A Molecular Tête-à-Tête Arranged by a Designed Adaptor Protein

Come together: The joining of two miniature proteins binding different protein targets in a synthetic adaptor protein is a novel way to induce proximity between proteins. The formation of a ternary complex was shown to cause the phosphorylation of a noninherent substrate (hDM2) by the kinase Hck. The approach holds promise to become a genetically encodable system to redirect enzyme activities in vivo.     

Anti-cancer activity of novel dibenzo[b,f]azepine tethered isoxazoline derivatives

Background  

Nef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK) strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Recently, we identified a series of kinase inhibitors that preferentially inhibit Hck in the presence of Nef. These compounds also block Nef-dependent HIV replication, validating the Nef-SFK signaling pathway as an antiretroviral drug target. Our findings also suggested that by binding to the Hck SH3 domain, Nef indirectly affects the conformation of the kinase active site to favor inhibitor association.     

Encodable activators of SRC family kinases

There is considerable current interest in the design of encodable molecules that regulate intracellular protein circuitry and/or activity, ideally with a high level of specificity. Src homology 3 (SH3) domains are ubiquitous components of multidomain signaling proteins, including many kinases, and are attractive drug targets because of the important role their interactions play in diseases as diverse as cancer, osteoporosis, and inflammation. Here we describe a set of miniature proteins that recognize distinct SH3 domains from Src family kinases with high affinity. Three of these molecules discriminate effectively between the SH3 domains of Src and Fyn, which are expressed ubiquitously, and two of these three activate Hck kinase with potencies that rival HIV Nef, one of the most potent kinase activators known. These results suggest that miniature proteins represent a viable, encodable strategy for selective activation of Src family kinases in a variety of cell types.     

Selection of v-abl tyrosine kinase substrate sequences from randomized peptide and cellular proteomic libraries using mRNA display

Methodologies for rapidly identifying cellular protein interactions resulting in posttranslational modification of one of the partners are lacking. Here, we select for substrates of the v-abl tyrosine kinase from two protein display libraries in which the protein is covalently linked to its encoding mRNA. Successive selection cycles from a randomized peptide library identified a consensus sequence closely matching that previously reported for the v-abl tyrosine kinase. Selections from a proteomic library derived from cellular mRNA identified several novel targets of v-abl, including a new member of a class of SH2 domain-containing adaptor proteins. Upon modification, several of the substrates obtained in these selections were found to be effective inhibitors of v-abl kinase activity in vitro. These experiments establish a novel method for identifying the substrates of tyrosine kinases from synthetic and cellular protein libraries.     

Formation and activity of template-assembled receptor signaling complexes

Problems in membrane biology require methods to recreate the interactions between receptors and cytoplasmic signaling proteins at the membrane surface. Here, unilamellar vesicles composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine and a nickel-chelating lipid were used as templates to direct the assembly of proteins from the Escherichia coli chemotaxis signaling pathway. The bacterial chemoreceptors are known to form clusters, which promote the binding of the adaptor protein (CheW) and the kinase (CheA). When CheA was incubated with vesicles, CheW, and a histidine-tagged cytoplasmic domain fragment of the aspartate chemoreceptor (CF), the kinase activity was stimulated approximately 300-fold. Activity and pull-down assays were used with dynamic light scattering and electron microscopy to characterize the protein-vesicle compositions that were correlated with the high levels of activity, which demonstrated that CF-CheW-CheA complexes on the vesicle surface were the active entities. Assembly and stimulation occurred with vesicles of different sizes and CFs in different extents of glutamine substitution (in place of glutamate) at physiologically relevant sites. An exception was the combination of sonicated vesicles with the unsubstituted CF, which displayed lower CheA activity. The lower activity was attributed to the high curvature of the sonicated vesicles and a weaker tendency of the unsubstituted CF to self-assemble. Electron micrographs of the vesicle-protein assemblies revealed that protein binding induced pronounced changes in vesicle shape, which was consistent with the introduction of positive curvature in the outer leaflet of the bilayer. Overall, vesicle-mediated template-directed assembly is shown to be an effective way to form functional complexes of membrane-associated proteins and suggests that significant changes in membrane shape can be involved in the process of transmembrane signaling.     

Molecular aptamers for real-time protein-protein interaction study

Protein-protein interactions play critical roles in cellular functions, but current techniques for real-time study of these interactions are limited. We report the real-time monitoring of protein-protein interactions without labeling either of the two interacting proteins; this procedure poses minimum effects on the binding properties of the proteins. Our strategy uses a protein/aptamer complex to probe the interactions in a competitive assay where the binding of an aptamer to its target protein is altered by a second protein that interacts with the target protein. Two signal transduction strategies, fluorescence resonance energy transfer (FRET) and fluorescence anisotropy, have been designed to study the interactions of human alpha-thrombin with different proteins by using two aptamers specific for two binding sites on alpha-thrombin. Our method has been shown to be simple and effective, does not require labeling of proteins, makes use of easily obtainable aptamers, provides detailed protein-protein interaction information and has excellent sensitivity for protein detection and protein-protein interaction studies. The FRET and the fluorescent anisotropy approaches complement each other in providing insight into the kinetics, mechanisms, binding sites and binding dynamics of the interacting proteins.     

Microseparation techniques for the study of the enantioselectivity of drug–plasma protein binding

Stereoselectivity in protein binding can have a significant effect on the pharmacokinetic and pharmacodynamic properties of chiral drugs. The investigation of enantioselectivity of drugs in their binding with human plasma proteins and the identification of the molecular mechanisms involved in the stereodiscrimination by the proteins represent a great challenge for clinical pharmacology. In this review, the separation techniques used for enantioselective protein binding experiments are described and compared. An overview of studies on enantiomer–protein interactions, enantiomer–enantiomer interactions as well as chiral drug–drug interactions, including allosteric effects, is presented. The contribution of individual plasma proteins to the overall enantioselective binding and the animal species variability in drug–plasma protein binding stereoselectivity are reviewed. Copyright © 2008 John Wiley & Sons, Ltd.     

Electrostatics in protein binding and function

Protein electrostatic properties stem from the proportion and distribution of polar and charged residues. Polar and charged residues regulate the electrostatic properties by forming short-range interactions, like salt-bridges and hydrogen-bonds, and by defining the over-all electrostatic environment in the protein. Electrostatics play a major role in defining the mechanisms of protein-protein complex formation, molecular recognitions, thermal stabilities, conformational adaptabilities and protein movements. For example:- Functional hinges, or flexible regions of the protein, lack short-range electrostatic interactions; Thermophilic proteins have higher electrostatic interactions than their mesophilic counter parts; Increase in binding specificity and affinity involve optimization of electrostatics; High affinity antibodies have higher, and stronger, electrostatic interactions with their antigens; Rigid parts of proteins have higher and stronger electrostatic interactions. In this review we address the significance of electrostatics in protein folding, binding and function. We discuss that the electrostatic properties are evolutionally selected by a protein to perform an specific function. We also provide bona fide examples to illustrate this. Additionally, using continuum electrostatic and molecular dynamics approaches we show that the "hot-spot" inter-molecular interactions in a very specific antibody-antigen binding are mainly established through charged residues. These "hot-spot" molecular interactions stay intact even during high temperature molecular dynamics simulations, while the other inter-molecular interactions, of lesser functional significance, disappear. This further corroborates the significance of charge-charge interactions in defining binding mechanisms. High affinity binding frequently involves "electrostatic steering". The forces emerge from over-all electrostatic complementarities and by the formation of charged and polar interactions. We demonstrate that although the high affinity binding of barnase-barstar and anti-hen egg white lysozyme (HEL) antibody-HEL complexes involve different molecular mechanisms, it is electrostatically regulated in both the cases. These observations, and several other studies, suggest that a fine tuning of local and global electrostatic properties are essential for protein binding and function.     

Affinity-guided protein conjugation: the trilogy of covalent protein labeling,assembly and inhibition

Specific and dynamic biological interactions pave the blueprint of signal networks in cell. For example, a great variety of specific protein-ligand interactions define how intracellular signals flow. Taking advantage of the specificity of these interactions, we postulate an “affinity-guided covalent conjugation” strategy to lock binding ligands through covalent reactions between the ligand and the receptor protein. The presence of a nucleophile close to the ligand binding site of a protein is sine qua none of this reaction. Specific noncovalent interaction of a ligand derivative (which contains an electrophile at a designed position) to the ligand binding site of the protein brings the electrophile to the close proximity of the nucleophile. Subsequently, a conjugation reaction spontaneously takes place between the nucleophile and the electrophile, and leads to an intermolecular covalent linkage. This strategy was first showcased in coiled coil peptides which include a cysteine mutation at a selected position. The short peptide sequence was used for covalent labeling of cell surface receptors. The same strategy was then used to guide the design of a set of protein Lego bricks for covalent assembly of protein complexes of unnatural geometry. We finally made “reactive peptides” for natural adaptor proteins that play significant roles in signal transduction. The peptides were designed to react with a single domain of the multidomain adaptor protein, delivered into the cytosol of neurons, and re-directed the intracellular signal of neuronal migration. The trilogy of protein labeling, assembly, and inhibition of intracellular signals, all through a specific covalent bond, fully demonstrated the generality and versatility of “affinity-guided covalent conjugation” in various applications.     

Investigating and characterizing the binding activity of the immobilized calmodulin to calmodulin-dependent protein kinase I binding domain with atomic force microscopy

Protein–protein interactions are responsible for many biological processes, and the study of how proteins undergo a conformational change induced by other proteins in the immobilized state can help us to understand a protein’s function and behavior, empower the current knowledge on molecular etiology of disease, as well as the discovery of putative protein targets of therapeutic interest. In this study, a bottom-up approach was utilized to fabricate micro/nanometer-scale protein patterns. One cysteine mutated calmodulin (CaM), as a model protein, was immobilized on thiol-terminated pattern surfaces. Atomic Force Microscopy (AFM) was then employed as a tool to investigate the interactions between CaM and CaM kinase I binding domain, and show that the immobilized CaM retains its activity to interact with its target protein. Our work demonstrate the potential of employing AFM to the research and assay works evolving surface-based protein–protein interactions biosensors, bioelectronics or drug screening.     

PLIMSTEX: a novel mass spectrometric method for the quantification of protein–ligand interactions in solution

Protein–ligand interactions by mass spectrometry, titration, and H/D exchange (PLIMSTEX) is a new mass spectrometric method for determining association constants and binding stoichiometry for interactions of proteins with various ligands, as well as for quantifying the conformational changes associated with ligand binding to proteins. The association constants determined with PLIMSTEX agree with literature values within a factor of six, establishing its validity for protein interactions involving metal ions, small organic molecules, peptides, and proteins. PLIMSTEX provides solution, not gas-phase, properties by taking advantage of ESI and MALDI mass spectrometry to measure accurately the mass of a protein as it undergoes amide H/D exchange. The approach sidesteps the problem of relating gas-phase abundances of the protein or protein–ligand complex ions to their solution concentrations. With on-column concentration and desalting, high picomole quantities of proteins are sufficient for reproducible mass detection, and the concentration of the protein can be as low as 10⁻⁸ M. It is amenable to different protein/ligand systems in physiologically relevant media. No specially labeled protein or ligand is needed. PLIMSTEX offers minimal perturbation of the binding equilibrium because it uses no denaturants, no additional spectroscopy or reaction probes, and no physical separation of ligand and protein during binding.     

Local factor analysis of rank-deficient reaction systems

A variety of biochemical and physical properties of proteins are regulated by calcium ion (Ca²⁺) binding with varying specificity and affinity. Calcium ion binding can adjust the phospholipid-protein interactions through changing the properties of phospholipid membrane. As an attractive detection technique, whole column imaging detection (WCID) coupled to capillary isoelectric focusing (cIEF) displays several advantages in the study of protein-ligand and protein-protein interactions, including fast and high-efficient separation, high resolution, and simple operation. In this study, a cIEF-WCID method was evaluated for studying the effect of Ca²⁺ binding on protein structural changes and phospholipid-protein interactions. Four proteins with different isoelectirc point (pI), trypsin inhibitor (pI = 4.5), β-lactoglobulin B (pI = 5.2), phosphorylase b (pI = 6.3), and trypsinogen (pI = 9.3), were used for this purpose. The targeted proteins exhibited altered cIEF profiles due to protein conformation changes resulting from the Ca²⁺ binding. The study showed that Ca²⁺ can be buried in the phospholipid membrane, modify the membrane property, and change the phospholipid-protein interactions. The utility of the cIEF-WCID technique demonstrates that the calcium binding plays a crucial role in the protein structural changes and the phospholipid-protein interactions, and elucidates the possible mechanisms involved in calcium-protein binding and calcium bound phospholipid-protein interactions.     

Identifying the inhibition of TIR proteins involved in TLR signalling as an anti-inflammatory strategy

Toll/IL1 receptor (TIR) adaptor proteins continue to be an integral part of Toll-like receptors’ (TLR) signalling involved in inflammation. Signalling is likely to be initiated by these TIR adaptors when they are recruited to a TIR–TIR interface formed by TLR dimerization. Among these, myeloid differentiation factor-88 (MyD88), MyD88 adapter-like protein (Mal), TIR domain-containing adaptor protein inducing interferon-β (TRIF) and TRIF-related adaptor molecule (TRAM) play pivotal roles at many steps in the signalling events leading to inflammation. The presence of the conserved BB loop residues in the TIR domain of all these important adaptor proteins make them possible targets for inhibition by synthetic compounds. We have designed compounds based on an already known MyD88 TIR dimerization inhibitor, T6167923, which binds well not only to the original target but also to the TIR domains of Mal, TRIF and TRAM. The designed inhibitors are based on modifications of the bromophenyl-sulphonyl-thiophenyl-piperazine-carboxamide series of compounds. We have further suggested modifications in these high-affinity compounds for efficient absorption inside the body. Further, a pharmacophore model highlighting important structural interaction features has been developed. The screened compounds are better in binding to the TIR proteins then the parent compound and hence are good starting points for multi-TIR inhibition.     

Self-oligomerization of ASC PYD Domain Prevents the Assembly of Inflammasome In Vitro

NALP3 inflammasome, which is an inflammatory caspase-activating complex, is composed of three proteins: NALP3 (an NOD-like receptor), an apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1. NALP3 senses danger signals, while ASC is an adaptor molecule containing two protein interaction modules: pyrin domain (PYD) and caspase recruitment domain (CARD). Caspase-1 is a cysteine protease that uses cysteine as a nucleophile and has a CARD domain for protein interaction. During inflammasome formation, the ASC adaptor acts as a bridge between caspase and NOD-like receptor (NLR) by offering the CARD for CARD–CARD interactions and PYD for PYD–PYD interactions. In the current study, we successfully purified and characterized NALP3 PYD and ASC PYD. The results showed that ASC PYD easily self-oligomerized under physiological conditions, and this self-oligomerization of the ASC PYD prevented complex formation with NALP3 PYD in vitro.     

Quantitative analysis of energy transfer between fluorescent proteins in CFP-GBP-YFP and its response to Ca2+

This article reports the full characterisation of the optical properties of a biosynthesised protein consisting of fused cyan fluorescent protein, glucose binding protein and yellow fluorescent protein. The cyan and yellow fluorescent proteins act as donors and acceptors for intramolecular fluorescence resonance energy transfer. Absorption, fluorescence, excitation and fluorescence decays of the compound protein were measured and compared with those of free fluorescent proteins. Signatures of energy transfer were identified in the spectral intensities and fluorescence decays. A model describing the fluorescence properties including energy transfer in terms of rate equations is presented and all relevant parameters are extracted from the measurements. The compound protein changes conformation on binding with calcium ions. This is reflected in a change of energy transfer efficiency between the fluorescent proteins. We track the conformational change and the kinetics of the calcium binding reaction from fluorescence intensity and decay measurements and interpret the results in light of the rate equation model. This visualisation of change in protein conformation has the potential to serve as an analytical tool in the study of protein structure changes in real time, in the development of biosensor proteins and in characterizing protein-drug interactions.     

色谱技术在药物-血浆蛋白相互作用研究中的应用进展

小分子药物进入人体血液循环系统后与人血清白蛋白(HSA)、α₁ -酸性糖蛋白(AGP)等血浆蛋白存在广泛的相互作用,这些相互作用深刻影响药物在体内的分布及其与靶标蛋白的结合,进而影响药物效应的发挥。深入探究药物与血浆蛋白间的相互作用对于候选药物的成药性优化、新药研发、联合用药的风险评控等意义重大。而发展高效、灵敏、准确的分析检测方法是开展药物-血浆蛋白相互作用研究的关键。近年来,色谱技术由于其高通量、高分离性能、高灵敏度等特点在该领域得到了广泛的应用,包括测定血浆蛋白翻译后修饰对药物结合的影响,多种药物的竞争性结合等。其中,高效亲和色谱(HPAC)和毛细管电泳(CE)应用最为广泛,能够通过多种分析方法获取结合常数、结合位点数、解离速率常数等相互作用信息。该文着重综述了HPAC和CE在药物-血浆蛋白相互作用研究中的常用策略及最新研究进展,包括HPAC中常用的前沿色谱法、竞争洗脱法、超快亲和提取法、峰值分析法和峰衰减分析法,以及CE中常用的亲和毛细管电泳法(ACE)和毛细管电泳前沿分析法(CE-FA)等。最后,该文还对当前色谱方法存在的不足进行了总结,并对色谱技术在药物-血浆蛋白相互作用研究领域的应用前景和发展方向进行了展望。     

Mapping of stereoselective recognition sites on human serum transferrin by capillary electrophoresis and molecular modelling

Stereoselective recognition of chiral compounds can be used for mapping of surface interaction sites on proteins. Iron-free human serum transferrin is a suitable chiral selector in capillary electrophoresis used in native form in solution. Separation of optical isomers of tryptophan-methylester, tryptophan-ethylester and tryptophan-butylester and various drugs were studied in capillary zone electrophoresis applying a distinct transferrin zone prior to sample injection. Changes in the electrophoretic patterns (i.e., in the migration properties) of the molecules reflected the possible interactions with the protein. The tryptophan derivatives and eight drugs possessed stereoselective interactions, seven drugs showed interactions without appreciable chiral separation, and the others did not present any direct complexation with the protein molecules. Molecular modelling was performed to characterize the binding areas at the iron binding site of iron-free transferrin. The docking of tryptophan derivatives on transferrin showed that the R-enantiomers possess a stronger complexation with transferrin, whereas the S-enantiomers are bound by weaker interactions, which is in excellent agreement with the capillary electrophoresis results, where the R-enantiomers were always retarded stronger by transferrin. A ranking of drugs by the lipo score parameter of the docking shows an accordance with the stereoselective interactions by the protein.     

Circumventing the problems caused by protein diversity in microarrays: implications for protein interaction networks

Protein microarrays provide a well-controlled, high-throughput way to uncover protein-protein interactions. One problem with this and other standardized assays, however, is that proteins vary considerably with respect to their physical properties. If a simple threshold-based approach is used to define protein-protein interactions, the resulting binary networks can be strongly biased. Here, we investigate the extent to which even closely related protein interaction domains vary when printed as microarrays. We find that, when a collection of well behaved, monomeric Src homology 2 (SH2) domains are printed at the same concentration, they vary by up to 50-fold with respect to the resulting surface density of active protein. When a threshold-based binding assay is performed on these domains using fluorescently labeled phosphopeptides, a misleading picture of the underlying biophysical interactions emerges. This problem can be circumvented, however, by obtaining saturation binding curves for each protein-peptide interaction. Importantly, the equilibrium dissociation constants obtained from these curves are independent of the surface density of active protein. We submit that an increased emphasis should be placed on obtaining quantitative information from protein microarrays and that this should serve as a more general goal in all efforts to define large-scale protein interaction networks.     

Inhibition of Ras Signaling by Blocking Ras–Effector Interactions with Cyclic Peptides

Ras genes are frequently activated in human cancers, but the mutant Ras proteins remain largely “undruggable” through the conventional small‐molecule approach owing to the absence of any obvious binding pockets on their surfaces. By screening a combinatorial peptide library, followed by structure–activity relationship (SAR) analysis, we discovered a family of cyclic peptides possessing both Ras‐binding and cell‐penetrating properties. These cell‐permeable cyclic peptides inhibit Ras signaling by binding to Ras‐GTP and blocking its interaction with downstream proteins and they induce apoptosis of cancer cells. Our results demonstrate the feasibility of developing cyclic peptides for the inhibition of intracellular protein–protein interactions and of direct Ras inhibitors as a novel class of anticancer agents.