Isothermal chemical denaturation to determine binding affinity of small molecules to G-protein coupled receptors

The determination of accurate binding affinities is critical in drug discovery and development. Several techniques are available for characterizing the binding of small molecules to soluble proteins. The situation is different for integral membrane proteins. Isothermal chemical denaturation has been shown to be a valuable biophysical method to determine, in a direct and label-free fashion, the binding of ligands to soluble proteins. In this study, the application of isothermal chemical denaturation was applied to an integral membrane protein, the A2a G-protein coupled receptor. Binding affinities for a set of 19 small molecule agonists/antagonists of the A2a receptor were determined and found to be in agreement with data from surface plasmon resonance and radioligand binding assays previously reported in the literature. Therefore, isothermal chemical denaturation expands the available toolkit of biophysical techniques to characterize and study ligand binding to integral membrane proteins, specifically G-protein coupled receptors in vitro.     

The lipid/protein interface as xenobiotic target site: kinetic analysis of the nicotinic acetylcholine receptor

Membrane proteins are known to be solvated and functionally activated by a fixed number of lipid molecules whose multiple binding can be described by Adair-type binding equations. Lipophilic xenobiotics such as general anesthetics may act by competitive displacement of protein-bound lipids. A kinetic equation is now presented for various binding stoichiometries of lipid and xenobiotic, and microscopic binding constants of anesthetics and organic solvents are derived from two independent assay systems for the enhancement of agonist binding to the nicotinic acetylcholine receptor. These constants lead to the first available free energy estimate (-6.4 kcal/mol) for the binding of membrane lipid to an integral membrane protein.     

Lipids in membrane protein structures

This review describes the recent knowledge about tightly bound lipids in membrane protein structures and deduces general principles of the binding interactions. Bound lipids are grouped in annular, nonannular, and integral protein lipids. The importance of lipid binding for vertical positioning and tight integration of proteins in the membrane, for assembly and stabilization of oligomeric and multisubunit complexes, for supercomplexes, as well as their functional roles are pointed out. Lipid binding is stabilized by multiple noncovalent interactions from protein residues to lipid head groups and hydrophobic tails. Based on analysis of lipids with refined head groups in membrane protein structures, distinct motifs were identified for stabilizing interactions between the phosphodiester moieties and side chains of amino acid residues. Differences between binding at the electropositive and electronegative membrane side, as well as a preferential binding to the latter, are observed. A first attempt to identify lipid head group specific binding motifs is made. A newly identified cardiolipin binding site in the yeast cytochrome bc(1) complex is described. Assignment of unsaturated lipid chains and evolutionary aspects of lipid binding are discussed.     

A novel flow cytometric assay to quantify interactions between proteins and membrane lipids

A diverse set of experimental systems has been developed to probe protein-lipid interactions. These include measurements with the headgroups of membrane lipids in solution, immobilized membrane lipids, and analysis of protein binding to membrane lipids reconstituted in liposomes. Each of these methodologies has strengths but also substantial limitations. For example, measurements between proteins and lipid headgroups or with immobilized membrane lipids do not probe interactions in their natural environment, the lipid bilayer. The use of liposomes, however, was so far mostly restricted to biochemical flotation experiments that do not provide quantitative and/or kinetic data. Here, we present a fast and sensitive flow cytometric method to detect protein-lipid interactions. This technique allows for quantitative measurements of interactions between multiple fluorescently labeled proteins and membrane lipids reconstituted in lipid bilayers. The assay can be used to quantify binding efficiencies and to determine kinetic constants. The method is further characterized by a short sampling time of only a few seconds that allows for high-content screening procedures. Finally, using light scatter measurements, the described method also allows for monitoring changes of membrane curvature as well as tethering of liposomes evoked by binding of proteins.     

Lysosomes and protein degradation

Evidence from studies on mouse peritoneal macrophages using the inhibitor pepstatin confirms lysosomal involvement in basal protein degradation, and extends its relevance to degradation of long half-life and analogue containing proteins. Studies on the ability of MRC-5 (a limited life-span fibroblast line) cells to selectively degrade analogue-containing proteins are described. These indicate that this capacity is retained even in very old cells; indeed such cells show an increased proportion of rapidly-degradable proteins. Analogue containing proteins bind preferentially to lysosomal membranes, and like liver cytosol proteins of short half-life, are selectively endocytosed and degraded by certain cells in culture. Thus membrane binding allowing selective entry to the lysosomal system may be important in controlling rate of degradation of both intracellular and extracellular protein. A method potentially allowing for determination of the rate of autophagy in cells, is described. This should enable further assessment of the quantitative involvement of lysosomes in protein degradation.     

Plasma membrane tubulin

The association of tubulin with the plasma membrane comprises multiple levels of penetration into the bilayer: from integral membrane protein, to attachment via palmitoylation, to surface binding, and to microtubules attached by linker proteins to proteins in the membrane. Here we discuss the soundness and weaknesses of the chemical and biochemical evidence marshaled to support these associations, as well as the mechanisms by which tubulin or microtubules may regulate functions at the plasma membrane.     

Scanning the DNA for damage by the nucleotide excision repair machinery

Damage detection during nucleotide excision repair requires the action of multiple proteins that probe the DNA for different parameters like disruption of basepairing, DNA bendability and presence of chemical modifications. In a recent study it has been shown that two of these probing events can be spatially separated on the DNA. Upon initial binding of the XPC protein to a region with disrupted basepairing a complex of XPC, TFIIH and XPA is translocated to a CPD lesion even when this chemical modification is located up to 160 nucleotides from the mispaired region.     

Polymersome surface decoration by an EGFP fusion protein employing Cecropin A as peptide "anchor"

Polymer based nanocompartments have potential applications in synthetic biology, medicine (drug release) and industrial biotechnology (chiral nanoreactors, multistep syntheses, selective product recovery). A step towards the aforementioned goals is the polymer membrane functionalization through covalent bonding of chemical anchors or insertion of proteins/peptides, to obtain specific properties like recognition, catalytic activity and facilitated diffusion, mimicking the complexity of a biological membrane. The use of genetic engineering techniques widens the possible applications of peptides and proteins specifically designed for polymer membrane interactions.A fusion protein (CecEGFP) based on the antibacterial peptide Cecropin A and the EGFP (Enhanced Green Fluorescent Protein) was designed, expressed and biophysically characterized. CecEGFP interaction with the tri-block copolymer PIB-PEG-PIB (PIB = polyisobutylene, PEG = polyethylene glycol) based polymersome membrane was analyzed by circular dichroism as well as EGFP and Trp fluorescence measurements. Results proved that Cecropin A is usable as a “membrane surface anchor” for water soluble proteins, as it inserts into the polymer membrane.The aim and novelty of this study is within the design of fusion proteins specifically developed for polymer membrane interactions. The use of amphiphilic Cecropin A “anchoring” water soluble proteins to the polymersome surface, avoids chemical coupling between polymers and proteins.     

Computational approaches for protein function prediction: A combined strategy from multiple sequence alignment to molecular docking-based virtual screening

The functional characterization of proteins represents a daily challenge for biochemical, medical and computational sciences. Although finally proved on the bench, the function of a protein can be successfully predicted by computational approaches that drive the further experimental assays. Current methods for comparative modeling allow the construction of accurate 3D models for proteins of unknown structure, provided that a crystal structure of a homologous protein is available. Binding regions can be proposed by using binding site predictors, data inferred from homologous crystal structures, and data provided from a careful interpretation of the multiple sequence alignment of the investigated protein and its homologs. Once the location of a binding site has been proposed, chemical ligands that have a high likelihood of binding can be identified by using ligand docking and structure-based virtual screening of chemical libraries. Most docking algorithms allow building a list sorted by energy of the lowest energy docking configuration for each ligand of the library. In this review the state-of-the-art of computational approaches in 3D protein comparative modeling and in the study of protein–ligand interactions is provided. Furthermore a possible combined/concerted multistep strategy for protein function prediction, based on multiple sequence alignment, comparative modeling, binding region prediction, and structure-based virtual screening of chemical libraries, is described by using suitable examples. As practical examples, Abl-kinase molecular modeling studies, HPV-E6 protein multiple sequence alignment analysis, and some other model docking-based characterization reports are briefly described to highlight the importance of computational approaches in protein function prediction.     

Cyclic dipeptide-based small molecules modulate zinc-mediated liquid–liquid phase separation of tau

Liquid–liquid phase separation (LLPS) is a complex physicochemical phenomenon mediated by multivalent transient weak interactions among macromolecules like polymers, proteins, and nucleic acids. It has implications in cellular physiology and disease conditions like cancer and neurodegenerative disorders. Many proteins associated with neurodegenerative disorders like RNA binding protein FUS (FUsed in Sarcoma), alpha-synuclein (α-Syn), TAR DNA binding protein 43 (TDP-43), and tau are shown to undergo LLPS. Recently, the tau protein responsible for Alzheimer's disease (AD) and other tauopathies is shown to phase separate into condensates in vitro and in vivo. The diverse noncovalent interactions among the biomolecules dictate the complex LLPS phenomenon. There are limited chemical tools to modulate protein LLPS which has therapeutic potential for neurodegenerative disorders. We have rationally designed cyclic dipeptide (CDP)-based small-molecule modulators (SMMs) by integrating multiple chemical groups that offer diverse chemical interactions to modulate tau LLPS. Among them, compound 1c effectively inhibits and dissolves Zn-mediated tau LLPS condensates. The SMM also inhibits tau condensate-to-fibril transition (tau aggregation through LLPS). This approach of designing SMMs of LLPS establishes a novel platform that has potential implication for the development of therapeutics for neurodegenerative disorders.     

Cell surface localization and processing of the ComG proteins, required for DNA binding during transformation of Bacillus subtilis

The comG operon of Bacillus subtilis encodes seven proteins essential for the binding of transforming DNA to the competent cell surface. We have explored the processing of the ComG proteins and the cellular localization of six of them. All of the proteins were found to be membrane associated. The four proteins with N-terminal sequence motifs typical of type 4 prepilins (ComGC, GD, GE and GG) are processed by a pathway that requires the product of comC , also an essential competence gene. The unprocessed forms of ComGC and GD behave like integral membrane proteins. Pre-ComGG differs from pre-ComGC and pre-ComGD, in that it is accessible to proteolysis only from the cytoplasmic face of the membrane and at least a portion of it behaves like a peripheral membrane protein. The mature forms of these proteins are translocated to the outer face of the membrane and are liberated when peptidoglycan is hydrolysed by lysozyme or mutanolysin. ComGG exists in part as a disulphide-cross-linked homodimer in vivo . ComGC was found to possess an intramolecular disulphide bond. The previously identified homodimer form of this protein is not stabilized by disulphide bond formation. ComGF behaves as an integral membrane protein, while ComGA, a putative ATPase, is located on the inner face of the membrane as a peripheral membrane protein. Possible roles of the ComG proteins in DNA binding to the competent cell surface are discussed in the light of these and other results.     

Interactions of lipids and proteins: Some general principles

Methods to describe the binding of phospholipids to membrane proteins are described. It is shown that it is difficult to obtain estimates of the number of phospholipids bound to the surface of a membrane protein from ESR experiments in which plots of free to bound spin label (y) vs. molar ratio of lipid to protein are extrapolated toy=0. The relative advantages and disadvantages of ESR and fluorescence methods for measuring relative binding constants of phospholipids to membrane proteins are discussed. The particular problems associated with comparing binding constants of molecules of very different sizes (e.g., fatty acids and cardiolipin) are described and equations are presented to account for these problems. The possible effects of membrane viscosity and thickness on activity of membrane proteins are discussed, but it is concluded that effects of phospholipid structure on activity can only be understood in terms of a reasonably complete kinetic model for the protein.     

Membrane binding of lipidated Ras peptides and proteins — The structural point of view

Biological membranes are interesting interfaces, at which important biological processes occur. In addition to integral membrane proteins, a number of proteins bind to the membrane surface and associate with it. Posttranslational lipid modification is one important mechanism, by which soluble molecules develop a propensity towards the membrane and reversibly bind to it. Membrane binding by insertion of hydrophobic lipid moieties is relevant for up to 10% of all cellular proteins. A particular interesting lipid-modified protein is the small GTPase Ras, which plays a key role in cellular signal transduction. Until recently, the structural basis for membrane binding of Ras was not well-defined. However, with the advent of new synthesis techniques and the advancement of several biophysical methods, a number of structural and dynamical features about membrane binding of Ras proteins have been revealed. This review will summarize the chemical biology of Ras and discuss in more detail the biophysical and structural features of the membrane bound C-terminus of the protein.     

Membrane binding of lipidated Ras peptides and proteins--the structural point of view

Biological membranes are interesting interfaces, at which important biological processes occur. In addition to integral membrane proteins, a number of proteins bind to the membrane surface and associate with it. Posttranslational lipid modification is one important mechanism, by which soluble molecules develop a propensity towards the membrane and reversibly bind to it. Membrane binding by insertion of hydrophobic lipid moieties is relevant for up to 10% of all cellular proteins. A particular interesting lipid-modified protein is the small GTPase Ras, which plays a key role in cellular signal transduction. Until recently, the structural basis for membrane binding of Ras was not well-defined. However, with the advent of new synthesis techniques and the advancement of several biophysical methods, a number of structural and dynamical features about membrane binding of Ras proteins have been revealed. This review will summarize the chemical biology of Ras and discuss in more detail the biophysical and structural features of the membrane bound C-terminus of the protein.     

Molecular dissection of the membrane aggregation mechanisms induced by monomeric annexin A2

Annexins are a multigene family of proteins involved in aggregation and fusion processes of biological membranes. One of its best-known members is annexin A2 (or p36), capable of binding to acidic phospholipids in a calcium-dependent manner, as occurs with other members of the same family. In its heterotetrameric form, especially with protein S100A10 (p11), annexin A2 has been involved as a determinant factor in innumerable biological processes like tumor development or anticoagulation. However, the subcellular coexistence of different pools of the protein, in which the monomeric form of annexin A2 is growing in functional relevance, is to date poorly described. In this work we present an exhaustive structural and functional characterization of monomeric human annexin A2 by using different recombinant mutants. The important role of the amphipathic N-terminal α-helix in membrane binding and aggregation has been analyzed. We have also studied the potential implication of lateral “antiparallel” protein dimers in membrane aggregation. In contrast to what was previously suggested, formation of these dimers negatively regulate aggregation. We have also confirmed the essential role of three lysine residues located in the convex surface of the molecule in calcium-free and calcium-dependent membrane binding and aggregation. Finally, we propose models for annexin A2-mediated vesicle aggregation mechanisms.     

Molecular architecture of glycinergic synapses

Synapses can be considered chemical machines, which are optimized for fast and repeated exocytosis of neurotransmitters from presynaptic nerve terminals and the reliable electrical or chemical transduction of neurotransmitter binding to the appropriate receptors in the postsynaptic membrane. Therefore, synapses share a common repertoire of proteins like, e.g., the release machinery and certain cell adhesion molecules. This basic repertoire must be extended in order to generate specificity of neurotransmission and allow plastic changes, which are considered the basis of developmental and/or learning processes. Here, we focus on these complementary molecules located in the presynaptic terminal and postsynaptic membrane specializations of glycinergic synapses. Moreover, as specificity of neurotransmission in this system is established by the specific binding of the neurotransmitter to its receptor, we review the molecular properties of glycine receptor subunits and their assembly into functional glycine receptors with different functional characteristics. The past years have revealed that the molecular machinery underlying inhibitory and especially glycinergic postsynaptic membrane specializations is more complex and dynamic than previously anticipated from morphological studies. The emerging features include structural components as well as signaling modules, which could confer the plasticity required for the proper function of distinct motor and sensory functions.     

Conformational plasticity of the influenza A M2 transmembrane helix in lipid bilayers under varying pH, drug binding, and membrane thickness

Membrane proteins change their conformations to respond to environmental cues, thus conformational plasticity is important for function. The influenza A M2 protein forms an acid-activated proton channel important for the virus lifecycle. Here we have used solid-state NMR spectroscopy to examine the conformational plasticity of membrane-bound transmembrane domain of M2 (M2TM). (13)C and (15)N chemical shifts indicate coupled conformational changes of several pore-facing residues due to changes in bilayer thickness, drug binding, and pH. The structural changes are attributed to the formation of a well-defined helical kink at G34 in the drug-bound state and in thick lipid bilayers, nonideal backbone conformation of the secondary-gate residue V27 in the presence of drug, and nonideal conformation of the proton-sensing residue H37 at high pH. The chemical shifts constrained the (?, ψ) torsion angles for three "basis" states, the equilibrium among which explains the multiple resonances per site in the NMR spectra under different combinations of bilayer thickness, drug binding, and pH conditions. Thus, conformational plasticity is important for the proton conduction and inhibition of M2TM. The study illustrates the utility of NMR chemical shifts for probing the structural plasticity and folding of membrane proteins.     

Domain Analyses Reveal That Chlamydia trachomatis CT694 Protein Belongs to the Membrane-localized Family of Type III Effector Proteins

The Chlamydia trachomatis type three-secreted effector protein CT694 is expressed during late-cycle development yet is secreted by infectious particles during the invasion process. We have previously described the presence of at least two functional domains within CT694. CT694 was found to interact with the human protein Ahnak through a C-terminal domain and affect formation of host-cell actin stress fibers. Immunolocalization analyses of ectopically expressed pEGFP-CT694 also revealed plasma membrane localization for CT694 that was independent of Ahnak binding. Here we provide evidence that CT694 contains multiple functional domains. Plasma membrane localization and CT694-induced alterations in host cell morphology are dependent on an N-terminal domain. We demonstrate that membrane association of CT694 is dependent on a domain resembling a membrane localization domain (MLD) found in anti-host proteins from Yersinia, Pseudomonas, and Salmonella spp. This domain is necessary and sufficient for localization and morphology changes but is not required for Ahnak binding. Further, the CT694 MLD is able to complement ExoS ΔMLD when ectopically expressed. Taken together, our data indicate that CT694 is a multidomain protein with the potential to modulate multiple host cell processes.     

Thematic Minireview Series: Cell Biology of G Protein Signaling

This thematic series is on the topic of cell signaling from a cell biology perspective, with a particular focus on G proteins. G protein-coupled receptors (GPCRs, also known as seven-transmembrane receptors) are typically found at the cell surface. Upon agonist binding, these receptors will activate a GTP-binding G protein at the cytoplasmic face of the plasma membrane. Additionally, there is growing evidence that G proteins can also be activated by non-receptor binding partners, and they can signal from non-plasma membrane compartments. The production of second messengers at multiple, spatially distinct locations represents a type of signal encoding that has been largely neglected. The first minireview in the series describes biosensors that are being used to monitor G protein signaling events in live cells. The second describes the implementation of antibody-based biosensors to dissect endosome signaling by G proteins and their receptors. The third describes the function of a non-receptor, cytoplasmic activator of G protein signaling, called GIV (Girdin). Collectively, the advances described in these articles provide a deeper understanding and emerging opportunities for new pharmacology.