Functionalization of a Rigid Divalent Ligand for LecA, a Bacterial Adhesion Lectin.

The bacterial adhesion lectin LecA is an attractive target for interference with the infectivity of its producer P. aeruginosa. Divalent ligands with two terminal galactoside moieties connected by an alternating glucose-triazole spacer were previously shown to be very potent inhibitors. In this study, we chose to prepare a series of derivatives with various new substituents in the spacer in hopes of further enhancing the LecA inhibitory potency of the molecules. Based on the binding mode, modifications were made to the spacer to enable additional spacer-protein interactions. The introduction of positively charged, negatively charged, and also lipophilic functional groups was successful. The compounds were good LecA ligands, but no improved binding was seen, even though altered thermodynamic parameters were observed by isothermal titration calorimetry (ITC).

Thiourea-based spacers in potent divalent inhibitors of Pseudomonas aeruginosa virulence lectin LecA.

A new divalent highly potent inhibitor of the Pseudomonas aeruginosa lectin and virulence factor LecA was prepared. It contains two thiourea linkages which were found to be in the Z,Z isomeric form. This brings the spacer into an elongated conformation required to bridge the two binding sites, which results in the chelating binding mode responsible for the high potency.

Tissue-type plasminogen activator binds to Aß and AIAPP amyloid fibrils with multiple domains.

Binding of tissue-type plasminogen activator (tPA) to amyloid and denatured proteins is reported in a number of studies. The binding site has been mapped previously to the finger domain of tPA. In this study, tPA and truncated tPA constructs, lacking the finger domain, were tested for their ability to bind to Aß and AIAPP amyloid-like fibrils. Surface plasmon resonance experiments and pull-down assays clearly show that indeed tPA binds, but that the finger domain is not essential. Another possible binding mechanism via the lysine binding site on the kringle 2 domain was also not crucial for the binding. Immuno-electron microscopy studies show that tPA binds to fibril sides. This study shows that, besides the finger domain, other domains in tPA are involved in amyloid binding.

Site-specific peptide and protein immobilization on surface plasmon resonance chips via strain-promoted cycloaddition.

Surface plasmon resonance (SPR) is a powerful label-free diagnostic tool to study biomolecular interactions. However, one of the drawbacks of SPR is the lack of controlled immobilization of ligands on the sensor surface. We have developed a modular platform for the fast, reagent-free and site-specific immobilization of azide-containing ligands by strain-promoted cycloaddition onto a cyclooctyne-modified SPR sensor surface. The usefulness of the concept was shown in a study with a papain model system, and up to 150 experiments were performed without loss of surface quality. Furthermore, azide-containing green fluorescent protein (GFP) was also effectively immobilized. Taken together, cyclooctyne-modified SPR chips enable smooth and site-selective immobilization of ligands and prove to be more robust than traditionally functionalized systems.

Unusual binding of Grb2 protein to a bivalent polyproline-ligand immobilized on a SPR sensor: intermolecular bivalent binding.

The Grb2 adapter protein is involved in the activation of the Ras signaling pathway. It recruits the Sos protein by binding of its two SH3 domains to Sos polyproline sequences. We observed that the binding of Grb2 to a bivalent ligand, containing two Sos-derived polyproline-sequences immobilized on a SPR sensor, shows unusual kinetic behavior. SPR-kinetic analysis and supporting data from other techniques show major contributions of an intermolecular bivalent binding mode. Each of the two Grb2 SH3 domains binds to one polyproline-sequence of two different ligand molecules, facilitating binding of a second Grb2 molecule to the two remaining free polyproline binding sites. A molecular model based on the X-ray structure of the Grb2 dimer shows that Grb2 is flexible enough to allow this binding mode. The results fit with a role of Grb2 in protein aggregation, achieving specificity by multivalent interactions, despite the relatively low affinity of single SH3 interactions.

Surface plasmon resonance for proteomics.

Surface plasmon resonance (SPR) is a well-established label-free technique to detect mass changes near an SPR surface. For 20 years the benefits of SPR have been proven in biomolecular interaction analysis, including measurements of affinity and kinetics. The emergence of proteomics and a need for high throughput analysis drives the development of SPR systems capable of analyzing microarrays. The use of SPR imaging (also known as SPR microscopy) makes it possible to use multiplexed arrays to follow binding reactions. As SPR only analyzes the binding process, but not the identity of captured molecules on the SPR surface, technologies have been developed to integrate SPR with mass spectrometric (MS) analysis. Such approaches involve the recovery of analytes from the SPR surface and subsequent MALDI-TOF MS analysis, or LC-MS/MS after tryptic digestion of recovered proteins. An approach compatible with SPR arrays is on-chip MALDI-TOF MS, from arrayed spots on an SPR surface. This review describes some exciting developments in the application of SPR to proteomics, using instruments which are on the market already, or are expected to be available in the years to come.

Cell permeable ITAM constructs for the modulation of mediator release in mast cells.

Spleen tyrosine kinase (Syk) is essential for high affinity IgE receptor (FcεRI) mediated mast cell degranulation. Once FcεRI is stimulated, intracellular ITAM motifs of the receptor are diphosphorylated (dpITAM) and Syk is recruited to the receptor by binding of the Syk tandem SH2 domain to dpITAM, resulting in activation of Syk and, eventually, degranulation. To investigate intracellular effects of ITAM mimics, constructs were synthesized with ITAM mimics conjugated to different cell penetrating peptides, i.e. Tat, TP10, octa-Arg and K(Myr)KKK, or a lipophilic C(12)-chain. In most constructs the cargo and carrier were linked to each other through a disulfide bridge, which is convenient for combining different cargos with different carriers and has the advantage that the cargo and the carrier may be separated by reduction of the disulfide once it is intracellular. The ability of these ITAM constructs to label RBL-2H3 cells was assessed using flow cytometry. Fluorescence microscopy showed that the octa-Arg-SS-Flu-ITAM construct was present in various parts of the cells, although it was not homogeneously distributed. In addition, cell penetrating constructs without fluorescent labels were synthesized to examine degranulation in RBL-2H3 cells. Octa-Arg-SS-ITAM stimulated the mediator release up to 140%, indicating that ITAM mimics may have the ability to activate non-receptor bound Syk.

Surface plasmon resonance: a general introduction.

Surface plasmon resonance (SPR) analysis is rather unique in that it allows assay of binding constants (affinity) and kinetic analysis of binding phenomena. This introductory chapter deals with some specific features that are relevant to many diverse applications. The role and impact of kinetics in biomolecular interactions is highlighted. A concise description of the physical principles of the SPR phenomenon is given from a practical point of view, such that some possibilities and limitations of the method can be rationalized, e.g., depth of the evanescent field. A specific condition that may come forward in kinetic analysis is mass transport limitation (MTL). A practical model is presented, which allows estimation of the extent of MTL. Based on this model it can be rationalized whether MTL can be avoided by experimental design. In this framework also rules are presented to convert SPR signals (RU or millidegree) to mass/surface unit. The chapter concludes with an overview of commercially available SPR equipment.

Affinity constants for small molecules from SPR competition experiments.

Direct assay of small molecules by SPR in general is troublesome and at least tedious procedures have to be applied. Competition experiments offer an attractive alternative. A small ligand known to bind to the analyte is immobilized on an SPR sensor surface, and the binding of the larger analyte in the presence of compounds under investigation in a concentration range is assayed. The resulting inhibition curves of the equilibrium SPR signal as function of the compound concentration can be analyzed to yield thermodynamic binding constants for the interaction in solution between analyte and the compounds under investigation. An additional advantage of this method is that series of compounds can be analyzed using the same sensor surface, so there is no immobilization needed for each compound. An adaptation of the method to analyze interactions with bivalent analytes (e.g., antibodies) is also included. Some observed different affinities in solution compared to that on the SPR surface are discussed.

ITAM-derived phosphopeptide-containing dendrimers as multivalent ligands for Syk tandem SH2 domain.

Spleen tyrosine kinase (Syk) is activated when its tandem SH2 domain (tSH2) binds to a diphosphorylated ITAM motif of e.g. the FcepsilonRI receptor. In this divalent interaction each SH2 domain binds to a phosphotyrosine-containing tetrapeptide motif in ITAM. One of those tetrapeptide sequences was synthesized and conjugated to dendrimers via 'click' chemistry to create a series of functional phosphopeptide-containing dendrimers ranging from a monovalent to an octavalent dendrimer. The affinity of the functionalized dendrimers for Syk tSH2 has been assayed in SPR competition experiments. Both the tetra- and octavalent dendrimer had an affinity in the high nanomolar range, which is approximately 100-fold enhanced compared to the monovalent tetrapeptide, indicating a multivalency effect.

Switching between low and high affinity for the Syk tandem SH2 domain by irradiation of azobenzene containing ITAM peptidomimetics.

Spleen tyrosine kinase (Syk) plays an essential role in IgE receptor signaling (FcepsilonRI), which leads to mast cell degranulation. Divalent binding of the tandem SH2 domain (tSH2) of Syk to the intracellular ITAM motif of FcepsilonRI activates the kinase domain of Syk, and thereby initiates cell degranulation. The inter SH2 domain distance in Syk tSH2 might be important for Syk kinase activation. In this study, photoswitchable ITAM peptidomimetics containing an azobenzene moiety were synthesized. Irradiation of these constructs changes the distance between the two SH2 binding epitopes and therefore, they may be used as photoswitches. The affinity of the cis- and trans-isomer for tSH2 was assayed with SPR. The ITAM peptidomimetic with the smallest linker displayed the largest difference in affinity between the two isomers (at least 100-fold), and the affinity of the cis-isomer was comparable to monovalent binding. The ITAM mimics with larger photoswitchable linkers displayed modest differences. These results indicate that Syk tSH2 is able to adapt the inter SH2 domain distance to ligands larger than native ITAM, but not to smaller ones.

A photoswitchable ITAM peptidomimetic: synthesis and real time surface plasmon resonance (SPR) analysis of the effects of cis-trans isomerization on binding.

The Syk protein plays an important role in immune receptor signaling. The Syk tandem SH2 domain (tSH2)-ITAM interaction is important for recruiting Syk to the receptor complex and for Syk kinase activation. A peptidomimetic ligand for tSH2 was synthesized in which a photoswitchable azobenzene moiety was incorporated. Such a photoswitchable moiety may regulate the distance between the two phosphotyrosine containing ITAM sequences, which bind to tSH2. Different affinities of the cis and trans isomer of the ligand were found by surface plasmon resonance (SPR). By in situ irradiation during SPR measurements the effect of the cis-trans isomerization on binding could be monitored in real time.

Protein flexibility and ligand rigidity: a thermodynamic and kinetic study of ITAM-based ligand binding to Syk tandem SH2.

The Syk tandem Src homology 2 domain (Syk tSH2) constitutes a flexible protein module involved in the regulation of Syk kinase activity. The Syk tSH2 domain is assumed to function by adapting the distance between its two SH2 domains upon bivalent binding to diphosphotyrosine ligands. A thermodynamic and kinetic analysis of ligand binding was performed by using surface plasmon resonance (SPR). Furthermore, the effect of binding on the Syk tSH2 structural dynamics was probed by hydrogen/deuterium exchange and electrospray mass spectrometry (ESI-MS). Two ligands were studied: 1, a flexible peptide derived from the tSH2 recognition ITAM sequence at the gamma chain of the FcepsilonRI-receptor, and 2, a ligand in which the amino acids between the two SH2 binding motifs in ligand 1 have been replaced by a rigid linker of comparable length. Both ligands display comparable affinity for Syk tSH2 at 25 degrees C, yet a major difference in thermodynamics is observed. Upon binding of the rigid ligand, 2, the expected entropy advantage is not realized. On the contrary, 2 binds with a considerably higher entropy price of approximately 9 kcal mol-1, which is attributed to a further decrease in protein flexibility upon binding to this rigid ligand. The significant reduction in deuterium incorporation in the Syk tSH2 protein upon binding of either 1 or 2, as monitored by ESI-MS, indicates a major reduction in protein dynamics upon binding. The results are consistent with a two-step binding model: after an initial binding step, a rapid structural change of the protein occurs, followed by a second binding step. Such a bivalent binding model allows high affinity and fast dissociation kinetics, which are very important in transient signal-transduction processes.

Surface plasmon resonance thermodynamic and kinetic analysis as a strategic tool in drug design. Distinct ways for phosphopeptides to plug into Src- and Grb2 SH2 domains.

Thermodynamic and kinetic studies of biomolecular interactions give insight into specificity of molecular recognition processes and advance rational drug design. Binding of phosphotyrosine (pY)-containing peptides to Src- and Grb2-SH2 domains was investigated using a surface plasmon resonance (SPR)-based method. This SPR assay yielded thermodynamic binding constants in solution, and the kinetic information contained in the SPR signal allowed kinetic analysis, which demonstrated distinct ways for pY ligands to interact with the SH2 domains. The results for binding to Src SH2 were consistent with sequestration of water molecules in the interface of the pYEEI peptide/Src SH2 complex. The results for a pYVNV peptide binding to Grb2 SH2 suggested a conformational change for Grb2 SH2 upon binding, which is not observed for Src SH2. Binding of a cyclic construct, allowing the pYVNV sequence in the bound conformation, did not have the expected entropy advantage. The results suggest an alternative binding mode for this construct, with the hydrophobic ring-closing part interacting with the protein. In all cases, except for full-length Grb2 protein, the affinity for the immobilized peptide at the SPR sensor and in solution was identical. This study demonstrates that SPR thermodynamic and kinetic analysis is a useful strategic tool in drug design.

Changes in structural dynamics of the Grb2 adaptor protein upon binding of phosphotyrosine ligand to its SH2 domain.

Growth factor receptor-bound protein 2 (Grb2) is an extensively studied adaptor protein involved in cell signaling. Grb2 is a highly flexible protein composed of a single SH2 domain flanked by two SH3 domains. Here we report on the structural dynamic effects upon interaction of a phosphopeptide ligand derived from the recognition sequence of the Shc adaptor protein with (i) the isolated SH2 domain of Grb2 (Grb2 SH2) and (ii) the full-length Grb2 protein. From kinetic studies using surface plasmon resonance, it was deduced that a conformation change occurred in the SH2 protein as well as the full-length Grb2 after binding. Measurements of hydrogen/deuterium exchange (HDX) in the isolated SH2 domain and full-length Grb2 protein as monitored by electrospray mass spectrometry, showed that binding reduces the overall flexibility of the proteins, possibly via slightly different mechanisms for the single SH2 domain and the full-length Grb2 protein.

Cyclic phosphopeptides for interference with Grb2 SH2 domain signal transduction prepared by ring-closing metathesis and phosphorylation.

Cyclic phosphopeptides were prepared using ring-closing metathesis followed by phosphorylation. These cyclic phosphopeptides were designed to interact with the SH2 domain of Grb2, which is a signal transduction protein of importance as a target for antiproliferative drug development. Binding of these peptides to the Grb2 SH2 domain was evaluated by a surface plasmon resonance assay. High affinity binding to the Grb2 SH2 domain was maintained upon macrocyclization, thus indicating that this method can be used to assemble high affinity cyclic phosphopeptides that interfere with signal transduction cascades.

Role of solution conformation and flexibility of short peptide ligands that bind to the p56(lck) SH2 domain.

A general approach in drug design is making ligands more rigid in order to avoid loss in conformational entropy (deltaS(conf)) upon receptor binding. We hypothesized that in the high affinity binding of pYEEI peptide ligands to the p56(lck) SH2 domain this loss in deltaS(conf) might be diminished due to preorganization of the fourfold negatively charged pYEEI peptide in the bound, extended, conformation. A thermodynamic analysis was performed on the peptides Ac-pYEEI-NH(2), Ac-pYAAI-NH(2) and Ac-pYGGI-NH(2) using surface plasmon resonance (SPR) competition experiments to assay affinity constants at different temperatures. To study the effect of solution conformation and flexibility a computational conformation analysis was performed from which low energy conformations in solution were calculated, and S(conf) estimated. It was found that the calculated low energy conformations for especially the pYE moiety in solution resemble that in the bound state. In the calculated minimum energy conformation in solution isoleucine is bent towards the pY aromatic ring, the occurrence of such conformation is experimentally confirmed by NMR. The estimated values for S(conf) of the EE- and AA-peptide were similar, suggesting no predominant role of preorganization of the solution conformation due to electrostatic repulsion. Apparently the thermodynamics obey the same entropy-enthalpy compensation relationship, which also was found to hold for other peptides and peptidomimetics binding to p60(src) family SH2 domains. The implications of the results for drug design are discussed.

Amino propynyl benzoic acid building block in rigid spacers of divalent ligands binding to the Syk SH2 domains with equally high affinity as the natural ligand.

The construction of rigid spacers composed of amino propynyl benzoic acid building blocks is described. These spacers were used to link two phosphopeptide ligand sites towards obtaining divalent ligands with a high affinity for Syk tandem SH2 domains, which are important in signal transduction. The spacer containing two of those rigid building blocks led to a ligand which was as active as the natural ligand, indicating that this building block can be used in the design and synthesis of high affinity divalent constructs that can successfully interfere with crucial protein-protein interactions.