A novel mechanism of ligand binding and release in the odorant binding protein 20 from the malaria mosquito Anopheles gambiae

Anopheles gambiae mosquitoes that transmit malaria are attracted to humans by the odor molecules that emanate from skin and sweat. Odorant binding proteins (OBPs) are the first component of the olfactory apparatus to interact with odorant molecules, and so present potential targets for preventing transmission of malaria by disrupting the normal olfactory responses of the insect. AgamOBP20 is one of a limited subset of OBPs that it is preferentially expressed in female mosquitoes and its expression is regulated by blood feeding and by the day/night light cycles that correlate with blood‐feeding behavior. Analysis of AgamOBP20 in solution reveals that the apo‐protein exhibits significant conformational heterogeneity but the binding of odorant molecules results in a significant conformational change, which is accompanied by a reduction in the conformational flexibility present in the protein. Crystal structures of the free and bound states reveal a novel pathway for entrance and exit of odorant molecules into the central‐binding pocket, and that the conformational changes associated with ligand binding are a result of rigid body domain motions in α‐helices 1, 4, and 5, which act as lids to the binding pocket. These structures provide new insights into the specific residues involved in the conformational adaptation to different odorants and have important implications in the selection and development of reagents targeted at disrupting normal OBP function.     

Structure and dynamics of cationic membrane peptides and proteins: insights from solid-state NMR

Many membrane peptides and protein domains contain functionally important cationic Arg and Lys residues, whose insertion into the hydrophobic interior of the lipid bilayer encounters significant energy barriers. To understand how these cationic molecules overcome the free energy barrier to insert into the lipid membrane, we have used solid-state NMR spectroscopy to determine the membrane-bound topology of these peptides. A versatile array of solid-state NMR experiments now readily yields the conformation, dynamics, orientation, depth of insertion, and site-specific protein-lipid interactions of these molecules. We summarize key findings of several Arg-rich membrane peptides, including β-sheet antimicrobial peptides, unstructured cell-penetrating peptides, and the voltage-sensing helix of voltage-gated potassium channels. Our results indicate the central role of guanidinium-phosphate and guanidinium-water interactions in dictating the structural topology of these cationic molecules in the lipid membrane, which in turn account for the mechanisms of this functionally diverse class of membrane peptides.     

Non-covalent binding of membrane lipids to membrane proteins

Polar lipids and membrane proteins are major components of biological membranes, both cell membranes and membranes of enveloped viruses. How these two classes of membrane components interact with each other to influence the function of biological membranes is a fundamental question that has attracted intense interest since the origins of the field of membrane studies. One of the most powerful ideas that driven the field is the likelihood that lipids bind to membrane proteins at specific sites, modulating protein structure and function. However only relatively recently has high resolution structure determination of membrane proteins progressed to the point of providing atomic level structure of lipid binding sites on membrane proteins. Analysis of X-ray diffraction, electron crystallography and NMR data over 100 specific lipid binding sites on membrane proteins. These data demonstrate tight lipid binding of both phospholipids and cholesterol to membrane proteins. Membrane lipids bind to membrane proteins by their headgroups, or by their acyl chains, or binding is mediated by the entire lipid molecule. When headgroups bind, binding is stabilized by polar interactions between lipid headgroups and the protein. When acyl chains bind, van der Waals effects dominate as the acyl chains adopt conformations that complement particular sites on the rough protein surface. No generally applicable motifs for binding have yet emerged. Previously published biochemical and biophysical data link this binding with function. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

NMR reveals molecular interactions and dynamics of fatty acid binding to albumin

Background

The molecular details of fatty acid (FA) interactions with albumin are fundamental to understanding transport in the plasma and cellular utilization of these key nutrients and building blocks of membranes.

Scope of review

This review focuses on the development and application of NMR methods to study FA binding to albumin [bovine (BSA) and human (HSA)]. The key strategy was to use ¹³C enrichment of a specific carbon in the FA as a non-perturbing probe to permit visualization of the small ligand complexed to the very large protein. NMR contributions to illuminating molecular interactions and FA dynamics are summarized from three decades of studies.

Major conclusions

Our early studies detected multiple binding sites that we hypothesized were distinguished because of the unique tertiary structure of the protein in close proximity to the FA labeled carbon in each site. Later crystallographic structures revealed the presence of polar and charged amino acid side chains near the carboxyl carbon of the FA and unique tertiary structures lining all of the FA binding pockets. In collaboration with the crystallography group, several FA sites in the crystalline state were matched with NMR resonances in the solution state. With the newest application of NMR, 2D NMR spectroscopy detected nine binding sites, and three were located in the crystal structure through displacement of drugs with identified sites.

General significance

NMR spectroscopy utilizing the FA as a probe allows characterization of site-specific interactions, molecular motions within binding sites, the order of filling and removal of FA from sites. This article is part of a Special Issue entitled Serum Albumin.     

Free energy of proton binding in proteins

In this article we use literature data on the titration of denatured ribonuclease to test the accuracy of proton-binding distributions obtained using our recent approach employing moments. We find that using only the local slope of the titration curve at a small number of points (five, for example) we can reproduce the detailed proton-binding distribution at all pH values. Our method gives the complete proton-binding polynomial for a given protein and each coefficient in this polynomial in turn yields the free energy for binding a given number of protons in all ways to the protein. Using these net free energies, we can then compute the average proton-binding free energy per proton as a function of the fraction of protons bound. We find that this function is remarkably similar for different proteins, even for proteins that exhibit quite different titration behavior. For the special case of binding to independent sites, we obtain simple relations for the first and last terms in the free energy per-proton function. For this special case we also can calculate the distribution functions giving the probability that a molecule has a given number of positive or negative charges and the joint distribution that a molecule simultaneously has a given number of positive and negative charge.     

NMR spectroscopy of peptides and proteins

High resolution nuclear magnetic resonance (NMR) spectroscopy is the only method available for determining the three-dimensional structures of peptides and proteins in solution at atomic resolution. This article deals with a range of practical considerations associated with such studies, including sample preparation, instrumental setup, one- and two-dimensional NMR methods, interpretation of spectral data, and structure calculations.     

Identification of Plasmodium falciparum RhopH3 protein peptides that specifically bind to erythrocytes and inhibit merozoite invasion

The identification of sequences involved in binding to erythrocytes is an important step for understanding the molecular basis of merozoite-erythrocyte interactions that take place during invasion of the Plasmodium falciparum malaria parasite into host cells. Several molecules located in the apical organelles (micronemes, rhoptry, dense granules) of the invasive-stage parasite are essential for erythrocyte recognition, invasion, and establishment of the nascent parasitophorous vacuole. Particularly, it has been demonstrated that rhoptry proteins play an important role in binding to erythrocyte surface receptors, among which is the PfRhopH3 protein, which triggers important immune responses in patients from endemic regions. It has also been reported that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes, further supporting its direct involvement in erythrocyte invasion processes. In this study, PfRhopH3 consecutive peptides were synthesized and tested in erythrocyte binding assays for identifying those regions mediating binding to erythrocytes. Fourteen PfRhopH3 peptides presenting high specific binding activity were found, whose bindings were saturable and presented nanomolar dissociation constants. These high-activity binding peptides (HABPs) were characterized by having alpha-helical structural elements, as determined by circular dichroism, and having receptors of a possible sialic acid-dependent and/or glycoprotein-dependent nature, as evidenced in enzyme-treated erythrocyte binding assays and further corroborated by cross-linking assay results. Furthermore, these HABPs inhibited merozoite in vitro invasion of normal erythrocytes at 200 microM by up to 60% and 90%, suggesting that some RhopH3 protein regions are involved in the P. falciparum erythrocyte invasion.     

DynaBiS: A hierarchical sampling algorithm to identify flexible binding sites for large ligands and peptides

Knowing the ligand or peptide binding site in proteins is highly important to guide drug discovery, but experimental elucidation of the binding site is difficult. Therefore, various computational approaches have been developed to identify potential binding sites in protein structures. However, protein and ligand flexibility are often neglected in these methods due to efficiency considerations despite the recognition that protein–ligand interactions can be strongly affected by mutual structural adaptations. This is particularly true if the binding site is unknown, as the screening will typically be performed based on an unbound protein structure. Herein we present DynaBiS, a hierarchical sampling algorithm to identify flexible binding sites for a target ligand with explicit consideration of protein and ligand flexibility, inspired by our previously presented flexible docking algorithm DynaDock. DynaBiS applies soft-core potentials between the ligand and the protein, thereby allowing a certain protein–ligand overlap resulting in efficient sampling of conformational adaptation effects. We evaluated DynaBiS and other commonly used binding site identification algorithms against a diverse evaluation set consisting of 26 proteins featuring peptide as well as small ligand binding sites. We show that DynaBiS outperforms the other evaluated methods for the identification of protein binding sites for large and highly flexible ligands such as peptides, both with a holo or apo structure used as input.     

Structural biology of transmembrane domains: efficient production and characterization of transmembrane peptides by NMR

Structural characterization of transmembrane peptides (TMPs) is justified because transmembrane domains of membrane proteins appear to often function independently of the rest of the protein. However, the challenge in obtaining milligrams of isotopically labeled TMPs to study these highly hydrophobic peptides by nuclear magnetic resonance (NMR) is significant. In the present work, a protocol is developed to produce, isotopically label, and purify TMPs in high yield as well as to initially characterize the TMPs with CD and both solution and solid-state NMR. Six TMPs from three integral membrane proteins, CorA, M2, and KdpF, were studied. CorA and KdpF are from Mycobacterium tuberculosis, while M2 is from influenza A virus. Several milligrams of each of these TMPs ranging from 25 to 89 residues were obtained per liter of M9 culture. The initial structural characterization results showed that these peptides were well folded in both detergent micelles and lipid bilayer preparations. The high yield, the simplicity of purification, and the convenient protocol represents a suitable approach for NMR studies and a starting point for characterizing the transmembrane domains of membrane proteins.     

Prediction of the binding energy for small molecules,peptides and proteins

A fast and reliable evaluation of the binding energy from a single conformation of a molecular complex is an important practical task. Knowledge‐based scoring schemes may not be sufficiently general and transferable, while molecular dynamics or Monte Carlo calculations with explicit solvent are too computationally expensive for many applications. Recently, several empirical schemes using finite difference Poisson–Boltzmann electrostatics to predict energies for particular types of complexes were proposed. Here, an improved empirical binding energy function has been derived and validated on three different types of complexes: protein–small ligand, protein–peptide and protein–protein. The function uses the boundary element algorithm to evaluate the electrostatic solvation energy. We show that a single set of parameters can predict the relative binding energies of the heterogeneous validation set of complexes with 2.5 kcal/mol accuracy. We also demonstrate that global optimization of the ligand and of the flexible side‐chains of the receptor improves the accuracy of the evaluation. Copyright © 1999 John Wiley & Sons, Ltd.     

Rational design of bifunctional chimeric peptides that combine antimicrobial and titanium binding activity

Bacterial biofilm formation remains a serious problem for clinical materials and often leads to implant failure. To counteract bacterial adhesion, which initiates biofilm formation, the development of antibiotic surface coating strategies is of high demand and warrants further investigations. In this study, we have created bifunctional chimeric peptides by fusing the recently developed antimicrobial peptide MGD2 (GLRKRLRKFFNKIKF) with different titanium-binding sequences. The novel peptides were investigated regarding their antibacterial potential against a set of different bacterial strains including drug-resistant Staphylococcus aureus. All peptides showed high antimicrobial activities both when in solution and when immobilized on titanium surfaces. Owing to the ease of synthesis and handling, the herein described peptides might be a true alternative to prevent bacterial biofilm formation.     

Immunological properties of low molecular-weight proteins binding heavy metals in rat kidney and liver

Two homogenous fractions of hepatic metallothioneins ((Cd,Zn) MT-1 and (Cd,Zn) MT-2) and renal metal binding proteins ((Bi,Cu) BP-1 and (Bi,Cu) BP-2) were isolated from rats exposed to heavy metals and specific antisera to them were produced in rabbits.These antisera were tested by immunodiffusion and immunoelectrophoresis for their ability to bind different fractions of hepatic Cd,Zn -metallothionein and renal (Bi,Cu)-, (Hg,Cu)- and (Cd,Cu)-binding proteins. It was found that anti (Bi,Cu) BP antisera did not cross-react with hepatic (Cd,Zn) MT-1 and (Cd,Zn) MT-2. Strong immunological cross-reactions were detected between anti (Bi,Cu) BP antisera and individual forms of (Cd,Cu)-, (Hg,Cu)- and (Bi,Cu)-binding proteins isolated from rat kidneys.     

Binding of phosphorylated peptides and inhibition of their interaction with disease‐relevant human proteins by synthetic metal‐chelate receptors

The modulation of biological signal transduction pathways by masking phosphorylated amino acid residues represents a viable route toward pharmacologic protein regulation. Binding of phosphorylated amino acid residues has been achieved with synthetic metal‐chelate receptors. The affinity and selectivity of such receptors can be enhanced if combined with a second binding site. We demonstrate this principle with a series of synthetic ditopic metal‐chelate receptors, which were synthesized and investigated for their binding affinity to phosphorylated short peptides under conditions of physiological pH. The compounds showing highest affinity were subsequently used to inhibit the interaction of the human STAT1 protein to a peptide derived from the interferon‐γ receptor, and between the checkpoint kinase Chk2 and its preferred binding motif. Two of the investigated ditopic synthetic receptors show a significant increase in inhibition activity. The results show that regulation of protein function by binding to phosphorylated amino acids is possible. The introduction of additional binding sites into the synthetic receptors increases their affinity, but the flexibility of the structures investigated so far prohibited stringent amino acid sequence selectivity in peptide binding. Copyright © 2009 John Wiley & Sons, Ltd.     

Kinetic and functional characterisation of the heparin‐binding peptides from human transglutaminase 2

Transglutaminase 2 (TG2) is an autoantigen in celiac disease (CD) and it has multiple biologic functions including involvement in cell adhesion through interactions with integrins, fibronectin (FN), and heparan sulfate proteoglycans. We aimed to delineate the heparin‐binding regions of human TG2 by studying binding kinetics of the predicted heparin‐binding peptides using surface plasmon resonance method. In addition, we characterized immunogenicity of the TG2 peptides and their effect on cell adhesion. The high‐affinity binding of human TG2 to the immobilized heparin was observed, and two TG2 peptides, P1 (amino acids 202–215) and P2 (261–274), were found to bind heparin. The amino acid sequences corresponding to the heparin‐binding peptides were located close to each other on the surface of the TG2 molecule as part of the α‐helical structures. The heparin‐binding peptides displayed increased immunoreactivity against serum IgA of CD patients compared with other TG2 peptides. The cell adhesion reducing effect of the peptide P2 was revealed in Caco‐2 intestinal epithelial cell attachment to the FN and FN‐TG2 coated surfaces. We propose that TG2 amino acid sequences 202–215 and 261–274 could be involved in binding of TG2 to cell surface heparan sulfates. High immunoreactivity of the corresponding heparin‐binding peptides of TG2 with CD patient's IgA supports the previously described role of anti‐TG2 autoantibodies interfering with this interaction. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) was used to measure the binding of Cu2+ ions to synthetic peptides corresponding to sections of the sequence of the mature prion protein (PrP). ESI-MS demonstrates that Cu2+ is unique among divalent metal ions in binding to PrP and defines the location of the major Cu2+ binding site as the octarepeat region in the N-terminal domain, containing multiple copies of the repeat ProHisGlyGlyGlyTrpGlyGln. The stoichiometries of the complexes measured directly by ESI-MS are pH dependent: a peptide containing four octarepeats chelates two Cu2+ ions at pH 6 but four at pH 7.4. At the higher pH, the binding of multiple Cu2+ ions occurs with a high degree of cooperativity for peptides C-terminally extended to incorporate a fifth histidine. Dissociation constants for each Cu2+ ion binding to the octarepeat peptides, reported here for the first time, are mostly in the low micromolar range; for the addition of the third and fourth Cu2+ ions to the extended peptides at pH 7.4, K(D)'s are <100 nM. N-terminal acetylation of the peptides caused some reduction in the stoichiometry of binding at both pH's. Cu2+ also binds to a peptide corresponding to the extreme N-terminus of PrP that precedes the octarepeats, arguing that this region of the sequence may also make a contribution to the Cu2+ complexation. Although the structure of the four-octarepeat peptide is not affected by pH changes in the absence of Cu2+, as judged by circular dichroism, Cu2+ binding induces a modest change at pH 6 and a major structural perturbation at pH 7.4. It is possible that PrP functions as a Cu2+ transporter by binding Cu2+ ions from the extracellular medium under physiologic conditions and then releasing some or all of this metal upon exposure to acidic pH in endosomes or secondary lysosomes.     

Sub‐angstrom modeling of complexes between flexible peptides and globular proteins

A wide range of regulatory processes in the cell are mediated by flexible peptides that fold upon binding to globular proteins. Computational efforts to model these interactions are hindered by the large number of rotatable bonds in flexible peptides relative to typical ligand molecules, and the fact that different peptides assume different backbone conformations within the same binding site. In this study, we present Rosetta FlexPepDock, a novel tool for refining coarse peptide–protein models that allows significant changes in both peptide backbone and side chains. We obtain high resolution models, often of sub‐angstrom backbone quality, over an extensive and general benchmark that is based on a large nonredundant dataset of 89 peptide–protein interactions. Importantly, side chains of known binding motifs are modeled particularly well, typically with atomic accuracy. In addition, our protocol has improved modeling quality for the important application of cross docking to PDZ domains. We anticipate that the ability to create high resolution models for a wide range of peptide–protein complexes will have significant impact on structure‐based functional characterization, controlled manipulation of peptide interactions, and on peptide‐based drug design. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Ligand binding and homology modelling of insect odorant‐binding proteins

This review describes the main characteristics of odorant‐binding proteins (OBPs) for homology modelling and presents a summary of structure prediction studies on insect OBPs, along with the steps involved and some limitations and improvements. The technique involves a computing approach to model protein structures and is based on a comparison between a target (unknown structure) and one or more templates (experimentally determined structures). As targets for structure prediction, OBPs are considered to play a functional role for recognition, desorption, scavenging, protection and transportation of hydrophobic molecules (odourants) across an aqueous environment (lymph) to olfactory receptor neurones (ORNs) located in sensilla, the main olfactory units of insect antennae. Lepidopteran pheromone‐binding proteins, a subgroup of OBPs, are characterized by remarkable structural features, in which high sequence identities (approximately 30%) among these OBPs and a large number of available templates can facilitate the prediction of precise homology models. Approximately 30 studies have been performed on insect OBPs using homology modelling as a tool to predict their structures. Although some of the studies have assessed ligand‐binding affinity using structural information and biochemical measurements, few have performed docking and molecular dynamic (MD) simulations as a virtual method to predict best ligands. Docking and MD simulations are discussed in the context of discovery of novel semiochemicals (super‐ligands) using homology modelling to conceive further strategies in insect management.     

Novel structures of self-associating stapled peptides

Hydrocarbon stapling of peptides is a powerful technique to transform linear peptides into cell-permeable helical structures that can bind to specific biological targets. In this study, we have used high resolution solution NMR techniques complemented by dynamic light scattering to characterize extensively a family of hydrocarbon stapled peptides with known inhibitory activity against HIV-1 capsid assembly to evaluate the various factors that modulate activity. The helical peptides share a common binding motif but differ in charge, the length, and position of the staple. An important outcome of the study was to show the peptides, share a propensity to self-associate into organized polymeric structures mediated predominantly by hydrophobic interactions between the olefinic chain and the aromatic side-chains from the peptide. We have also investigated in detail the structural significance of the length and position of the staple, and of olefinic bond isomerization in stabilizing the helical conformation of the peptides as potential factors driving polymerization. This study presents the numerous challenges of designing biologically active stapled peptides and the conclusions have broad implications for optimizing a promising new class of compounds in drug discovery.     

Solution structure of barley lipid transfer protein complexed with palmitate. Two different binding modes of palmitate in the homologous maize and barley nonspecific lipid transfer proteins.

The structure of a nonspecific lipid transfer protein from barley (ns-LTPbarley) in complex with palmitate has been determined by NMR spectroscopy. The structure has been compared to the structure of ns-LTPbarley in the absence of palmitate, to the structure of ns-LTPbarley in complex with palmitoyl coenzyme A, to the structure of ns-LTPmaize in its free form, and to the maize protein complexed with palmitate. Binding of palmitate only affects the structure of ns-LTPbarley moderately in contrast to the binding of palmitoyl coenzyme A, which leads to a considerable expansion of the protein. The modes of binding palmitate to the maize and barley protein are different. Although in neither case there are major conformational changes in the protein, the orientation of the palmitate in the two proteins is exactly opposite.