Localization Of Binding Sites Research Articles

Propofol modulates the lipid phase transition and localizes near the headgroup of membranes

The compound 2,6-diisopropylphenol (Propofol, PRF) is widely used for inducing general anesthesia, but the mechanism of PRF action remains relatively poorly understood at the molecular level. This work examines the possibility that a potential mode of action of PRF is to modulate the lipid order in target membranes. The effect on monolayers and bilayers of dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) was probed using Langmuir monolayer isotherms, differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC) and molecular dynamics (MD) simulations. Increasing amounts of PRF in a DPPC monolayer causes a decrease in isothermal compressibility modulus at the phase transition. A partition constant for PRF in DPPC liposomes on the order of K≈1500M−1 was found, and the partitioning was found to be enthalpy-driven above the melting temperature (Tm). A decrease in Tm with PRF content was found whereas the bilayer melting enthalpy ΔHm remains almost constant. The last finding indicates that PRF incorporates into the membrane at a depth near the phosphatidylcholine headgroup, in agreement with our MD-simulations. The simulations also reveal that PRF partitions into the membrane on a timescale of 0.5μs and has a cholesterol-like ordering effect on DPPC in the fluid phase. The vertical location of the PRF binding site in a bacterial ligand-gated ion channel coincides with the location found in our MD-simulations. Our results suggest that multiple physicochemical mechanisms may determine anesthetic potency of PRF, including effects on proteins that are mediated through the bilayer.

Na+–Substrate Coupling in the Multidrug Antiporter NorM Probed with a Spin-Labeled Substrate

NorM of the multidrug and toxic compound extrusion (MATE) family of transporters couples the efflux of a broad range of hydrophobic molecules to an inward Na⁺ gradient across the cell membrane. Several crystal structures of MATE transporters revealed distinct substrate binding sites leading to differing models of the mechanism of ion-coupled substrate extrusion. In the experiments reported here, we observed that a spin-labeled derivative of daunorubicin, Ruboxyl, is transported by NorM from Vibrio cholerae. It is therefore ideal for characterizing mechanistically relevant binding interactions with NorM and directly addressing the coupling of ion and drug binding. Fluorescence and electron paramagnetic resonance experiments revealed that Ruboxyl binds to NorM with micromolar affinity and becomes immobilized upon binding, even in the presence of Na⁺. Using double electron-electron resonance spectroscopy, we determined that Ruboxyl binds to a single site on the periplasmic side of the protein. The presence of Na⁺ did not translocate the substrate to a second site as previously proposed. These experiments surprisingly show that Na⁺ does not affect the affinity or location of the substrate binding site on detergent-solubilized NorM, thus suggesting that additional factors beyond simple mutual exclusivity of binding, such as the presence of a Na⁺ gradient across the native membrane, govern Na⁺-drug coupling during antiport.

Identification of LIN28B-bound mRNAs reveals features of target recognition and regulation

The conserved human LIN28 RNA-binding proteins function in development, maintenance of pluripotency and oncogenesis. We used PAR-CLIP and a newly developed variant of this method, iDo-PAR-CLIP, to identify LIN28B targets as well as sites bound by the individual RNA-binding domains of LIN28B in the human transcriptome at nucleotide resolution. The position of target binding sites reflected the known structural relative orientation of individual LIN28B-binding domains, validating iDo-PAR-CLIP. Our data suggest that LIN28B directly interacts with most expressed mRNAs and members of the let-7 microRNA family. The Lin28-binding motif detected in pre-let-7 was enriched in mRNA sequences bound by LIN28B. Upon LIN28B knockdown, cell proliferation and the cell cycle were strongly impaired. Quantitative shotgun proteomics of LIN28B depleted cells revealed significant reduction of protein synthesis from its RNA targets. Computational analyses provided evidence that the strength of protein synthesis reduction correlated with the location of LIN28B binding sites within target transcripts.

Identification of thyroid hormone receptor binding sites in developing mouse cerebellum

BackgroundThyroid hormones play an essential role in early vertebrate development as well as other key processes. One of its modes of action is to bind to the thyroid hormone receptor (TR) which, in turn, binds to thyroid response elements (TREs) in promoter regions of target genes. The sequence motif for TREs remains largely undefined as does the precise chromosomal location of the TR binding sites. A chromatin immunoprecipitation on microarray (ChIP-chip) experiment was conducted using mouse cerebellum post natal day (PND) 4 and PND15 for the thyroid hormone receptor (TR) beta 1 to map its binding sites on over 5000 gene promoter regions. We have performed a detailed computational analysis of these data.ResultsBy analysing a recent spike-in study, the optimal normalization and peak identification approaches were determined for our dataset. Application of these techniques led to the identification of 211 ChIP-chip peaks enriched for TR binding in cerebellum samples. ChIP-PCR validation of 25 peaks led to the identification of 16 true positive TREs. Following a detailed literature review to identify all known mouse TREs, a position weight matrix (PWM) was created representing the classic TRE sequence motif. Various classes of promoter regions were investigated for the presence of this PWM, including permuted sequences, randomly selected promoter sequences, and genes known to be regulated by TH. We found that while the occurrence of the TRE motif is strongly correlated with gene regulation by TH for some genes, other TH-regulated genes do not exhibit an increased density of TRE half-site motifs. Furthermore, we demonstrate that an increase in the rate of occurrence of the half-site motifs does not always indicate the specific location of the TRE within the promoter region. To account for the fact that TR often operates as a dimer, we introduce a novel dual-threshold PWM scanning approach for identifying TREs with a true positive rate of 0.73 and a false positive rate of 0.2. Application of this approach to ChIP-chip peak regions revealed the presence of 85 putative TREs suitable for further in vitro validation.ConclusionsThis study further elucidates TRβ gene regulation in mouse cerebellum, with 211 promoter regions identified to bind to TR. While we have identified 85 putative TREs within these regions, future work will study other mechanisms of action that may mediate the remaining observed TR-binding activity.

Please, Get in My Way So That I Can Be More Efficient!

Please, Get in My Way So That I Can Be More Efficient!

Computational repositioning of ethno medicine elucidated gB-gH-gL complex as novel anti herpes drug target

BackgroundHerpes viruses are important human pathogens that can cause mild to severe lifelong infections with high morbidity. They remain latent in the host cells and can cause recurrent infections that might prove fatal. These viruses are known to infect the host cells by causing the fusion of viral and host cell membrane proteins. Fusion is achieved with the help of conserved fusion machinery components, glycoproteins gB, heterodimer gH-gL complex along with other non-conserved components. Whereas, another important glycoprotein gD without which viral entry to the cell is not possible, acts as a co-activator for the gB-gH-gL complex formation. Thus, this complex formation interface is the most promising drug target for the development of novel anti-herpes drug candidates. In the present study, we propose a model for binding of gH-gL to gB glycoprotein leading from pre to post conformational changes during gB-gH-gL complex formation and reported the key residues involved in this binding activity along with possible binding site locations. To validate the drug targetability of our proposed binding site, we have repositioned some of the most promising in vitro, in vivo validated anti-herpes molecules onto the proposed binding site of gH-gL complex in a computational approach.MethodsHex 6.3 standalone software was used for protein-protein docking studies. Arguslab 4.0.1 and Accelrys® Discovery Studio 3.1 Visualizer softwares were used for semi-flexible docking studies and visualizing the interactions respectively. Protein receptors and ethno compounds were retrieved from Protein Data Bank (PDB) and Pubchem databases respectively. Lipinski’s Filter, Osiris Property Explorer and Lazar online servers were used to check the pharmaceutical fidelity of the drug candidates.ResultsThrough protein-protein docking studies, it was identified that the amino acid residues VAL342, GLU347, SER349, TYR355, SER388, ASN395, HIS398 and ALA387 of gH-gL complex play an active role in its binding activity with gB. Semi flexible docking analysis of the most promising in vitro, in vivo validated anti-herpes molecules targeting the above mentioned key residues of gH-gL complex showed that all the analyzed ethno medicinal compounds have successfully docked into the proposed binding site of gH-gL glycoprotein with binding energy range between -10.4 to -6.4 K.cal./mol.ConclusionsSuccessful repositioning of the analyzed compounds onto the proposed binding site confirms the drug targetability of gH-gL complex. Based on the free binding energy and pharmacological properties, we propose (3-chloro phenyl) methyl-3,4,5 trihydroxybenzoate as worth a small ethno medicinal lead molecule for further development as potent anti-herpes drug candidate targeting gB-gH-gL complex formation interface.

Characterization of the ADRP Fatty Acid Binding Site: A Fluorescence Binding and Circular Dichroic Study

Although ADRP (adipose differentiation‐related protein, also known as perilipin 2) has been shown to bind fatty acids (FA) and other lipids with high affinity, the structure and location of the FA binding site has not been examined in detail. This is of some importance since ADRP's affinity for lipids and especially FA may be related to lipid droplet formation, triacylglycerol accumulation, and FA uptake and metabolism in the cell. The purpose of the present investigation was to characterize ADRP and several truncated mutants using fluorescence binding assays and circular dichroism (CD) to assess the structural requirements for binding. Results presented here show that removal of 120 amino acids from the N‐terminus, a high homology region common to other lipid droplet proteins increased the FA binding affinity (Kd) 43%. In contrast, deleting 254 residues decreased the Kd 24%. Removal of 173 amino acids from the C‐terminus, however did not significantly change the protein's ability to bind FA. Conformational changes upon binding were confirmed by CD. Taken together, these results indicate that residues 121–254 appear essential for highest affinity binding and show that the ability of ADRP to bind FA may depend on the proper folding of several domains, the partial deletion of which leads to altered, but not ablated ligand binding. This work was supported by NIH Grant DK70965 (BPA).

Chiroptical Properties of Bilirubin‐Serum Albumin Binding Sites

Although the interactions between bilirubin and serum albumin are among the most studied serum albumin-ligand interactions, the binding-site location and the participation of bilirubin-serum albumin complexes in pathological and physiological processes are under debate. In this article, we have benefited from the chiral structure of bilirubin and used CD spectroscopy to characterize the structure of bilirubin bound to human and bovine serum albumins. We determined that in a phosphate buffer at pH 7.8 there are three binding sites in both human and bovine serum albumins. While the primary binding sites in human and bovine serum albumins bind bilirubin with P- and M-helical conformations, respectively, the secondary binding sites in both albumins bind bilirubin in the P-helical conformation. We have shown that the bonding of bilirubin to the serum albumin matrix is a more favorable process than the self-association of bilirubin under the studied conditions, with a maximum of three bound bilirubins per serum albumin molecule. Although bilirubin bound to the primary binding site has attracted the most attention, the presented results have documented the impact of the secondary binding sites which are relevant in the displacement reactions between BR and drugs and in the phenomena where bilirubin plays antioxidant, antimutagenic, and anti-inflammatory roles.

Mapping the Nucleotide Binding Site of Uncoupling Protein 1 Using Atomic Force Microscopy

A tight regulation of proton transport in the inner mitochondrial membrane is crucial for physiological processes such as ATP synthesis, heat production, or regulation of the reactive oxygen species as proposed for the uncoupling protein family members (UCP). Specific regulation of proton transport is thus becoming increasingly important in the therapy of obesity and inflammatory, neurodegenerative, and ischemic diseases. We and other research groups have shown previously that UCP1- and UCP2-mediated proton transport is inhibited by purine nucleotides. Several hypotheses have been proposed to explain the inhibitory effect of ATP, although structural details are still lacking. Moreover, the unresolved mystery is how UCP operates in vivo despite the permanent presence of high (millimolar) concentrations of ATP in mitochondria. Here we use the topographic and recognition (TREC) mode of an atomic force microscope to visualize UCP1 reconstituted into lipid bilayers and to analyze the ATP–protein interaction at a single molecule level. The comparison of recognition patterns obtained with anti-UCP1 antibody and ATP led to the conclusion that the ATP binding site can be accessed from both sides of the membrane. Using cantilever tips with different cross-linker lengths, we determined the location of the nucleotide binding site inside the membrane with 1 Å precision. Together with the recently published NMR structure of a UCP family member (Berardi et al. Nature, 2011, 476, 109–113), our data provide a valuable insight into the mechanism of the nucleotide binding and pave the way for new pharmacological approaches against the diseases mentioned above.

Genome-scale bacterial transcriptional regulatory networks: reconstruction and integrated analysis with metabolic models

Advances in sequencing technology are resulting in the rapid emergence of large numbers of complete genome sequences. High-throughput annotation and metabolic modeling of these genomes is now a reality. The high-throughput reconstruction and analysis of genome-scale transcriptional regulatory networks represent the next frontier in microbial bioinformatics. The fruition of this next frontier will depend on the integration of numerous data sources relating to mechanisms, components and behavior of the transcriptional regulatory machinery, as well as the integration of the regulatory machinery into genome-scale cellular models. Here, we review existing repositories for different types of transcriptional regulatory data, including expression data, transcription factor data and binding site locations and we explore how these data are being used for the reconstruction of new regulatory networks. From template network-based methods to de novo reverse engineering from expression data, we discuss how regulatory networks can be reconstructed and integrated with metabolic models to improve model predictions and performance. We also explore the impact these integrated models can have in simulating phenotypes, optimizing the production of compounds of interest or paving the way to a whole-cell model.

A nicotinic acetylcholine receptor transmembrane point mutation (G275E) associated with resistance to spinosad in Frankliniella occidentalis.

High levels of resistance to spinosad, a macrocyclic lactone insecticide, have been reported previously in western flower thrips, Frankliniella occidentalis, an economically important insect pest of vegetables, fruit and ornamental crops. We have cloned the nicotinic acetylcholine receptor (nAChR) α6 subunit from F. occidentalis (Foα6) and compared the nucleotide sequence of Foα6 from susceptible and spinosad-resistant insect populations (MLFOM and R1S respectively). A single nucleotide change has been identified in Foα6, resulting in the replacement of a glycine (G) residue in susceptible insects with a glutamic acid (E) in resistant insects. The resistance-associated mutation (G275E) is predicted to lie at the top of the third α-helical transmembrane domain of Foα6. Although there is no direct evidence identifying the location of the spinosad binding site, the analogous amino acid in the C. elegans glutamate-gated chloride channel lies in close proximity (4.4 Å) to the known binding site of ivermectin, another macrocyclic lactone pesticide. The functional consequences of the resistance-associated mutation have been examined in the human nAChR α7 subunit. Introduction of an analogous (A272E) mutation in α7 abolishes the modulatory effects of spinosad whilst having no significant effect upon activation by acetylcholine, consistent with spinosad having an allosteric mechanism of action.

Identifying the Propofol Binding Site(S) in Heterologously Expressed Human Alpha1 Beta3 Gaba-A Receptors using a Photoreactive Propofol Analog

Propofol, a widely used intravenous general anesthetic, potentiates GABA-A receptor (GABAAR) function at anesthetic concentrations, and “knock-in” mice expressing a single mutation at position 15 in the β3 subunit M2 helix (βM2-15) have reduced sensitivity to many of the anesthetic effects of propofol. However, the location of propofol's binding site(s) remains to be determined. Here we use a photoactive analog of propofol, [3H]m-azipropofol(2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol), an aryl diazirine with broad amino acid side chain reactivity, to identify propofol binding site(s) in affinity-purified, expressed human α1β3 GABAARs in detergent/asolectin solution. m-Azipropofol acts as a tadpole anesthetic with potency (EC50 = 3μM) similar to propofol, and it acts as a low efficacy GABAAR potentiator (Hall et al. 2010, J Med. Chem. 53, 5667). Irradiation of GABAAR for 30 min at 365nm resulted in [3H]m-azipropofol photoincorporation into both the α1 and β3 subunits that was inhibitable by propofol and non-radioactive m-azipropofol with IC50s of 28±3 μM and 6.7±1 μM, respectively. Individual amino acids photolabeled by [3H]m-azipropofol were identified by protein microsequencing of subunit fragments generated by enzymatic or chemical digestion. In β3, there was propofol-inhibitable photolabeling of β3Met-286, an amino acid that is photolabeled by [3H]azietomidate in the etomidate binding site at the β3M3-α1M1 interface (Li et al. 2006, J Neurosci. 26, 11599, Chiara et al. 2012, Biochem. 51, 836). In α1, [3H]m-azipropofol photolabeled α1Ile-239 within α1M1, located one helical turn below α1Met-236 that is photolabeled by [3H]azietomidate. Consistent with propofol and m-azipropofol binding to this etomidate site, they completely inhibit [3H]azietomidate photolabeling with IC50s of 46±3 μM and 23±3 μM, respectively. Both β3Met-286 and α1Met-236 are in close proximity to βM2-15, which docking calculations suggest may also be part of this propofol binding site.

Computational Analysis of Integrin Subunit Glycosylation: Investigations on its Role in Orienting Ligand-Receptor Interaction

Integrins serve as the primary mediators of cellular adhesion to the extracellular matrix (ECM). Several cellular processes (e.g. apoptosis) are controlled through their interactions with ligands in the ECM. Integrins bind to their ECM ligands through their head domains. The crystal structure of an integrin heterodimer (αVβ3) reveals the location of the ECM ligand binding site. Integrin function may be altered by the disruption of the ligand binding site either through changes in protein conformation or heterodimer binding orientation. Previous studies have suggested a link between disruptions of integrin function to changes in glycosylation. This study focuses on characterizing the effect of altering individual glycosylation sites on the interaction between integrin head domains. Part of the analysis was conducted using the molecular soft docking program, BiGGER (Krippahl, 2003). Docking simulations were done using structure files generated from integrin subunit head domain entries in the Protein Data Bank (www.pdb.org). Dock assessment involved the measurement of subunit orientations based on user-defined anchor residues in the interacting subunits. Initial analysis was conducted on the Integrin αVβ3 heterodimer. The integrin αVβ3 head domain has four glycosylation sites, three for the αV beta propeller and one for the β3 A-domain. Sugar residues were iteratively removed from each of these glycosylation sites to determine their effects on integrin subunit interaction. Our results suggest the importance of glycosylation at αV N266 for the formation of the ligand binding site. Removal of these sugar residues resulted in the greatest deviation in predicted docking orientation. A ∼90° rotation, counter-clockwise from the reference position, was predicted for the docking of the β3 subunit to the N266-deglycosylated αV subunit. This altered conformation is predicted to effectively destroy the ligand binding site and thus, disrupt integrin αVβ3 function.

Immunoglobulin E and G4 epitopes of the major allergen of birch pollen Bet v 1 share residues critical for antibody binding

Background Millions of patients with allergy to birch pollen develop clinically cross-reactive IgE against Bet v 1-like proteins in plant foods. Specific immunotherapy (SIT) with birch pollen extracts induces the biosynthesis of Bet v 1-specific immunoglobulin (Ig)G4. IgG4 is believed to act as a blocking antibody preventing IgE binding to Bet v 1, thus alleviating allergic symptoms. Only little information on the location and relationship of IgE and IgG4 binding sites of Bet v 1 is available. In this study we seek to identify epitopes of IgE and IgG4 antibodies on Bet v 1.

Anesthetic Binding Sites within the Transmembrane Domain of the α7 Nicotinic Acetylcholine Receptor

General anesthetics inhibit the α7 neuronal nicotinic acetylcholine receptor (nAChR) with different sensitivities, but the location of anesthetic binding sites remains unclear. Here we used high-resolution solution NMR to study the interaction of general anesthetics ketamine and halothane with the transmembrane domain (TMD) of the human α7 nAChR. The TMD was expressed in E. coli and purified into detergent LDAO micelles. The specific residues involved in anesthetic binding were identified by anesthetics-induced chemical shift changes in 1H-15N HSQC NMR spectra. Ketamine binds to an intrasubunit site involving residues of all four helices at the intracellular end of the TMD. Two halothane interaction sites were detected. The first site was located near the intracellular end of the TMD, similar to the ketamine site, but involved only residues in TM1 and TM2. The second halothane site was formed by residues in the middle of TM1 and TM3. This site was not observed for ketamine binding, probably because ketamine was too large to penetrate deep into the TMD. Interestingly, unlike the previous finding in the α4β2 nAChR, anesthetic binding to the extracellular end of the TMD was not observed in the α7 nAChR. This difference may be responsible for their distinct sensitivity to halothane. In addition, halothane modulated motion less in α7 than in α4β2 on the micro- to milli-second (μs-ms) timescale, as demonstrated by changes in peak intensity, line width, and peak splitting for some residues. This work was supported by NIH grants: R01GM066358, R01GM056257, and R37GM049202.

Building multi-component crystals from cations and co-crystals: the use of chaperones

Ternary crystalline complexes consisting of both salts and ionic co-crystals have been created through the crystallisation of the binary co-crystal 3,5-dinitrobenzoic acid–4-(dimethylamino)benzoic acid with group 1 or ammonium cations. The size and charge density of the cation can be used to adjust the protonation level and local geometry of the acid pair. The selectivity and coordination geometry of the chaperone cation may be further adjusted by the inclusion of a crown ether to reduce the number and location of potential binding sites.

Exploring the ATP-binding site of P2X receptors

P2X receptors are ATP-gated non-selective cation channels involved in many different physiological processes, such as synaptic transmission, inflammation, and neuropathic pain. They form homo- or heterotrimeric complexes and contain three ATP-binding sites in their extracellular domain. The recent determination of X-ray structures of a P2X receptor solved in two states, a resting closed state and an ATP-bound, open-channel state, has provided unprecedented information not only regarding the three-dimensional shape of the receptor, but also on putative conformational changes that couple ATP binding to channel opening. These data provide a structural template for interpreting the huge amount of functional, mutagenesis, and biochemical data collected during more than fifteen years. In particular, the interfacial location of the ATP binding site and ATP orientation have been successfully confirmed by these structural studies. It appears that ATP binds to inter-subunit cavities shaped like open jaws, whose tightening induces the opening of the ion channel. These structural data thus represent a firm basis for understanding the activation mechanism of P2X receptors.

Competing hydrogen-bond and halogen-bond donors in crystal engineering

In order to study the structure-directing competition between hydrogen- and halogen-bond donors we have synthesized two ligands, 3,3′-azobipyridine and 4,4′-azobipyridine, and co-crystallized them with a series of bi-functional donor molecules comprising an activated halogen-bond donor (I or Br) as well as a hydrogen-bond donor (acid, phenol or oxime) on the same backbone. Based on the subsequent single-crystal analysis, 5 of 6 co-crystals of 3,3′-azobipyridine are assembled using hydrogen bonds as the primary driving force accompanied by weaker secondary (C–X⋯O) interactions. However, in 5 out of the 6 co-crystals of 4,4′-azobipyridine, both hydrogen bonds (O–H⋯N) and halogen bonds (C–X⋯N) are present as structure-directing interactions leading to 1-D chains. Since the charges on the acceptor sites in 3,3′- and 4,4′-azobipyridine are very similar, the observed difference in binding behaviour highlights the importance of binding-site location on the acceptor molecules (anti-parallel in 3,3′-azobipyridine and co-linear in 4,4′-azobipyridine) as a direct influence over the structural balance between hydrogen- and halogen-bond donors.

Pitx1 broadly associates with limb enhancers and is enriched on hindlimb cis-regulatory elements

Extensive functional analyses have demonstrated that the pituitary homeodomain transcription factor Pitx1 plays a critical role in specifying hindlimb morphology in vertebrates. However, much less is known regarding the target genes and cis-regulatory elements through which Pitx1 acts. Earlier studies suggested that the hindlimb transcription factors Tbx4, HoxC10, and HoxC11 might be transcriptional targets of Pitx1, but definitive evidence for direct regulatory interactions has been lacking. Using ChIP-Seq on embryonic mouse hindlimbs, we have pinpointed the genome-wide location of Pitx1 binding sites during mouse hindlimb development and identified potential gene targets for Pitx1. We determined that Pitx1 binding is significantly enriched near genes involved in limb morphogenesis, including Tbx4, HoxC10, and HoxC11. Notably, Pitx1 is bound to the previously identified HLEA and HLEB hindlimb enhancers of the Tbx4 gene and to a newly identified Tbx2 hindlimb enhancer. Moreover, Pitx1 binding is significantly enriched on hindlimb relative to forelimb-specific cis-regulatory features that are differentially marked by H3K27ac. However, our analysis revealed that Pitx1 also strongly associates with many functionally verified limb enhancers that exhibit similar levels of activity in the embryonic mesenchyme of forelimbs and hindlimbs. We speculate that Pitx1 influences hindlimb morphology both through the activation of hindlimb-specific enhancers as well as through the hindlimb-specific modulation of enhancers that are active in both sets of limbs.

Insights into the Glycosaminoglycan-Mediated Cytotoxic Mechanism of Eosinophil Cationic Protein Revealed by NMR

Protein-glycosaminoglycan interactions are essential in many biological processes and human diseases, yet how their recognition occurs is poorly understood. Eosinophil cationic protein (ECP) is a cytotoxic ribonuclease that interacts with glycosaminoglycans at the cell surface; this promotes the destabilization of the cellular membrane and triggers ECP's toxic activity. To understand this membrane destabilization event and the differences in the toxicity of ECP and its homologues, the high resolution solution structure of the complex between full length folded ECP and a heparin-derived trisaccharide (O-iPr-α-D-GlcNS6S-α(1-4)-L-IdoA2S-α(1-4)-D-GlcNS6S) has been solved by NMR methods and molecular dynamics simulations. The bound protein retains the tertiary structure of the free protein. The (2)S(0) conformation of the IdoA ring is preferably recognized by the protein. We have identified the precise location of the heparin binding site, dissected the specific interactions responsible for molecular recognition, and defined the structural requirements for this interaction. The structure reveals the contribution of Arg7, Gln14, and His15 in helix α1, Gln40 in strand β1, His64 in loop 4, and His128 in strand β6 in the recognition event and corroborates the previously reported participation of residues Arg34-Asn39. The participation of the catalytic triad (His15, Lys38, His128) in recognizing the heparin mimetic reveals, at atomic resolution, the mechanism of heparin's inhibition of ECP's ribonucleolytic activity. We have integrated all the available data to propose a molecular model for the membrane interaction process. The solved NMR complex provides the structural model necessary to design inhibitors to block ECP's toxicity implicated in eosinophil pathologies.