Tyrosine-based "activatable pro-tag": enzyme-catalyzed protein capture and release

Protein recovery is often achieved by a series of capture and release steps that often involve chromatographic binding and elution. We report an alternative, non-chromatographic, capture and release approach that employs enzymes and the stimuli-responsive polysaccharide chitosan. We capture our protein using the enzyme tyrosinase that oxidizes accessible tyrosine residues of the protein and "activates" these residues for covalent capture (i.e., conjugation) onto chitosan. Using fusions of green fluorescent protein (GFP) we observed that: (i) enzymatic activation is required for protein capture to chitosan; and (ii) capture is enhanced (approximately five-fold) by engineering the protein to have a penta-tyrosine fusion tag that provides additional accessible tyrosine residues for enzymatic activation. Because the fusion tag appears to be the primary site for capture, and capture requires activation, we designate penta-tyrosine as a "pro-tag." The captured GFP-chitosan conjugate possesses the pH-responsive solubility that is characteristic of chitosan. We exploit this pH-responsive solubility to facilitate purification of the captured protein. Two enzymatic methods were explored to release the captured GFP from the chitosan conjugate. The first method employs enterokinase (EK) to cleave the protein at an engineered EK-cleavage site. The second method employs chitosanase to hydrolyze the chitosan backbone. Using GFP as a model protein, we demonstrated that enzymatic capture and release provides a simple, non-chromatographic means to recover proteins directly from cell lysates.     

End-point immobilization of recombinant thrombomodulin via sortase-mediated ligation

We report an enzymatic end-point modification and immobilization of recombinant human thrombomodulin (TM), a cofactor for activation of anticoagulant protein C pathway via thrombin. First, a truncated TM mutant consisting of epidermal growth factor-like domains 4-6 (TM(456)) with a conserved pentapeptide LPETG motif at its C-terminal was expressed and purified in E. coli. Next, the truncated TM(456) derivative was site-specifically modified with N-terminal diglycine containing molecules such as biotin and the fluorescent probe dansyl via sortase A (SrtA) mediated ligation (SML). The successful ligations were confirmed by SDS-PAGE and fluorescence imaging. Finally, the truncated TM(456) was immobilized onto an N-terminal diglycine-functionalized glass slide surface via SML directly. Alternatively, the truncated TM(456) was biotinylated via SML and then immobilized onto a streptavidin-functionalized glass slide surface indirectly. The successful immobilizations were confirmed by fluorescence imaging. The bioactivity of the immobilized truncated TM(456) was further confirmed by protein C activation assay, in which enhanced activation of protein C by immobilized recombinant TM was observed. The sortase A-catalyzed surface ligation took place under mild conditions and occurs rapidly in a single step without prior chemical modification of the target protein. This site-specific covalent modification leads to molecules being arranged in a definitively ordered fashion and facilitating the preservation of the protein's biological activity.     

Site-specific, covalent attachment of proteins to a solid surface

Immobilized and site-specifically labeled proteins are becoming invaluable tools in proteomics. Here, we describe a strategy to attach a desired protein to a solid surface in a covalent, site-specific manner. This approach employs an enzymatic posttranslational modification method to site-specifically label a target protein with an azide; an alternative substrate for protein farnesyl transferase containing an azide group was developed for this purpose. A bio-orthogonal Cu(I)-catalyzed cycloaddition reaction is then used to covalently attach the protein to agarose beads bearing an alkyne functional group. We demonstrate that both the azide incorporation and the capture steps can be performed on either a purified protein target or on a protein present within a complex mixture. This approach involves the use of a four-residue tag which is significantly smaller than most other tags reported to date and results in covalent immobilization of the target protein. Hence it should have significant applicability in protein science.     

Computational design of high-affinity epitope scaffolds by backbone grafting of a linear epitope

Computational grafting of functional motifs onto scaffold proteins is a promising way to engineer novel proteins with pre-specified functionalities. Typically, protein grafting involves the transplantation of protein side chains from a functional motif onto structurally homologous regions of scaffold proteins. Using this approach, we previously transplanted the human immunodeficiency virus 2F5 and 4E10 epitopes onto heterologous proteins to design novel "epitope-scaffold" antigens. However, side-chain grafting is limited by the availability of scaffolds with compatible backbone for a given epitope structure and offers no route to modify backbone structure to improve mimicry or binding affinity. To address this, we report here a new and more aggressive computational method-backbone grafting of linear motifs-that transplants the backbone and side chains of linear functional motifs onto scaffold proteins. To test this method, we first used side-chain grafting to design new 2F5 epitope scaffolds with improved biophysical characteristics. We then independently transplanted the 2F5 epitope onto three of the same parent scaffolds using the newly developed backbone grafting procedure. Crystal structures of side-chain and backbone grafting designs showed close agreement with both the computational models and the desired epitope structure. In two cases, backbone grafting scaffolds bound antibody 2F5 with 30- and 9-fold higher affinity than corresponding side-chain grafting designs. These results demonstrate that flexible backbone methods for epitope grafting can significantly improve binding affinities over those achieved by fixed backbone methods alone. Backbone grafting of linear motifs is a general method to transplant functional motifs when backbone remodeling of the target scaffold is necessary.     

The role of CyaY in iron sulfur cluster assembly on the E. coli IscU scaffold protein

Progress in understanding the mechanism underlying the enzymatic formation of iron-sulfur clusters is difficult since it involves a complex reaction and a multi-component system. By exploiting different spectroscopies, we characterize the effect on the enzymatic kinetics of cluster formation of CyaY, the bacterial ortholog of frataxin, on cluster formation on the scaffold protein IscU. Frataxin/CyaY is a highly conserved protein implicated in an incurable ataxia in humans. Previous studies had suggested a role of CyaY as an inhibitor of iron sulfur cluster formation. Similar studies on the eukaryotic proteins have however suggested for frataxin a role as an activator. Our studies independently confirm that CyaY slows down the reaction and shed new light onto the mechanism by which CyaY works. We observe that the presence of CyaY does not alter the relative ratio between [2Fe2S](2+) and [4Fe4S](2+) but directly affects enzymatic activity.     

Protein scaffolds can enhance the bistability of multisite phosphorylation systems

The phosphorylation of a substrate at multiple sites is a common protein modification that can give rise to important structural and electrostatic changes. Scaffold proteins can enhance protein phosphorylation by facilitating an interaction between a protein kinase enzyme and its target substrate. In this work we consider a simple mathematical model of a scaffold protein and show that under specific conditions, the presence of the scaffold can substantially raise the likelihood that the resulting system will exhibit bistable behavior. This phenomenon is especially pronounced when the enzymatic reactions have sufficiently large K(M), compared to the concentration of the target substrate. We also find for a closely related model that bistable systems tend to have a specific kinetic conformation. Using deficiency theory and other methods, we provide a number of necessary conditions for bistability, such as the presence of multiple phosphorylation sites and the dependence of the scaffold binding/unbinding rates on the number of phosphorylated sites.     

Engineering of an anti-epidermal growth factor receptor antibody to single chain format and labeling by Sortase A-mediated protein ligation

Sortase-mediated protein ligation is a biological covalent conjugation system developed from the enzymatic cell wall display mechanism found in Staphylococcus aureus. This three-component system requires: (i) purified Sortase A (SrtA) enzyme; (ii) a substrate containing the LPXTG peptide recognition sequence; and (iii) an oligo-glycine acceptor molecule. We describe cloning of the single-chain antibody sc528, which binds to the extracellular domain of the epidermal growth factor receptor (EGFR), from the parental monoclonal antibody and incorporation of a LPETGG tag sequence. Utilizing recombinant SrtA, we demonstrate successful incorporation of biotin from GGG-biotin onto sc528. EGFR is an important cancer target and is over-expressed in human tumor tissues and cancer lines, such as the A431 epithelial carcinoma cells. SrtA-biotinylated sc528 specifically bound EGFR expressed on A431 cells, but not negative control lines. Similarly, when sc528 was labeled with fluorescein we observed antigen-specific labeling. The ability to introduce functionality into recombinant antibodies in a controlled, site-specific manner has applications in experimental, diagnostic, and potentially clinical settings. For example, we demonstrate addition of all three reaction components in situ within a biosensor flow cell, resulting in oriented covalent capture and presentation of sc528, and determination of precise affinities for the antibody-receptor interaction.     

Revisited BIA-MS combination: entire "on-a-chip" processing leading to the proteins identification at low femtomole to sub-femtomole levels

We present the results of a study in which biomolecular interaction analysis (BIA, Biacoretrade mark 2000) was combined with mass spectrometry (MS) using entire "on-a-chip" procedure. Most BIA-MS studies included an elution step of the analyte prior MS analysis. Here, we report a low-cost approach combining Biacore analysis with homemade chips and MS in situ identification onto the chips without elution step. First experiments have been made with rat serum albumin to determine the sensitivity and validation of the concept has been obtained with an antibody/antigen couple. Our "on-a-chip" procedure allowed complete analysis by MS/MS(2) of the biochip leading to protein identifications at low femtomole to sub-femtomole levels. Using this technique, identification of protein complexes were routinely obtained giving the opportunity to the "on-a-chip" processing to complete the BIA-MS approach in the discovery and analysis of protein complexes.     

Engineering botulinum neurotoxin domains for activation by toxin light chain

Targeted secretion inhibitors (TSI) are a new class of biopharmaceuticals designed from a botulinum neurotoxin protein scaffold. The backbone consists of the 50-kDa endopeptidase light chain and translocation domain (N-terminal portion of the heavy chain), lacks neuronal toxicity, but retains the ability to target cytoplasmic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. TSI are produced as single-chain proteins and then cleaved post-translationally to generate functional heterodimers. Precise proteolytic cleavage is essential to activate the protein to a dichain form. TSI are themselves highly specific proteases. We have exploited this activity to create self-activating enzymes by replacing the native proteolytic site with a substrate SNARE peptide for the TSI protease. We have also created cross-activating backbones. By replacing the proteolytic activation site in one backbone with the substrate SNARE peptide for another serotype, controlled activation is achieved. SNARE peptides encompassing the whole of the coiled-coil region enabled complete activation and assembly of the dichain backbone. These engineered TSI backbones are capable of translocating their enzymatic domains to target intracellular SNARE proteins. They are also investigative tools with which to further the understanding of endopeptidase activity of light chain in SNARE interactions.     

EphrinB ligands recruit GRIP family PDZ adaptor proteins into raft membrane microdomains

Transmembrane ephrinB proteins have important functions during embryonic patterning as ligands for Eph receptor tyrosine kinases and presumably as signal-transducing receptor-like molecules. Consistent with "reverse" signaling, ephrinB1 is localized in sphingo-lipid/cholesterol-enriched raft microdomains, platforms for the localized concentration and activation of signaling molecules. Glutamate receptor-interacting protein (GRIP) and a highly related protein, which we have termed GRIP2, are recruited into these rafts through association with the C-terminal PDZ target site of ephrinB1. Stimulation of ephrinB1 with soluble EphB2 receptor ectodomain causes the formation of large raft patches that also contain GRIP proteins. Moreover, a GRIP-associated serine/threonine kinase activity is recruited into ephrinB1-GRIP complexes. Our findings suggest that GRIP proteins provide a scaffold for the assembly of a multiprotein signaling complex downstream of ephrinB ligands.     

Presence of the epsilon-(gamma-glutamic)lysine crosslink in cellular proteins.

The epsilon-(gamma-glutamic)lysine bond is a covalent interaction which has been found to crosslink polypeptide chains of a number of extracellular proteins. Among known covalent bonds crosslinking protein chains, it is unique in that it is formed directly by enzymatic catalysis, a property which may also endow Glu-Lys crosslink formation with important intracellular functions. We found glutamic-lysine bonds to be present in the procaryote, Escherichia coli, in primitive eucaryotes such as the slime mold, Physarum polycephalum, and the ciliate, Paramecium aurelia, and in muscle cells of a bird and a mammal. Our data show that, although Glu-Lys bonds occur in low concentrations in cellular proteins, they are nevertheless widely distributed. Evidence is also presented indicating that the low levels of the Glu-Lys bonds we measure in the proteins of various cells types are not artifacts of our analytical procedures.     

Protein threading by recursive dynamic programming.

We present the recursive dynamic programming (RDP) method for the threading approach to three-dimensional protein structure prediction. RDP is based on the divide-and-conquer paradigm and maps the protein sequence whose backbone structure is to be found (the protein target) onto the known backbone structure of a model protein (the protein template) in a stepwise fashion, a technique that is similar to computing local alignments but utilising different cost functions. We begin by mapping parts of the target onto the template that show statistically significant similarity with the template sequence. After mapping, the template structure is modified in order to account for the mapped target residues. Then significant similarities between the yet unmapped parts of the target and the modified template are searched, and the resulting segments of the target are mapped onto the template. This recursive process of identifying segments in the target to be mapped onto the template and modifying the template is continued until no significant similarities between the remaining parts of target and template are found. Those parts which are left unmapped by the procedure are interpreted as gaps.The RDP method is robust in the sense that different local alignment methods can be used, several alternatives of mapping parts of the target onto the template can be handled and compared in the process, and the cost functions can be dynamically adapted to biological needs.Our computer experiments show that the RDP procedure is efficient and effective. We can thread a typical protein sequence against a database of 887 template domains in about 12 hours even on a low-cost workstation (SUN Ultra 5). In statistical evaluations on databases of known protein structures, RDP significantly outperforms competing methods. RDP has been especially valuable in providing accurate alignments for modeling active sites of proteins.RDP is part of the ToPLign system (GMD Toolbox for protein alignment) and can be accessed via the WWW independently or in concert with other ToPLign tools at http://cartan.gmd.de/ToPLign.html.     

Genome scale enzyme-metabolite and drug-target interaction predictions using the signature molecular descriptor

MOTIVATION: Identifying protein enzymatic or pharmacological activities are important areas of research in biology and chemistry. Biological and chemical databases are increasingly being populated with linkages between protein sequences and chemical structures. There is now sufficient information to apply machine-learning techniques to predict interactions between chemicals and proteins at a genome scale. Current machine-learning techniques use as input either protein sequences and structures or chemical information. We propose here a method to infer protein-chemical interactions using heterogeneous input consisting of both protein sequence and chemical information. RESULTS: Our method relies on expressing proteins and chemicals with a common cheminformatics representation. We demonstrate our approach by predicting whether proteins can catalyze reactions not present in training sets. We also predict whether a given drug can bind a target, in the absence of prior binding information for that drug and target. Such predictions cannot be made with current machine-learning techniques requiring binding information for individual reactions or individual targets.     

Development of a new class of liver receptor homolog-1 (LRH-1) agonists by photoredox conjugate addition

LRH-1 is a nuclear receptor that regulates lipid metabolism and homeostasis, making it an attractive target for the treatment of diabetes and non-alcoholic fatty liver disease. Building on recent structural information about ligand binding from our labs, we have designed a series of new LRH-1 agonists that further engage LRH-1 through added polar interactions. While the current synthetic approach to this scaffold has, in large part, allowed for decoration of the agonist core, significant variation of the bridgehead substituent is mechanistically precluded. We have developed a new synthetic approach to overcome this limitation, identified that bridgehead substitution is necessary for LRH-1 activation, and described an alternative class of bridgehead substituents for effective LRH-1 agonist development. We determined the crystal structure of LRH-1 bound to a bridgehead-modified compound, revealing a promising opportunity to target novel regions of the ligand binding pocket to alter LRH-1 target gene expression.     

A novel unstructured scaffold based on 4EBP1 enables the functional display of a wide range of bioactive peptides

Target validation using protein aptamers enables the characterization of a specific function of a target protein in an environment that resembles native conditions as closely as possible. A major obstacle to the use of this technology has been the generation of bioactive aptamers, which is dependent on the choice of scaffold. Constraining binding peptides within a particular scaffold does not necessarily result in binding aptamers, as suboptimal presentation of peptides can occur. It is therefore understandable that different peptides might require different scaffolds for optimal presentation. In this article, we describe a novel scaffold protein that bypasses the conventional requirement for scaffolds to have known rigid structures and yet successfully presents several peptides that need to adopt a wide range of conformations for binding to their target protein. Using an unstructured protein, 4EBP1, as scaffold, we successfully construct binding aptamers to three different target proteins: Mdm2, proliferating cell nuclear antigen, and cyclin A. The Mdm2-binding aptamer constructed using 4EBP1 as scaffold demonstrates better stability and bioactivity compared to that constructed using thioredoxin as scaffold. This new scaffold protein, which makes it relatively easy to create bioactive aptamers based on known interaction sequences, will greatly facilitate the aptamer approach to target validation.     

Targeted protein functionalization using His-tags

With the impressive growth in gene sequence data that has become available, recombinant proteins represent an increasingly vast source of molecular components, with unique functional and structural properties, for use in biotechnological applications and devices. To facilitate the use, manipulation, and integration of such molecules into devices, a controllable method for their chemical modification was developed. In this approach, a trifunctional labeling reagent first recognizes and binds a His-tag on the target protein's surface. After binding, a photoreactive group on the trifunctional molecule is triggered to create a covalent linkage between the reagent and the target protein. The third moiety on the labeling reagent can be varied to bring unique chemical functionality to the target protein. This approach provides: (1) specificity in that only His-tagged targets are modified, (2) regio-specific control in that the target is modified proximal to the His-tag, the position of which can be varied, and (3) stoichiometric control in that the number modifications is limited by the binding capacity of the His-tag. Two such labeling reagents were designed, synthesized, and used to modify both N- and C-terminally His-tagged versions of the enzyme murine dihydrofolate reductase (mDHFR). The first reagent biotinylated the enzyme,while the second served to attach an oligonucleotide to yield a protein-DNA conjugate. In all cases, modification in this manner brings new functionality to the protein while leaving the enzymatic activity intact. The protein-DNA conjugate was used to specifically immobilize the active enzyme through DNA hybridization onto polystyrene microspheres, a step toward creating a functional protein microarray.     

Reversible targeting of noncatalytic cysteines with chemically tuned electrophiles

Targeting noncatalytic cysteine residues with irreversible acrylamide-based inhibitors is a powerful approach for enhancing pharmacological potency and selectivity. Nevertheless, concerns about off-target modification motivate the development of reversible cysteine-targeting strategies. Here we show that electron-deficient olefins, including acrylamides, can be tuned to react with cysteine thiols in a rapidly reversible manner. Installation of a nitrile group increased the olefins' intrinsic reactivity, but, paradoxically, eliminated the formation of irreversible adducts. Incorporation of these electrophiles into a noncovalent kinase-recognition scaffold produced slowly dissociating, covalent inhibitors of the p90 ribosomal protein S6 kinase RSK2. A cocrystal structure revealed specific noncovalent interactions that stabilize the complex by positioning the electrophilic carbon near the targeted cysteine. Disruption of these interactions by protein unfolding or proteolysis promoted instantaneous cleavage of the covalent bond. Our results establish a chemistry-based framework for engineering sustained covalent inhibition without accumulating permanently modified proteins and peptides.     

Construction of protein binding sites in scaffold structures

We have developed a strategy for grafting a protein-protein interface based on the known crystal structure of a native ligand and receptor proteins in a complex. The key interaction residues at the ligand protein binding interface are transferred onto a scaffold protein so that the mutated scaffold protein will bind the receptor protein in the same manner as the ligand protein. First, our method identifies key residues and atoms in the ligand protein, which strongly interact with the receptor protein. Second, this method searches the scaffold protein for combinations of candidate residues, among which the distance between any two candidate residues is similar to that between relevant key interaction residues in the ligand protein. These candidate residues are mutated to key interaction residues in the ligand protein respectively. The scaffold protein is superposed onto the ligand protein based upon the coordinates of corresponding atoms, which are assumed to strongly interact with the receptor protein. Complementarity between scaffold and receptor proteins is evaluated. Scaffold proteins with a low superposing rms difference and high complementary score are accepted for further analysis. Then, the relative position of the scaffold protein is adjusted so that the interfaces between the scaffold and receptor proteins have a reasonable packing density. Other mutations are also considered to reduce the desolvation energy or bad steric contacts. Finally, the scaffold protein is cominimized with the receptor protein and evaluated. To test the method, the binding interface of barstar, the inhibitor of barnase, was grafted onto small proteins. Four scaffold proteins with high complementary scores are accepted.