl chain of Src does not match the host lipid bilayer in terms of chain conformation and dynamics, which is attributed to a tradeoff between hydrophobic burial of the myristoyl chain and repulsion of the peptidic moiety from the phospholipid headgroup region. Here, we combine thermodynamic information obtained from isothermal titration calorimetry with structural data derived from (2)H, (13)C, and (31)P solid-state nuclear magnetic resonance spectroscopy to decipher the hydrophobic and electrostatic contributions governing the interactions of a myristoylated Src peptide with zwitterionic and anionic membranes made from lauroyl (C12:0) or myristoyl (C14:0) lipids. Although the latter are expected to enable better hydrophobic matching, the Src peptide partitions more avidly into the shorter-chain lipid analog because this does not require the myristoyl chain to stretch extensively to avoid unfavorable peptide/headgroup interactions. Moreover, we find that Coulombic and intrinsic contributions to membrane binding are not additive, because the presence of anionic lipids enhances membrane binding more strongly than would be expected on the basis of simple Coulombic attraction.

Integrating solid-state NMR and computational modeling to investigate the structure and dynamics of membrane-associated ghrelin.

The peptide hormone ghrelin activates the growth hormone secretagogue receptor 1a, also known as the ghrelin receptor. This 28-residue peptide is acylated at Ser3 and is the only peptide hormone in the human body that is lipid-modified by an octanoyl group. Little is known about the structure and dynamics of membrane-associated ghrelin. We carried out solid-state NMR studies of ghrelin in lipid vesicles, followed by computational modeling of the peptide using Rosetta. Isotropic chemical shift data of isotopically labeled ghrelin provide information about the peptide's secondary structure. Spin diffusion experiments indicate that ghrelin binds to membranes via its lipidated Ser3. Further, Phe4, as well as electrostatics involving the peptide's positively charged residues and lipid polar headgroups, contribute to the binding energy. Other than the lipid anchor, ghrelin is highly flexible and mobile at the membrane surface. This observation is supported by our predicted model ensemble, which is in good agreement with experimentally determined chemical shifts. In the final ensemble of models, residues 8-17 form an α-helix, while residues 21-23 and 26-27 often adopt a polyproline II helical conformation. These helices appear to assist the peptide in forming an amphipathic conformation so that it can bind to the membrane.

The orientation and dynamics of estradiol and estradiol oleate in lipid membranes and HDL disc models.

Estradiol (E2) and E2 oleate associate with high-density lipoproteins (HDLs). Their orientation in HDLs is unknown. We studied the orientation of E2 and E2 oleate in membranes and reconstituted HDLs, finding that E2 and E2 oleate are membrane-associated and highly mobile. Our combination of NMR measurements, molecular dynamics simulation, and analytic theory identifies three major conformations where the long axis of E2 assumes a parallel, perpendicular, or antiparallel orientation relative to the membrane's z-direction. The perpendicular orientation is preferred, and furthermore, in this orientation, E2 strongly favors a particular roll angle, facing the membrane with carbons 6, 7, 15, and 16, whereas carbons 1, 2, 11, and 12 point toward the aqueous phase. In contrast, the long axis of E2 oleate is almost exclusively oriented at an angle of ∼60° to the z-direction. In such an orientation, the oleoyl chain is firmly inserted into the membrane. Thus, both E2 and E2 oleate have a preference for interface localization in the membrane. These orientations were also found in HDL discs, suggesting that only lipid-E2 interactions determine the localization of the molecule. The structural mapping of E2 and E2 oleate may provide a design platform for specific E2-HDL-targeted pharmacological therapies.

Interaction of the human N-Ras protein with lipid raft model membranes of varying degrees of complexity.

Ternary lipid mixtures composed of cholesterol, saturated (frequently with sphingosine backbone), and unsaturated phospholipids show stable phase separation and are often used as model systems of lipid rafts. Yet, their ability to reproduce raft properties and function is still debated. We investigated the properties and functional aspects of three lipid raft model systems of varying degrees of biological relevance--PSM/POPC/Chol, DPPC/POPC/Chol, and DPPC/DOPC/Chol--using 2H solid-state nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, and atomic force microscopy. While some minor differences were observed, the general behavior and properties of all three model mixtures were similar to previously investigated influenza envelope lipid membranes, which closely mimic the lipid composition of biological membranes. For the investigation of the functional aspects, we employed the human N-Ras protein, which is posttranslationally modified by two lipid modifications that anchor the protein to the membrane. It was previously shown that N-Ras preferentially resides in liquid-disordered domains and exhibits a time-dependent accumulation in the domain boundaries of influenza envelope lipid membranes. For all three model mixtures, we observed the same membrane partitioning behavior for N-Ras. Therefore, we conclude that even relatively simple models of raft membranes are able to reproduce many of their specific properties and functions.

Oxidative in vitro folding of a cysteine deficient variant of the G protein-coupled neuropeptide Y receptor type 2 improves stability at high concentration.

In vitro folding of G protein-coupled receptors into a detergent environment represents a promising strategy for obtaining sufficient amounts of functional receptor molecules for structural studies. Typically, these preparations exhibit a poor long-term stability especially at the required high protein concentration. Here, we report a protocol for the stabilization of the Escherichia coli-expressed and subsequently folded neuropeptide Y receptor type 2. We identified the free cysteines in the receptor as one major reason for intermolecular protein aggregation. Therefore, six out of the eight cysteine residues were mutated to alanine or serine without any significant loss of functionality of the receptor as demonstrated in cell culture models. Furthermore, the disulfide bond between the remaining two cysteines was irreversibly formed by applying oxidative in vitro folding. Applying this strategy, the stability of the functionally folded Y2 receptor could be increased to 20 days at a concentration of 15 µm in a micelle environment consisting of 1,2-diheptanoyl-sn-glycero-3-phosphocholine and n-dodecyl-ß-D-maltoside.

Over-sulfated chondroitin sulfate derivatives induce osteogenic differentiation of hMSC independent of BMP-2 and TGF-ß1 signalling.

Natural glycosaminoglycans (GAGs) and chemically modified GAG derivatives are known to support osteogenic differentiation of mesenchymal stromal cells (MSC). This effect has mainly been described to be mediated by increasing the effectiveness of bone anabolic growth factors such as bone morphogenetic proteins (BMPs) due to the binding and presentation of the growth factor or by modulating its signal transduction pathway. In the present study, the influence of chondroitin sulfate (CS) and two chemically over-sulfated CS derivatives on osteogenic differentiation of human mesenchymal stromal cells (hMSC) and on BMP-2 and transforming growth factor ß1 (TGF-ß1) signalling was investigated. Over-sulfated CS derivatives induced an increase of tissue non-specific alkaline phosphatase (TNAP) activity and calcium deposition, whereas collagen synthesis was slightly decreased. The BMP-2-induced Smad1/5 activation was inhibited in the presence of over-sulfated CS derivatives leading to a loss of BMP-2-induced TNAP activity and calcium deposition. In contrast, the TGF-ß1-induced activation of Smad2/3 and collagen synthesis were not affected by the over-sulfated CS derivatives. BMP-2 and TGF-ß1 did not activate the extracellular signal-regulated kinase 1/2 or mitogen-activated protein kinase p38 in hMSC. These data suggest that over-sulfated CS derivatives themselves are able to induce osteogenic differentiation, probably independent of BMP-2 and TGF-ß1 signalling, and offer therefore an interesting approach for the improvement of bone healing.

Assessment of a fully active class A G protein-coupled receptor isolated from in vitro folding.

We provide a protocol for the preparation of fully active Y2 G protein-coupled receptors (GPCRs). Although a valuable target for pharmaceutical research, information about the structure and dynamics of these molecules remains limited due to the difficulty in obtaining sufficient amounts of homogeneous and fully active receptors for in vitro studies. Recombinant expression of GPCRs as inclusion bodies provides the highest protein yields at lowest costs. But this strategy can only successfully be applied if the subsequent in vitro folding results in a high yield of active receptors and if this fraction can be isolated from the nonactive receptors in a homogeneous form. Here, we followed that strategy to provide large quantities of the human neuropeptide Y receptor type 2 and determined the folding yield before and after ligand affinity chromatography using a radioligand binding assay. Directly after folding, we achieved a proportion of ~25% active receptor. This value could be increased to ~96% using ligand affinity chromatography. Thus, a very homogeneous sample of the Y2 receptor could be prepared that exhibited a K(D) value of 0.1 ± 0.05 nM for the binding of polypeptide Y, which represents one of the natural ligands of the Y2 receptor.

Quantitative analysis of denatured collagen by collagenase digestion and subsequent MALDI-TOF mass spectrometry.

Collagens are the most abundant proteins in vertebrate tissues and constitute significant moieties of the extracellular matrix (ECM). The determination of the collagen content is of relevance not only in the field of native tissue research, but also regarding the quality assessment of bioengineered tissues. Here, we describe a quantitative method to assess small amounts of collagen based on MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry (MS) subsequent to digestion of collagen with clostridial collagenase (clostridiopeptidase A) in order to obtain characteristic oligopeptides. Among the resulting peptides, Gly-Pro-Hyp, which is highly indicative of collagen, has been used to assess the amount of collagen by comparing the Gly-Pro-Hyp peak intensities with the intensities of a spiked tripeptide (Arg-Gly-Asp). The approach presented herein is both simple and convenient and allows the determination of collagen in microgram quantities. In tissue samples such as cartilage, the actual collagen content has additionally been determined for comparative purposes by nuclear magnetic resonance spectroscopy subsequent to acidic hydrolysis. Both methods give consistent data within an experimental error of ±10%. Although the differentiation of the different collagen types cannot be achieved by this approach, the overall collagen contents of tissues can be easily determined.

Investigating the membrane orientation and transversal distribution of 17beta-estradiol in lipid membranes by solid-state NMR.

17beta-Estradiol (E(2)) is a potent estrogen, which modulates many important cellular functions by binding to specific estrogen receptors located in the cell nucleus and also on the plasma membrane. We have studied the membrane interaction of E(2) using a combination of solid-state NMR methods. (2)H NMR results indicate that E(2) does not cause a condensation effect of the surrounding phospholipids, which is contrary to the effects of cholesterol, and only very modest E(2) induced alterations of the membrane structure were detected. (1)H magic-angle spinning NMR showed well resolved signals from E(2) as well as of POPC in the membrane-lipid layer. Two-dimensional NOESY spectra revealed intense cross-peaks between E(2) and the membrane lipids indicating that E(2) is stably inserted into the membrane. The determination of intermolecular cross-relaxation rates revealed that E(2) is broadly distributed in the membrane with a maximum of the E(2) distribution function in the upper chain region of the membrane. We conclude that E(2) is highly dynamic in lipid membranes and may undergo rotations as it exhibits two polar hydroxyl groups on either side of the molecule.