A multivalent ligand-protein complex features multiple simultaneous, independent, noncovalent interactions. In general, a bivalent ligand shows an enhanced affinity compared to the affinity of the corresponding monovalent ligands. In chemical biology, multivalent binding can be used to enhance both the affinity and specificity of chemical probes, which are useful tools applied to the study of the function of proteins and their roles in disease. Indeed, various post-translational modifications (PTMs) on histones are often specifically recognized by chromatin reader proteins in a multivalent manner. Thus, multivalent interactions between reader proteins and PTMs are a hallmark of chromatin regulation.<br/>While the effect of binding enhancement for bivalent ligands has been theoretically characterized, the practical, physical, and structural characterization of bivalent binding encounters multiple technical difficulties. In particular, both the bivalent ligand and protein have flexible linkers that makes them difficult to study by X-ray crystallography and NMR. Moreover, multiple possible types of binding events, especially at high protein concentrations required for experimental techniques such as isothermal titration calorimetry (ITC), may confound the analysis. Here, we present a case study utilizing a combination of experimental techniques and explicit computational simulations to study the dual bromodomain protein, TAF1, which was chosen based on the availability of structural information, prior characterization of multivalent engagement, TAF1’s known role in transcription initiation, and the availability of functionalizable small molecule bromodomain ligands.<br/>We report our efforts to investigate the dynamics of TAF1 recognition using multivalent ligands. Several experimental techniques including ITC, X-ray crystallography and surface plasmon resonance were used to comprehensively characterize both the binding of monomeric ligands, as well as to assess apparent affinities for bivalent ligands with varying linker lengths. The experimental data for the monomeric ligands were fed into explicit computational simulations, in which ligand and protein species were represented in a broad range of concentrations. The simulations provided accurate estimates for both apparent affinities and individual binding constants for each type of ligand-protein complex. Finally, we hope to apply this approach to other dual-domain chromatin reader proteins.

Quantitative characterization of bivalent probes for the dual bromodomain protein transcription initiation factor TFIID subunit 1, TAF1

256th National Meeting (2018)

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