Arrays for the combinatorial exploration of cell adhesion

A new method for the fabrication of arrays of self-assembled monolayers (SAMs) of alkane thiols (ATs) on gold to combinatorially assay surfaces for cell adhesion is reported. A fluorous SAM, which is both cytophobic and solvophobic, was used as the background between the array features. The resulting solvophobic background permits the application of an assembly after conjugation strategy for fabrication. SAMs containing mixtures of ATs and peptide-terminated ATs were generated. Multiple cell types demonstrated differential and specific binding to these surfaces. Additionally, pluripotent human embryonic stem cells proliferated on surfaces generated by this method.     

Simple and versatile methods for the fabrication of arrays of live mammalian cells

Single-step methods for the generation of patterned surfaces on hydrogels are presented. Poly(vinyl alcohol) films covalently bonded on glass cover slips and commercially available hydrogel-coated polystyrene plates were used as cell-repellent surfaces. Cell-adhesive domains were created by spotting dilute solutions of sodium hypochlorite onto the surfaces. Alternatively, domains supporting cell attachment were created by exposure to UV light from a xenon excimer lamp, employing a contact mask. Rat skeletal myoblast cells, HEK 293 human embryonic kidney cells and Caco-2 colon carcinoma cells adhered and spread exclusively on modified areas. The surfaces are durable for weeks under cell culture conditions and re-usable after removal of the cells by trypsin treatment. Arrays of adhesive spots seeded with cells at a low density permitted dynamic monitoring of cell proliferation. Selected colonies can be harvested from the surfaces by means of local trypsination. Thus, these techniques may provide useful tools for the isolation of clonal cell populations. Additionally, we demonstrate the possibility of surface-mediated gene delivery from the micro patterns. We show that DNA, complexed with a lipid reagent, can be adsorbed on modified poly(vinyl alcohol) coatings, resulting in spatially controlled adhesion and reverse transfection of HEK 293 cells.     

Structure-based design and confirmation of peptide ligands for neuronal polo-like kinase to promote neuroregeneration

Neuronal polo-like kinase (nPLK) is an essential regular of cell cycle and differentiation in nervous system, and targeting nPLK has been established as a promising therapeutic strategy to treat neurological disorders and to promote neuroregeneration. The protein contains an N-terminal kinase domain (KD) and a C-terminal Polo-box domain (PBD) that are mutually inhibited by each other. Here, the intramolecular KD–PBD complex in nPLK was investigated at structural level via bioinformatics analysis, molecular dynamics (MD) simulation and binding affinity scoring. From the complex interface two regions representing separately two continuous peptide fragments in PBD domain were identified as the hot spots of KD–PBD interaction. Structural and energetic analysis suggested that one (PBD peptide 1) of the two peptides can bind tightly to a pocket nearby the active site of KD domain, which is thus potential as self-inhibitory peptide to target and suppress nPLK kinase activity. The knowledge harvesting from computational studies were then used to guide the structural optimization and mutation of PBD peptide 1. Consequently, two of three peptide mutants separately exhibited moderately and considerably increased affinity as compared to the native peptide. The computationally modeled complex structures of KD domain with these self-inhibitory peptides were also examined in detail to unravel the structural basis and energetic property of nPLK-peptide recognition and interaction.     

Tunable micropatterned substrates based on poly(dopamine) deposition via microcontact printing

A simple technique was developed to fabricate tunable micropatterned substrates based on mussel-inspired surface modification. Polydopamine (PDA) was developed on polydimethylsiloxane (PDMS) stamps and was easily imprinted to several substrates such as glass, silicon, gold, polystyrene, and poly(ethylene glycol) via microcontact printing. The imprinted PDA retained its unique reactivity and could modulate the chemical properties of micropatterns via secondary reactions, which was illustrated in this study. PDA patterns imprinted onto a cytophobic and nonfouling substrates were used to form patterns of cells or proteins. PDA imprints reacted with nucleophilic amines or thiols to conjugate molecules such as poly(ethylene glycol) for creating nonfouling area. Gold nanoparticles were immobilized onto PDA-stamped area. The reductive ability of PDA transformed silver ions to elemental metals as an electroless process of metallization. This facile and economic technique provides a powerful tool for development of a functional patterned substrate for various applications.     

An improved method for the synthesis of cellulose membrane-bound peptides with free C termini is useful for PDZ domain binding studies

SPOT synthesis permits parallel synthesis and screening of thousands of cellulose membrane-bound peptides to study protein-protein interactions in a proteomic context. Recognition of C-terminal residues is one of the most common binding features of PDZ domains. Unfortunately, most solid support-bound peptide libraries lack a free C terminus due to C-terminal fixation on the solid support. To overcome this restriction, we developed a robust methodology based on our previous strategy for generating peptides with authentic C termini. To validate this improved method, we screened a human peptide library of 6223 C termini with the syntrophin PDZ domain. Furthermore, using the same library, new peptide ligands derived from membrane proteins and receptors were found for the ERBIN PDZ domain. Finally, we identified the protein kinase breakpoint cluster region, which is known as a negative regulator of cell proliferation and oncogenic transformation, as an ERBIN ligand.     

Calorimetric and structural studies of 1,2,3-trisubstituted cyclopropanes as conformationally constrained peptide inhibitors of Src SH2 domain binding.

Isothermal titration calorimetry and X-ray crystallography have been used to determine the structural and thermodynamic consequences associated with constraining the pTyr residue of the pYEEI ligand for the Src Homology 2 domain of the Src kinase (Src SH2 domain). The conformationally constrained peptide mimics that were used are cyclopropane-derived isosteres whereby a cyclopropane ring substitutes to the N-Calpha-Cbeta atoms of the phosphotyrosine. Comparison of the thermodynamic data for the binding of the conformationally constrained peptide mimics relative to their equivalent flexible analogues as well as a native tetrapeptide revealed an entropic advantage of 5-9 cal mol(-1) K(-1) for the binding of the conformationally constrained ligands. However, an unexpected drop in enthalpy for the binding of the conformationally constrained ligands relative to their flexible analogues was also observed. To evaluate whether these differences reflected conformational variations in peptide binding modes, we have determined the crystal structure of a complex of the Src SH2 domain bound to one of the conformationally constrained peptide mimics. Comparison of this new structure with that of the Src SH2 domain bound to a natural 11-mer peptide (Waksman et al. Cell 1993, 72, 779-790) revealed only very small differences. Hence, cyclopropane-derived peptides are excellent mimics of the bound state of their flexible analogues. However, a rigorous analysis of the structures and of the surface areas at the binding interface, and subsequent computational derivation of the energetic binding parameters, failed to predict the observed differences between the binding thermodynamics of the rigidified and flexible ligands, suggesting that the drop in enthalpy observed with the conformationally constrained peptide mimic arises from sources other than changes in buried surface areas, though the exact origin of the differences remains unclear.     

Acquisition of Fyn-selective SH3 domain ligands via a combinatorial library strategy

A stepwise library-based strategy has been employed to acquire a potent ligand for the SH3 domain of Fyn, a Src kinase family member that plays a key role in T cell activation. The easily automated methodology is designed to identify potential interaction sites that circumscribe the protein/peptide binding region on the SH3 domain. The library protocol creates peptide/nonpeptide chimeras that are able to bind to these interaction sites that are otherwise inaccessible to natural amino acid residues. The peptide-derived lead and the Fyn-SH3 domain form a complex that exhibits a K(D) of 25 +/- 5 nM, approximately 1000-fold more potent than that displayed by the corresponding conventional peptide ligand. Furthermore, the lead ligand exhibits selectivity against SH3 domains derived from other Src kinases, in spite of a sequence identity of approximately 80%.     

Rewiring cell adhesion

The adhesion of cells is mediated by the binding of several cell-surface receptors to ligands found in the extracellular matrix. These receptors often have overlapping specificities for the peptide ligands, making it difficult to understand the roles for discrete receptors in cell adhesion, migration, and differentiation as well as to direct the selective adhesion of cell types in tissue-engineering applications. To overcome these limitations, we developed a strategy to rewire the receptor-ligand interactions between a cell and substrate to ensure that adhesion is mediated by a single receptor with unique specificity. The strategy combines a genetic approach to engineer the cell surface with a chimeric integrin receptor having a unique ligand binding domain with a surface chemistry approach to prepare substrates that present ligands that are bound by the new binding domain. We show that Chinese hamster ovary cells that are engineered with a chimeric beta1 integrin adhere, signal, and even migrate on a synthetic matrix.     

Simultaneous prediction of binding free energy and specificity for PDZ domain–peptide interactions

Interactions between protein domains and linear peptides underlie many biological processes. Among these interactions, the recognition of C-terminal peptides by PDZ domains is one of the most ubiquitous. In this work, we present a mathematical model for PDZ domain–peptide interactions capable of predicting both affinity and specificity of binding based on X-ray crystal structures and comparative modeling with Rosetta. We developed our mathematical model using a large phage display dataset describing binding specificity for a wild type PDZ domain and 91 single mutants, as well as binding affinity data for a wild type PDZ domain binding to 28 different peptides. Structural refinement was carried out through several Rosetta protocols, the most accurate of which included flexible peptide docking and several iterations of side chain repacking and backbone minimization. Our findings emphasize the importance of backbone flexibility and the energetic contributions of side chain-side chain hydrogen bonds in accurately predicting interactions. We also determined that predicting PDZ domain–peptide interactions became increasingly challenging as the length of the peptide increased in the N-terminal direction. In the training dataset, predicted binding energies correlated with those derived through calorimetry and specificity switches introduced through single mutations at interface positions were recapitulated. In independent tests, our best performing protocol was capable of predicting dissociation constants well within one order of magnitude of the experimental values and specificity profiles at the level of accuracy of previous studies. To our knowledge, this approach represents the first integrated protocol for predicting both affinity and specificity for PDZ domain–peptide interactions.     

Design of peptide that recognizes double-stranded DNA.

A novel molecular tool for double-stranded (ds) DNA detection using synthetic peptide is described. The peptide was designed based on the DNA binding domain of the lambda phage CRO repressor (CRO). The designed peptides contain helix-turn-helix (HTH), which is DNA binding motif. A cyclic peptide and a mutant peptide based on CRO were also designed, and the resulting affinity for dsDNA was increased. Furthermore, native amino acids of the peptide were replaced with arginine to increase the affinity for dsDNA. The affinity of these peptides for DNA binding was assessed by surface plasmon resonance (SPR) technique.     

Molecular recognition properties of FN3 monobodies that bind the Src SH3 domain

We have constructed a phage-displayed library based on the human fibronectin tenth type III domain (FN3) scaffold by randomizing residues in its FG and BC loops. Screening against the SH3 domain of human c-Src yielded six different clones. Five of these contained proline-rich sequences in their FG loop that resembled class I (i.e., +xxPxxP) peptide ligands for the Src SH3 domain. The sixth clone lacked the proline-rich sequence and showed particularly high binding specificity to the Src SH3 domain among various SH3 domains tested. Competitive binding, loop replacement, and NMR perturbation experiments were conducted to analyze the recognition properties of selected binders. The strongest binder was able to pull down full-length c-Src from murine fibroblast cell extracts, further demonstrating the potential of this scaffold for use as an antibody mimetic.     

Design of a hybrid biosensor for enhanced phosphopeptide recognition based on a phosphoprotein binding domain coupled with a fluorescent chemosensor

Protein-based fluorescent biosensors with sufficient sensing specificity are useful analytical tools for detection of biologically important substances in complicated biological systems. Here, we present the design of a hybrid biosensor, specific for a bis-phosphorylated peptide, based on a natural phosphoprotein binding domain coupled with an artificial fluorescent chemosensor. The hybrid biosensor consists of a phosphoprotein binding domain, the WW domain, into which has been introduced a fluorescent stilbazole having Zn(II)-dipicolylamine (Dpa) as a phosphate binding motif. It showed strong binding affinity and high sensing selectivity toward a specific bis-phosphorylated peptide in the presence of various phosphate species such as the monophosphorylated peptide, ATP, and others. Detailed fluorescence titration experiments clearly indicate that the binding-induced fluorescence enhancement and the sensing selectivity were achieved by the cooperative action of both binding sites of the hybrid biosensor, i.e., the WW domain and the Zn(II)-Dpa chemosensor unit. Thus, it is clear that the tethered Zn(II)-Dpa-stilbazole unit operated not only as a fluorescence signal transducer, but also as a sub-binding site in the hybrid biosensor. Taking advantage of its selective sensing property, the hybrid biosensor was successfully applied to real-time and label-free fluorescence monitoring of a protein kinase-catalyzed phosphorylation.     

Revealing two-state protein-protein interactions of calmodulin by single-molecule spectroscopy

We report a single-molecule fluorescence resonance energy transfer (FRET) and polarization study of conformational dynamics of calmodulin (CaM) interacting with a target peptide, C28W of a 28 amino acid oligomer. The C28W peptide represents the essential binding sequence domain of the Ca-ATPase protein interacting with CaM, which is important in cellular signaling for the regulation of energy in metabolism. However, the mechanism of the CaM/C28W recognition complex formation is still unclear. The amino-terminal (N-terminal) domain of the CaM was labeled with a fluorescein-based arsenical hairpin binder (FlAsH) that enables our unambiguous probing of the CaM N-terminal target-binding domain motions on a millisecond time scale without convolution of the probe-dye random motions. By analyzing the distribution of FRET efficiency between FlAsH labeled CaM and Texas Red labeled C28W and the polarization fluctuation dynamics and distributions of the CaM N-terminal domain, we reveal binding-unbinding motions of the N-terminal domain of the CaM in CaM/C28W complexes, which is strong evidence of a two-state binding interaction of CaM-mediated cell signaling.     

Predicting the effects of amino acid replacements in peptide hormones on their binding affinities for class B GPCRs and application to the design of secretin receptor antagonists

Computational prediction of the effects of residue changes on peptide-protein binding affinities, followed by experimental testing of the top predicted binders, is an efficient strategy for the rational structure-based design of peptide inhibitors. In this study we apply this approach to the discovery of competitive antagonists for the secretin receptor, the prototypical member of class B G protein-coupled receptors (GPCRs). Proteins in this family are involved in peptide hormone-stimulated signaling and are implicated in several human diseases, making them potential therapeutic targets. We first validated our computational method by predicting changes in the binding affinities of several peptides to their cognate class B GPCRs due to alanine replacement and compared the results with previously published experimental values. Overall, the results showed a significant correlation between the predicted and experimental ΔΔG values. Next, we identified candidate inhibitors by applying this method to a homology model of the secretin receptor bound to an N-terminal truncated secretin peptide. Predictions were made for single residue replacements to each of the other nineteen naturally occurring amino acids at peptide residues within the segment binding the receptor N-terminal domain. Amino acid replacements predicted to most enhance receptor binding were then experimentally tested by competition-binding assays. We found two residue changes that improved binding affinities by almost one log unit. Furthermore, a peptide combining both of these favorable modifications resulted in an almost two log unit improvement in binding affinity, demonstrating the approximately additive effect of these changes on binding. In order to further investigate possible physical effects of these residue changes on receptor binding affinity, molecular dynamics simulations were performed on representatives of the successful peptide analogues (namely A17I, G25R, and A17I/G25R) in bound and unbound forms. These simulations suggested that a combination of the α-helical propensity of the unbound peptide and specific interactions between the peptide and the receptor extracellular domain contribute to their higher binding affinities.     

Immunomodulatory peptides from IgSF proteins: a review

The immunoglobulin (Ig) domain is a highly conserved domain predominantly observed in cell surface proteins due to its ability to resist proteolysis. By mutation and selection the Ig domain has evolved to serve diverse biological functions including growth and development, signaling, adhesion and protein-carbohydrate interactions. Collectively, proteins with Ig-like domain constitute the immunoglobulin superfamily (IgSF). The IgSF proteins make up over 2% of human genes constituting the largest gene family in the human genome. Analogous to the complementarity determining regions (CDR)s that form the antigen combining sites of the antibody, the high specificity of the IgSF receptor-ligand interaction is attributed to the sequence and structure of the CDR-like regions unique to each IgSF protein. Hence, CDR-like regions provide ideal templates for the design of mimetics that can potentially perturb specific IgSF receptor/ligand interactions. The determinants of binding are localized near the CDR-like regions, conformation is determined locally and is unique for each loop. In structure based drug design one of the approaches to identify lead agents is to map the receptor/ligand binding epitope onto a small peptide. Data from theoretical, structural and functional studies have been adopted in the design of novel peptide antagonists of the IgSF protein-protein interactions. Many peptide antagonists have shown significant therapeutic potential in multiple animal models. The design of the IgSF peptide analogs, rationale as therapeutic targets, functional efficacy and the clinical benefits are reviewed here.     

Development and characterization of a high-throughput system for assessing cell-surface receptor-ligand engagement

A nonfouling interfacial interpenetrating polymer network (IPN) of poly(acrylamide-co-ethylene glycol/acrylic acid) [p(AAm-co-EG/AAc)] was grafted to polystyrene for use as a novel platform for the development of high-throughput assays for screening of specific bimolecular interactions (i.e., receptor-ligand engagement). For the development of the IPN, a water-soluble hydrogen-abstracting photoinitiator was investigated: (4-benzoylbenzyl)trimethylammonium chloride. IPN-modified polystyrene surfaces were characterized using XPS, contact angle goniometry, and protein adsorption analysis. These IPN surfaces minimized fibrinogen adsorption compared to tissue culture polystyrene (>96% reduction), prevented mammalian cell adhesion, and served as nonfouling surfaces to graft biological ligands. For bimolecular interaction studies, a model peptide ligand from bone sialoprotein (Ac-CGGNGEPRGDTYRAY-NH(2)) was grafted to p(AAm-co-EG/AAc) via a 3400 M(w) linear pEG spacer. Ligand density measurements, cell culture, and a centrifugal adhesion assay were used to study cell adhesion to peptide-modified IPNs (i.e., receptor-ligand engagement). Ligand density (Gamma) was controllable from approximately 1 to 20 pmol/cm(2) by modulating the peptide input concentration (0.02-20 microM). Cell adhesion was directly dependent on the ligand density. This technology creates a powerful high-throughput system to simultaneously probe a myriad of cell-surface receptor-ligand interactions.     

Spatiotemporal sub-cellular biopatterning using an AFM-assisted electrochemical system

The integration of electrochemical-based biolithography (ECBL) with an ordinary atomic force microscope (AFM) enables in situ lithography adjacent to a single, cultured cell, consequently allowing the morphological shape of the cell to be manipulated. The tip of a commercially available AFM cantilever was modified to serve as an electrode that could generate the oxidant HBrO for local, controlled etching of a cytophobic material (heparin or albumin) previously layered adjacent to a living cell. A NIH-3T3 fibroblast, initially confined to a patterned area, extended along a bioadhesive surface that had been newly exposed using the ECBL-AFM system.     

Thermodynamic basis for promiscuity and selectivity in protein-protein interactions: PDZ domains, a case study

Like other protein-protein interaction domains, PDZ domains are involved in many key cellular processes. These processes often require that specific multiprotein complexes be assembled, a task that PDZ domains accomplish by binding to specific peptide motifs in target proteins. However, a growing number of experimental studies show that PDZ domains (like other protein-protein interaction domains) can engage in a variety of interactions and bind distinct peptide motifs. Such promiscuity in ligand recognition raises intriguing questions about the molecular and thermodynamic mechanisms that can sustain it. To identify possible sources of promiscuity and selectivity underlying PDZ domain interactions, we performed molecular dynamics simulations of 20 to 25 ns on a set of 12 different PDZ domain complexes (for the proteins PSD-95, Syntenin, Erbin, GRIP, NHERF, Inad, Dishevelled, and Shank). The electrostatic, nonpolar, and configurational entropy binding contributions were evaluated using the MM/PBSA method combined with a quasi-harmonic analysis. The results revealed that PDZ domain interactions are characterized by overwhelmingly favorable nonpolar contributions and almost negligible electrostatic components, a mix that may readily sustain promiscuity. In addition, despite the structural similarity in fold and in recognition modes, the entropic and other dynamical aspects of binding were remarkably variable not only across PDZ domains but also for the same PDZ domain bound to distinct ligands. This variability suggests that entropic and dynamical components can play a role in determining selectivity either of PDZ domain interactions with peptide ligands or of PDZ domain complexes with downstream effectors.