Wendy A. Lea Ph.D. NIH

Two articles report on two methods that provided different levels of information on the differentiation of binding sites available on protein targets of interest.

Characterization of a biomolecular interaction usually ranges from obtaining basic binding parameters, such as binding affinities, to the understanding of more advanced binding signatures which can often provide additional insight on mode of action (MoA). Examples in the latter category include binding cooperatively and binding kinetics.

The study of binding kinetics can quantify the concentrations of binding species as a function of time based on initial total concentration and rate constants. Association rates (“on rates,” Ka) and dissociation rates (“off rates,” Kd) are frequently measured by optical sensor-based techniques, such as surface plasmon resonance (SPR), and a recent article made a welcomed comparison on the performance on reporting Ka and Kd between SPR and a TR-FRET based assay (LanthaScreen Eu kinase binding assay; Mason et al., Assay Drug Dev Technol 2012;10:468–475).

The concept of binding cooperativity has been featured in our August 2012 issue’s “Literature Search and Review” (see “Evaluation of binding cooperativity by MST,” Assay Drug Dev Technol 2012;10:306–309), and it entails the interaction among different binding sites, provided that the binding sites for specifically studied ligands have already been known. Herein, two different articles are highlighted because they reported on two different methods that provided different levels of information on the differentiation of binding sites that were available on protein targets of interest.

In the work done by Seidel et al.*, the authors successfully applied a true label-free version of microscale thermophoresis (MST) for the characterization of small molecule binding to a membrane receptor and a kinase. Compared to standard MST in which one binding partner has to be labeled with a fluorophore through covalent coupling, label-free MST takes advantage of protein intrinsic fluorescence, which is usually dominated by the presence of tryptophan (Trp). As a result, polarity of the local environment and proximity of other residues are main factors to influence fluorescence emission peak and strength.

Using the nonactivated form of mitogen-activated protein kinase (MAP kinase) p38 that contains five Trp residues, two structurally similar p38 inhibitors were found to exhibit the same directions in their thermophoretic profiles while a structurally different p38 inhibitor was found to exhibit opposite thermophoretic profile with respect to the former two compounds. Specifically, relative to the unbound protein, the former two compounds led to less depletion for the protein complexes (positive MST) and the latter compound resulted in greater depletion for the protein–compound complex (see Figure 1). In addition to the close agreement obtained between the affinities derived from label-free MST and previously published inhibitory activities, MST presented its potential for binding site discrimination through the direction of molecular movement under a controlled temperature gradient. In order to obtain a full picture of the inhibitor MoA, additional characterization, such as functional assays or competition assays, could be considered for further studies.

Several criteria need to be considered when applying label-free MST: autofluorescent buffer components should be avoided because they could interfere with protein Trp emission measurement; protein concentration should be chosen at the lowest possible level at which its fluorescence still exceeds background fluorescence significantly. When the titrated binding partner also possesses Trp (such as another protein) or strong autofluorescence (such as certain small molecules), label-free MST can be difficult to perform or careful control experiments are required to remove the fluorescence contribution from the titrated partner. Depending on the number and local environment of Trp residues, protein consumption can vary from case to case, and this could limit the detection range by at least one order of magnitude compared to the standard MST approach. Nevertheless, the applicability potential of MST, be it standard or label free, has been on a steep ascension since its commercialization.

Label-free MST complements standard MST because some proteins may suffer from labeling by losing activity. The ability of MST to answer advanced kinetics questions awaits further investigation and evaluation. In a separate work done by Xu et al.**, the authors applied standard MST to evaluate binding affinities for a panel of fibroblast growth factors (FGFs) with heparan sulfate (HS) and its derivatives. The Kds obtained from standard MST were derived at equilibrium, and they were consistent with Kds derived from binding kinetics parameters, Kon and Koff, validating the set-up of both heterogeneous and solution-based methods.

Figure 1. Screening of small-molecule kinase inhibitors. Three selective inhibitors were successfully tested for binding to the nonactivated form of MAP kinase p38a (c = 100 nM). Corresponding to structural differences, the binding of SB202190 and PD169316 has the opposite effect on the thermophoretic movement compared to SB239063. SB202190 binds with a Kd value of 48 ± 21 nM. The upper limits of affinity for PD169316 and SB239063 were determined as 33 nM and 20 nM, respectively. These results are in good agreement with previously reported values. Thermally denatured p38a did not show binding (control). MAP, mitogen-activated protein.

In order to gain a deeper understanding of the molecular determinants for the binding interactions between FGFs and heparin, Xu et al. also utilized a number of complementary techniques. In particular, they highlighted the usage of a validated “Protect and Label” approach for the identification and characterization of heparin binding sites (HBS) in a family of FGFs. Specifically, lysine side chains exposed to solvent when protein was bound to heparin was first protected by sulfo-N-hydroxysulfosuccinimide acetate, and was subsequently labeled with biotin after protein dissociated from heparin. Thus, biotinylated peptides could be further identified by tandem mass spectrometry (MS) after digestion, providing fingerprints for HBS. For example, in addition to a canonical HBS that had been recapitulated using this method, two other secondary sites were found for FGF1 (see Figure 2).

Through this approach, additional HBS has been recognized for a number of FGFs that were studied. Heparin binding induced protein structural changes, structural elements required in heparin derivatives for binding, and binding specificities were further studied using circular dichroism spectroscopy and differential scanning fluorimetry. With due optimization of the reaction conditions, this Protect and Label approach presents a good level of sensitivity and selectivity towards lysine-containing binding sites. Followed by MS analysis, the method has the potential to be transformed into high-throughput formats. One pitfall of the method is its inability to recognize non–lysine-containing binding sites, and novel protein modification reagents that could specifically target different amino acids are welcomed.

Figure 2. Position of biotinylated peptides in FGF-1 (residues 22–154) identified by structural proteomics mapped onto the predicted 3-dimensional structure (PDB 2ERM; Canales et al., FEBS J 2006;273:4716–4727). Labeled peptides are colored in blue, and peptides overlapping with literature-annotated and aligned canonical HBS lysines are colored in green (DiGabriele et al., Nature 1998;393:812–817; Pellegrini et al., Nature 2000;407:1029–1034). (A, B) Ribbon diagram. (C, D) Corresponding molecular surface. FGF-1 is shown using schematic representation. (B, D) 180° back view of (A) and (C). HBS, heparin binding site.

*Graphical Abstract from Angewandte Chemie International Edition 2012, Vol. 51: 10,656–10,659

Look, no label! Microscale thermophoresis makes use of the intrinsic fluorescence of proteins to quantify the binding affinities of ligands and discriminate between binding sites. This method is suitable for studying binding interactions of very small amounts of protein in solution. The binding of ligands to iGluR membrane receptors, small-molecule inhibitorss to kinase p38, aptamers to thrombin, and Ca2+ ions to synaptotagmin was quantified.

Graphical abstract from Seidel et al.

**Abstract from The Journal of Biological Chemistry 2012, Sept 27 [Epub ahead of issue]; DOI: 10.1074/jbc.M112.398826

Background. Heparan sulfate (HS) regulates the transport and signaling activities of fibroblast growth factors (FGF).

Results. The molecular determinants of the interactions of FGFs and heparin were identified.

Conclusion. There are clear molecular specificities determining the interactions of FGFs with the polysaccharide.

Significance. The expansion of the FGFs in metazoan evolution parallels the diversification of the specificity of their interactions with heparin.

Summary. The functions of a large number (>435) of extracellular regulatory proteins are controlled by their interactions with heparan sulfate (HS). In the case of fibroblast growth factors (FGFs), HS binding determines their transport between cells and is required for the assembly of high affinity signalling complexes with their cognate FGF receptor. However, the specificity of the interaction of FGFs with HS is still debated.

Here, we use a panel of FGFs (FGF-1, FGF-2, FGF-7, FGF-9, FGF-18, and FGF-21) spanning five FGF subfamilies to probe their specificities for HS at different levels: binding parameters, identification of heparin binding sites (HBSs) in the FGFs, changes in their secondary structure caused by heparin binding, and structures in the sugar required for binding. For interaction with heparin, the FGFs exhibit KD values varying between 38 nM (FGF-18) and 620 nM (FGF-9) and association rate constants spanning over 20-fold (FGF-1, 2,900,000 M-1s-1, FGF-9, 130,000 M-1s-1). The canonical HBS in FGF-1, FGF-2, FGF-7, FGF-9, and FGF-18 differs in its size and these FGFs have a different complement of secondary HBS, ranging from none (FGF-9) to two (FGF-1). Differential scanning fluorimetry identified clear preferences in these FGFs for distinct structural features in the polysaccharide.

These data suggest that the differences in heparin binding sites in both the protein and the sugar are greatest between sub-families and may be more restricted within a FGF sub-family in accord with the known conservation of function within FGF sub-families.

Wendy A. Lea, Ph.D., works at the NIH.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a “Literature Search and Review” column that identifies published papers of note and discusses their importance. GEN presents here one article from the “Literature Search and Review” column that analyzes two papers: one published in Angewandte Chemie International Edition titled “Label-free microscale thermophoresis discriminates sites and affinity of protein-ligand binding,” authored by Seidel SA, Wienken CJ, Geissler S, Jerabek-Willemsen M, Duhr S, Reiter A, Trauner D, Braun D, and Baaske P.; and the other published in Journal of Biological Chemistry titled “Diversification of the structural determinants of fibroblast growth factor-heparin interactions: implications for binding specificity,” authored by Xu R, Ori A, Rudd TR, Uniewicz KA, Ahmed YA, Guimond SE, Skidmore MA, Siligardi G, Yates EA, and Fernig DG.

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