A Mass‐Spectrometry‐Based Approach to Distinguish Annular and Specific Lipid Binding to Membrane Proteins

Membrane proteins engage in a variety of contacts with their surrounding lipids, but distinguishing between specifically bound lipids, and non‐specific, annular interactions is a challenging problem. Applying native mass spectrometry to three membrane protein complexes with different lipid‐binding properties, we explore the ability of detergents to compete with lipids bound in different environments. We show that lipids in annular positions on the presenilin homologue protease are subject to constant exchange with detergent. By contrast, detergent‐resistant lipids bound at the dimer interface in the leucine transporter show decreased k_off rates in molecular dynamics simulations. Turning to the lipid flippase MurJ, we find that addition of the natural substrate lipid‐II results in the formation of a 1:1 protein–lipid complex, where the lipid cannot be displaced by detergent from the highly protected active site. In summary, we distinguish annular from non‐annular lipids based on their exchange rates in solution.     

Structural Characterization of Act c 10.0101 and Pun g 1.0101—Allergens from the Non-Specific Lipid Transfer Protein Family

(1) Background: Non-specific lipid transfer proteins (nsLTPs), which belong to the prolamin superfamily, are potent allergens. While the biological role of LTPs is still not well understood, it is known that these proteins bind lipids. Allergen nsLTPs are characterized by significant stability and resistance to digestion. (2) Methods: nsLTPs from gold kiwifruit (Act c 10.0101) and pomegranate (Pun g 1.0101) were isolated from their natural sources and structurally characterized using X-ray crystallography (3) Results: Both proteins crystallized and their crystal structures were determined. The proteins have a very similar overall fold with characteristic compact, mainly α-helical structures. The C-terminal sequence of Act c 10.0101 was updated based on our structural and mass spectrometry analysis. Information on proteins’ sequences and structures was used to estimate the risk of cross-reactive reactions between Act c 10.0101 or Pun g 1.0101 and other allergens from this family of proteins. (4) Conclusions: Structural studies indicate a conformational flexibility of allergens from the nsLTP family and suggest that immunoglobulin E binding to some surface regions of these allergens may depend on ligand binding. Both Act c 10.0101 and Pun g 1.0101 are likely to be involved in cross-reactive reactions involving other proteins from the nsLTP family.     

Do Intrinsically Disordered Proteins Possess High Specificity in Protein–Protein Interactions?

Specific protein–protein interactions are critical to cellular function. Structural flexibility and disorder‐to‐order transitions upon binding enable intrinsically disordered proteins (IDPs) to overcome steric restrictions and form complementary binding interfaces, and thus, IDPs are widely considered to have high specificity and low affinity for molecular recognition. However, flexibility may also enable IDPs to form complementary binding interfaces with misbinding partners, resulting in a great number of nonspecific interactions. Consequently, it is questionable whether IDPs really possess high specificity. In this work, we investigated this question from a thermodynamic viewpoint. We collected mutant thermodynamic data for 35 ordered protein complexes and 43 disordered protein complexes. We found that the enthalpy–entropy compensation for disordered protein complexes was more complete than that for ordered protein complexes. We further simulated the binding processes of ordered and disordered protein complexes under mutations. Simulation data confirmed the observation of experimental data analyses and further revealed that disordered protein complexes possessed smaller changes in binding free energy than ordered protein complexes under the same mutation perturbations. Therefore, interactions of IDPs are more malleable than those of ordered proteins due to their structural flexibility in the complex. Our results provide new clues for exploring the relationship between protein flexibility, adaptability, and specificity.     

Evaluation of the searching abilities of HBOP and HBSITE for binding pocket detection

We evaluated the pocket‐searching abilities of the computer programs HBOP and HBSITE, in which hydrophobic potentials calculated on grid points generated around a protein function as an indicator of the pocket‐like property‐using a test set of 458 experimentally observed structures of protein–ligand complexes. The results were compared with those obtained using the alternative algorithms PASS and SiteID, which only consider geometric properties, and Q‐SiteFinder, which considers not only geometry but also physicochemical properties. The comparison revealed that HBOP and HBSITE could detect experimentally observed ligand‐binding pockets for more test complexes than PASS and SiteID. In addition, the success rate of HBOP for detecting binding pockets was higher than that of Q‐SiteFinder, and that of HBSITE was comparable with that of Q‐SiteFinder. Results of tests for ligand‐unbound state proteins indicated that HBSITE was more appropriate than Q‐SiteFinder for pocket searches of ligand‐unbound proteins, and HBSITE was more robust than Q‐SiteFinder against structural differences between ligand‐bound and ‐unbound state proteins. © 2009 Wiley Periodicals, Inc. J Comput Chem 2009     

A novel biological role for nsLTP2 from Oriza sativa: Potential incorporation with anticancer agents,nucleosides and their analogues

Development of a protein-based drug delivery system has major impact on the efficacy and bioavailability of unstable and water insoluble drugs. In the present study, the binding modes of a nonspecific lipid transfer protein (nsLTP2) from Oryza sativa with various nucleosides and analogous molecules were identified. The 3-D structure of the protein was designed and validated using modeler 9.13, Molegro virtual docker and procheck tool, respectively. The binding affinity and strength of interactions, key contributing residues and specificity toward the substrates were accomplished by computational docking and model prediction. The protein presented high affinity to acyclovir and vidarabine as purine-analogous drugs. Binding affinity is influenced by the core template and functional groups of the ligands which are structurally different cause the variation of interaction energies with nsLTP2. Nonetheless, all the evaluated analogous drugs occupy the proximity space at the nsLTP active site with high similarity in their binding modes. Our findings hold great promise for the future applications of nsLTPs in various aspects of pharmaceutical science and molecular biology.     

Anisotropic solvent model of the lipid bilayer. 2. Energetics of insertion of small molecules, peptides, and proteins in membranes

A new computational approach to calculating binding energies and spatial positions of small molecules, peptides, and proteins in the lipid bilayer has been developed. The method combines an anisotropic solvent representation of the lipid bilayer and universal solvation model, which predicts transfer energies of molecules from water to an arbitrary medium with defined polarity properties. The universal solvation model accounts for hydrophobic, van der Waals, hydrogen-bonding, and electrostatic solute-solvent interactions. The lipid bilayer is represented as a fluid anisotropic environment described by profiles of dielectric constant (ε), solvatochromic dipolarity parameter (π*), and hydrogen bonding acidity and basicity parameters (α and β). The polarity profiles were calculated using published distributions of quasi-molecular segments of lipids determined by neutron and X-ray scattering for DOPC bilayer and spin-labeling data that define concentration of water in the lipid acyl chain region. The model also accounts for the preferential solvation of charges and polar groups by water and includes the effect of the hydrophobic mismatch for transmembrane proteins. The method was tested on calculations of binding energies and preferential positions in membranes for small-molecules, peptides and peripheral membrane proteins that have been experimentally studied. The new theoretical approach was implemented in a new version (2.0) of our PPM program and applied for the large-scale calculations of spatial positions in membranes of more than 1000 peripheral and integral proteins. The results of calculations are deposited in the updated OPM database ( http://opm.phar.umich.edu ).     

Modeling,docking and dynamics simulations of a non-specific lipid transfer protein from Peganum harmala L.

Non-specific lipid transfer proteins (ns-LTPs), ubiquitously found in various types of plants, have been well-known to transfer amphiphilic lipids and promote the lipid exchange between mitochondria and microbody. In this study, an in silico analysis was proposed to study ns-LTP in Peganum harmala L., which may belong to ns-LTP1 family, aiming at constructing its three-dimensional structure. Moreover, we adopted MEGA to analyze ns-LTPs and other species phylogenetically, which brought out an initial sequence alignment of ns-LTPs. In addition, we used molecular docking and molecular dynamics simulations to further investigate the affinities and stabilities of ns-LTP with several ligands complexes. Taken together, our results about ns-LTPs and their ligand-binding activities can provide a better understanding of the lipid–protein interactions, indicating some future applications of ns-LTP-mediated transport.     

High Relaxivity Supramolecular Adducts Between Human‐Liver Fatty‐Acid‐Binding Protein and Amphiphilic GdIII Complexes: Structural Basis for the Design of Intracellular Targeting MRI Probes

Gadolinium complexes linked to an apolar fragment are known to be efficiently internalized into various cell types, including hepatocytes. Two lipid‐functionalized gadolinium chelates have been investigated for the targeting of the human liver fatty acid binding protein (hL‐FABP) as a means of increasing the sensitivity and specificity of intracellular‐directed MRI probes. hL‐FABP, the most abundant cytosolic lipid binding protein in hepatocytes, displays the ability to interact with multiple ligands involved in lipid signaling and is believed to be an obligate carrier to escort lipidic drugs across the cell. The interaction modes of a fatty acid and a bile acid based gadolinium complex with hL‐FABP have been characterized by relaxometric and NMR experiments in solution with close‐to‐physiological protein concentrations. We have introduced the analysis of paramagnetic‐induced protein NMR signal intensity changes as a quantitative tool for the determination of binding stoichiometry and of precise metal‐ion‐center positioning in protein–ligand supramolecular adducts. A few additional NMR‐derived restraints were then sufficient to locate the ligand molecules in the protein binding sites by using a rapid data‐driven docking method. Relaxometric and ¹³C NMR competition experiments with oleate and the gadolinium complexes revealed the formation of heterotypic adducts, which indicates that the amphiphilic compounds may co‐exist in the protein cavity with physiological ligands. The differences in adduct formation between fatty acid and bile acid based complexes provide the basis for an improved molecular design of intracellular targeted probes.     

Bioactive Structure of Membrane Lipids and Natural Products Elucidated by a Chemistry‐Based Approach

Determining the bioactive structure of membrane lipids is a new concept, which aims to examine the functions of lipids with respect to their three‐dimensional structures. As lipids are dynamic by nature, their “structure” does not refer solely to a static picture but also to the local and global motions of the lipid molecules. We consider that interactions with lipids, which are completely defined by their structures, are controlled by the chemical, functional, and conformational matching between lipids and between lipid and protein. In this review, we describe recent advances in understanding the bioactive structures of membrane lipids bound to proteins and related molecules, including some of our recent results. By examining recent works on lipid‐raft‐related molecules, lipid–protein interactions, and membrane‐active natural products, we discuss current perspectives on membrane structural biology.     

Computational prediction of monosaccharide binding free energies to lectins with linear interaction energy models

The linear interaction energy (LIE) method to compute binding free energies is applied to lectin‐monosaccharide complexes. Here, we calculate the binding free energies of monosaccharides to the Ralstonia solanacearum lectin (RSL) and the Pseudomonas aeruginosa lectin‐II (PA‐IIL). The standard LIE model performs very well for RSL, whereas the PA‐IIL system, where ligand binding involves two calcium ions, presents a major challenge. To overcome this, we explore a new variant of the LIE model, where ligand–metal ion interactions are scaled separately. This model also predicts the saccharide binding preference of PA‐IIL on mutation of the receptor, which may be useful for protein engineering of lectins. © 2012 Wiley Periodicals, Inc.     

Water molecules inside protein structure affect binding of monosaccharides with HIV‐1 antibody 2G12

Water molecules inside biomolecules constitute integral parts of their structure and participate in the functions of the proteins. Some of the X‐ray crystallographic data are insufficient for analyzing a series of ligand–protein complexes in the same condition. We theoretically investigated antibody binding abilities of saccharide ligands and the effects of the inner water molecules of ligand–antibody complexes. Classical molecular dynamics and quantum chemical simulations using a model with possible water molecules inside the protein were performed with saccharide ligands and Human Immunodeficiency Virus 1 neutralizing antibody 2G12 complexes to estimate how inner water molecules of the protein affect the dynamics of the complexes as well as the ligand–antibody interaction. Our results indicate the fact that d ‐fructose's strong affinity to the antibody was partly due to the good retentiveness of solvent water molecules of the ligand and its stability of the ligand's conformation and relative position in the active site. © 2016 Wiley Periodicals, Inc.     

Native Desorption Electrospray Ionization Liberates Soluble and Membrane Protein Complexes from Surfaces

Mass spectrometry (MS) applications for intact protein complexes typically require electrospray (ES) ionization and have not been achieved via direct desorption from surfaces. Desorption ES ionization (DESI) MS has however transformed the study of tissue surfaces through release and characterisation of small molecules. Motivated by the desire to screen for ligand binding to intact protein complexes we report the development of a native DESI platform. By establishing conditions that preserve non‐covalent interactions we exploit the surface to capture a rapid turnover enzyme–substrate complex and to optimise detergents for membrane protein study. We demonstrate binding of lipids and drugs to membrane proteins deposited on surfaces and selectivity from a mix of related agonists for specific binding to a GPCR. Overall therefore we introduce this native DESI platform with the potential for high‐throughput ligand screening of some of the most challenging drug targets including GPCRs.     

Membrane Proteins Have Distinct Fast Internal Motion and Residual Conformational Entropy

The internal motions of integral membrane proteins have largely eluded comprehensive experimental characterization. Here the fast side‐chain dynamics of the α‐helical sensory rhodopsin II and the β‐barrel outer membrane protein W have been investigated in lipid bilayers and detergent micelles by solution NMR relaxation techniques. Despite their differing topologies, both proteins have a similar distribution of methyl‐bearing side‐chain motion that is largely independent of membrane mimetic. The methyl‐bearing side chains of both proteins are, on average, more dynamic in the ps–ns timescale than any soluble protein characterized to date. Accordingly, both proteins retain an extraordinary residual conformational entropy in the folded state, which provides a counterbalance to the absence of the hydrophobic effect. Furthermore, the high conformational entropy could greatly influence the thermodynamics underlying membrane‐protein functions, including ligand binding, allostery, and signaling.     

Site‐Specific Labeling of Proteins with a Chemically Stable,High‐Affinity Tag for Protein Study

Site‐specific labeling of proteins with paramagnetic lanthanides offers unique opportunities by virtue of NMR spectroscopy in structural biology. In particular, these paramagnetic data, generated by the anisotropic paramagnetism including pseudocontact shifts (PCS), residual dipolar couplings (RDC), and paramagnetic relaxation enhancement (PRE), are highly valuable in structure determination and mobility studies of proteins and protein–ligand complexes. Herein, we present a new way to label proteins in a site‐specific manner with a high‐affinity and chemically stable tag, 4‐vinyl(pyridine‐2,6‐diyl)bismethylenenitrilo tetrakis(acetic acid) (4VPyMTA), through thiol alkylation. Its performance has been demonstrated in G47C and E64C mutants of human ubiquitin both in vitro and in a crowded environment. In comparison with the published tags, 4VPyMTA has several interesting features: 1) it has a very high binding affinity for lanthanides (higher than EDTA), 2) there is no heterogeneity in complexes with lanthanides, 3) the derivatized protein is stable and potentially applicable to the in situ analysis of proteins.     

Time to Face the Fats: What Can Mass Spectrometry Reveal about the Structure of Lipids and Their Interactions with Proteins?

Since the 1950s, X-ray crystallography has been the mainstay of structural biology, providing detailed atomic-level structures that continue to revolutionize our understanding of protein function. From recent advances in this discipline, a picture has emerged of intimate and specific interactions between lipids and proteins that has driven renewed interest in the structure of lipids themselves and raised intriguing questions as to the specificity and stoichiometry in lipid-protein complexes. Herein we demonstrate some of the limitations of crystallography in resolving critical structural features of ligated lipids and thus determining how these motifs impact protein binding. As a consequence, mass spectrometry must play an important and complementary role in unraveling the complexities of lipid-protein interactions. We evaluate recent advances and highlight ongoing challenges towards the twin goals of (1) complete structure elucidation of low, abundant, and structurally diverse lipids by mass spectrometry alone, and (2) assignment of stoichiometry and specificity of lipid interactions within protein complexes.     

Kinetic control of histidine-tagged protein surface density on supported lipid bilayers

Nickel-chelating lipids are general tools for anchoring polyhistidine-tagged proteins to supported lipid bilayers (SLBs), but controversy exists over the stability of the protein-lipid attachment. Here, we show that chelator lipids are suitable anchors for building stable, biologically active surfaces but that a simple Langmuirian model is insufficient to describe their behavior. Desorption kinetics from chelator lipids are governed by the valency of surface binding: monovalently bound proteins desorb within minutes (t1/2 approximately 6 min), whereas polyvalently bound species remain bound for hours (t1/2 approximately 12 h). Evolution between surface states is slow, so equilibrium is unlikely to be reached on experimental timescales. However, by tuning incubation conditions, the populations of each species can be kinetically controlled, providing a wide range of protein densities on SLBs with a single concentration of chelator lipid. We propose guidelines for the assembly of SLB surfaces functionalized with specific protein densities and demonstrate their utility in the formation of hybrid immunological synapses.     

Simultaneous removal of thiolated membrane proteins resulting in nanostructured lipid layers

Self-organization of membrane-embedded peptides and proteins causes the formation of lipid mesostructures in the membranes. One example is purple membranes (PM), which consist of lipids and bacteriorhodopsin (BR) as the only protein component. The BRs form a hexagonal crystalline lattice. A complementary structure is formed by the lipids. Employing BR and PM as an example, we report a method where major parts of the mesoscopic self-assembled protein structures can be extracted from the lipid bilayer membrane. A complementary lipid nanostructure remains on the substrate. To remove such a large number of thiolated proteins simultaneously by applying a mechanical force, they are first reacted at physiological conditions with gold nanoparticles, and then a thin gold film is sputtered onto them that fuses with the gold nanoparticles forming a uniform layer, which finally can be lifted off. In this step, all of the previously gold-labeled proteins are pulled out of the membrane simultaneously. A stable lipid nanostructure is obtained on the mica substrate. Its stability is due to either binding of the lipids to the substrate through ionic bonds or to enough residual proteins to stabilize the lipid nanostructure against reorganization. This method may be applied easily and efficiently wherever thiolated proteins or peptides are employed as self-assembling and structure-inducing units in lipid membranes.     

Fast and accurate predictions of binding free energies using MM‐PBSA and MM‐GBSA

In the drug discovery process, accurate methods of computing the affinity of small molecules with a biological target are strongly needed. This is particularly true for molecular docking and virtual screening methods, which use approximated scoring functions and struggle in estimating binding energies in correlation with experimental values. Among the various methods, MM‐PBSA and MM‐GBSA are emerging as useful and effective approaches. Although these methods are typically applied to large collections of equilibrated structures of protein‐ligand complexes sampled during molecular dynamics in water, the possibility to reliably estimate ligand affinity using a single energy‐minimized structure and implicit solvation models has not been explored in sufficient detail. Herein, we thoroughly investigate this hypothesis by comparing different methods for the generation of protein‐ligand complexes and diverse methods for free energy prediction for their ability to correlate with experimental values. The methods were tested on a series of structurally diverse inhibitors of Plasmodium falciparum DHFR with known binding mode and measured affinities. The results showed that correlations between MM‐PBSA or MM‐GBSA binding free energies with experimental affinities were in most cases excellent. Importantly, we found that correlations obtained with the use of a single protein‐ligand minimized structure and with implicit solvation models were similar to those obtained after averaging over multiple MD snapshots with explicit water molecules, with consequent save of computing time without loss of accuracy. When applied to a virtual screening experiment, such an approach proved to discriminate between true binders and decoy molecules and yielded significantly better enrichment curves. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2010     

Efficient molecular mechanics simulations of the folding,orientation, and assembly of peptides in lipid bilayers using an implicit atomic solvation model

Membrane proteins comprise a significant fraction of the proteomes of sequenced organisms and are the targets of approximately half of marketed drugs. However, in spite of their prevalence and biomedical importance, relatively few experimental structures are available due to technical challenges. Computational simulations can potentially address this deficit by providing structural models of membrane proteins. Solvation within the spatially heterogeneous membrane/solvent environment provides a major component of the energetics driving protein folding and association within the membrane. We have developed an implicit solvation model for membranes that is both computationally efficient and accurate enough to enable molecular mechanics predictions for the folding and association of peptides within the membrane. We derived the new atomic solvation model parameters using an unbiased fitting procedure to experimental data and have applied it to diverse problems in order to test its accuracy and to gain insight into membrane protein folding. First, we predicted the positions and orientations of peptides and complexes within the lipid bilayer and compared the simulation results with solid-state NMR structures. Additionally, we performed folding simulations for a series of host–guest peptides with varying propensities to form alpha helices in a hydrophobic environment and compared the structures with experimental measurements. We were also able to successfully predict the structures of amphipathic peptides as well as the structures for dimeric complexes of short hexapeptides that have experimentally characterized propensities to form beta sheets within the membrane. Finally, we compared calculated relative transfer energies with data from experiments measuring the effects of mutations on the free energies of translocon-mediated insertion of proteins into lipid bilayers and of combined folding and membrane insertion of a beta barrel protein.     

Prediction of the interaction of metallic moieties with proteins: An update for protein‐ligand docking techniques

In this article, we present a new approach to expand the range of application of protein‐ligand docking methods in the prediction of the interaction of coordination complexes (i.e., metallodrugs, natural and artificial cofactors, etc.) with proteins. To do so, we assume that, from a pure computational point of view, hydrogen bond functions could be an adequate model for the coordination bonds as both share directionality and polarity aspects. In this model, docking of metalloligands can be performed without using any geometrical constraints or energy restraints. The hard work consists in generating the convenient atom types and scoring functions. To test this approach, we applied our model to 39 high‐quality X‐ray structures with transition and main group metal complexes bound via a unique coordination bond to a protein. This concept was implemented in the protein‐ligand docking program GOLD. The results are in very good agreement with the experimental structures: the percentage for which the RMSD of the simulated pose is smaller than the X‐ray spectra resolution is 92.3% and the mean value of RMSD is < 1.0 Å. Such results also show the viability of the method to predict metal complexes–proteins interactions when the X‐ray structure is not available. This work could be the first step for novel applicability of docking techniques in medicinal and bioinorganic chemistry and appears generalizable enough to be implemented in most protein‐ligand docking programs nowadays available. © 2017 Wiley Periodicals, Inc.