Abstract
Many transcriptional activators are intrinsically unstructured yet display unique, defined conformations when bound to target proteins. Target-induced folding provides a mechanism by which activators could form specific interactions with an array of structurally unrelated target proteins. Evidence for such a binding mechanism has been reported previously in the context of the interaction between the cancer-related c-Myc protein and the TATA-binding protein, which can be modeled as a two-step process in which a rapidly forming, low affinity complex slowly converts to a more stable form, consistent with a coupled binding and folding reaction. To test the generality of the target-induced folding model, we investigated the binding of two widely studied acidic activators, Gal4 and VP16, to a set of target proteins, including TATA-binding protein and the Swi1 and Snf5 subunits of the Swi/Snf chromatin remodeling complex. Using surface plasmon resonance, we show that these activator-target combinations also display bi-phasic kinetics suggesting two distinct steps. A fast initial binding phase that is inhibited by high ionic strength is followed by a slow phase that is favored by increased temperature. In all cases, overall affinity increases with temperature and, in most cases, with increased ionic strength. These results are consistent with a general mechanism for recruitment of transcriptional components to promoters by naturally occurring acidic activators, by which the initial contact is mediated predominantly through electrostatic interactions, whereas subsequent target-induced folding of the activator results in a stable complex.
Highlights
Many transcriptional activators are intrinsically unstructured yet display unique, defined conformations when bound to target proteins
We have previously reported that the activation domain from the cancer-related c-Myc protein interacts with the TATA-binding protein (TBP) in two steps, such that folding of the c-Myc activation domain is coupled to its interaction with the surface of TBP [8]
TBP interaction, we measured the kinetics of VP16AD binding to TBP as well as protein segments constituting the ABDs of
Summary
Plasmids and Proteins—pRSET-Snf, which encodes amino acids 1–334 of yeast Snf with an N-terminal His tag, has been described previously [34] (referred to hereafter as Snf5ABD). pRSET-Swi encodes amino acids 329 –547 of yeast Swi with an N-terminal His tag and was constructed as described for pRSET-Snf5 [34] (Swi1ABD). pT7yD encodes full-length yeast TBP [35]. pGST-VP16 encodes amino acids 413– 490 of VP16 (VP16AD), deletion mutant pGST-VP16⌬456 lacks residues 456 – 490 (VP16⌬456) [18], and pGST-Gal encodes amino acids 769 – 881 of Gal (Gal4AD) [32, 36]. Cell pellets were resuspended in 100 mM Tris, pH 7.4, and 100 mM NaCl supplemented with Complete Protease Inhibitor Mixture They were subsequently treated essentially as described above, except that the soluble fraction was purified by affinity chromatography using glutathione-agarose (Sigma). All response data were collected at a rate of 1 Hz, manually aligned along the x-axis and y-axis, and processed by double referencing (i.e. subtraction of background binding to the affinity tag (GST), as well as adjustment for flow cell-specific bulk effects by subtraction of buffer injection). Characterization of the Gal4AD-TBP, Gal4AD-Swi1ABD, and VP16ADSwi1ABD interactions was performed on flow cell 2, using flow cell 1 for the GST reference, with the following concentrations of target proteins: 0.21, 0.43, 0.85, 1.7, and 3.4 M Swi1ABD; and 0.21, 0.41, 0.83, 1.7, and 3.3 M TBP. For duplicates on parallel flow cells in VP16AD-TBP experiments, results generally varied by Ͻ11% for the fast phase, 26% for the slow phase, and 26% for overall affinity
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