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. 2018 Sep 4;115(36):8960-8965.
doi: 10.1073/pnas.1806202115. Epub 2018 Aug 20.

Conservation of coactivator engagement mechanism enables small-molecule allosteric modulators

Andrew R Henderson  1   2 , Madeleine J Henley  1   2 , Nicholas J Foster  1   2 , Amanda L Peiffer  1   2 , Matthew S Beyersdorf  1   2 , Kevon D Stanford  1   3 , Steven M Sturlis  1 , Brian M Linhares  4 , Zachary B Hill  5 , James A Wells  5 , Tomasz Cierpicki  2   4 , Charles L Brooks 3rd  2   3   4 , Carol A Fierke  2   3 , Anna K Mapp  6   2   3
Affiliations

Conservation of coactivator engagement mechanism enables small-molecule allosteric modulators

Andrew R Henderson et al. Proc Natl Acad Sci U S A. .

Abstract

Transcriptional coactivators are a molecular recognition marvel because a single domain within these proteins, the activator binding domain or ABD, interacts with multiple compositionally diverse transcriptional activators. Also remarkable is the structural diversity among ABDs, which range from conformationally dynamic helical motifs to those with a stable core such as a β-barrel. A significant objective is to define conserved properties of ABDs that allow them to interact with disparate activator sequences. The ABD of the coactivator Med25 (activator interaction domain or AcID) is unique in that it contains secondary structural elements that are on both ends of the spectrum: helices and loops that display significant conformational mobility and a seven-stranded β-barrel core that is structurally rigid. Using biophysical approaches, we build a mechanistic model of how AcID forms binary and ternary complexes with three distinct activators; despite its static core, Med25 forms short-lived, conformationally mobile, and structurally distinct complexes with each of the cognate partners. Further, ternary complex formation is facilitated by allosteric communication between binding surfaces on opposing faces of the β-barrel. The model emerging suggests that the conformational shifts and cooperative binding is mediated by a flexible substructure comprised of two dynamic helices and flanking loops, indicating a conserved mechanistic model of activator engagement across ABDs. Targeting a region of this substructure with a small-molecule covalent cochaperone modulates ternary complex formation. Our data support a general strategy for the identification of allosteric small-molecule modulators of ABDs, which are key targets for mechanistic studies as well as therapeutic applications.

Keywords: Med25; allosteric modulator; protein–protein interactions; transcriptional coactivator.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Med25 AcID forms complexes with transcriptional activators of distinct sequences. (A) The AcID is the binding partner of a growing number of transcriptional activators and contains at least two binding surfaces, termed H1 and H2. The sequences of the transcriptional activation domains of the three Med25-dependent activators used in this study are shown below the protein structure (PDB ID code 2XNF). (B) Equilibrium dissociation constants for each of Med25 AcID–activator complexes, measured through fluorescence anisotropy experiments using fluorescein-labeled peptides. These values are the average of at least three independent measurements with the error indicated (standard deviation of the mean).
Fig. 2.
Fig. 2.
ATF6α binds to the H2 surface of Med25 AcID. (A) Results of chemical shift perturbation experiments superimposed upon the Med25 AcID structure (PDB ID code 2XNF). Residues displaying chemical shift perturbation greater than 2 SD upon ATF6α binding are depicted in rust spheres. (B) Scatter plot illustrating correlations between the chemical shift perturbations (CSPs) of individual Med25 AcID residues from HSQC experiments with ERM, ATF6α, and VP16. The position of each maize square represents the CSP of an individual residue in Med25 AcID upon binding to ERM (y axis) and VP16 (x axis). Thus, squares along the dotted diagonal are residues that shift similarly in both ERM–AcID and VP16–AcID complexes. The same analysis for ATF6α is shown in rust circles. Specifically labeled are the positions of three residues that are on the H1 face of AcID (T542) and H2 face of AcID (R466, Q456), highlighting the distinct pattern of correlated CSPs for ERM and ATF6α, consistent with the model in which the two activators do not interact with the same binding site. Full CSP datasets for each of the three activator–Med25 AcID complexes are shown in SI Appendix. (C) Results of direct binding experiments with fluorescein-labeled activators and the indicated mutants of Med25 AcID as measured by fluorescence polarization expressed the fold change relative to the dissociation constant of each activator for the WT AcID. The indicated error is propagated from three independent dissociation constant measurements.
Fig. 3.
Fig. 3.
Transient kinetic experiments define minimal mechanism of activator–AcID complexation. (A) Structure of the fluorophore used in these experiments, 4-N,N-dimethylamino-1,8-napththalimide (4-DMN). (B) Representative kinetic trace of association experiment with DMN-VP16 and AcID. The red line is the fit to a two-step binding model (see SI Appendix, Figs. S2 and S3 for additional data). (C) Dependence of the two observed rate constants for the fast (white circles) and slow (gray circles) kinetic phases on the concentration of AcID for association experiments of VP16 with AcID. (D) Sample kinetic trace of a dissociation experiment in which unlabeled VP16 was added in excess to preformed DMN-VP16–AcID. (E) General kinetic mechanism for TAD–AcID complex formation as determined by these experiments for all activators. Microscopic equilibrium constants (KC,n) are defined as the ratio of the respective forward and reverse rate constants. (F) Representation of equilibrium population distributions of bound states, calculated from equilibrium constants in 3G. Transparency of each state is scaled according to the indicated percentage population. When one equilibrium constant is too small to measure, the values are given as ranges. (G) Measured kinetic and equilibrium constants for all of the activators. Kinetic constants kF,2 and kR,2 are unable to be reliably calculated. a, The conformational change equilibrium constant is too small to be measured with precision. b, The overall equilibrium constant from Med25–ATF6a to Med25–ATF6a** is estimated to be ≤0.1 based on the limits of precision of our experiments, thus given that KC,1 ≤ 0.1, KC,2 must be ≤1.
Fig. 4.
Fig. 4.
Dissociation experiments reveal allosteric communication between two binding sites. (A) Schematic of the experiment. (B) Comparison of koff for VP16 (467–488) (blue bars) for Med25 AcID, Med25 AcID with VP16 (438–454)G450C covalently Tethered, and Med25 AcID with ERM prebound; the red bars summarize data from analogous experiments with ATF6α. The values shown are the average of 2–3 independent experiments with the indicated errors (SD). All changes from the binary complex were statistically significant (P < 0.01), except for the Tethered complex bound to ATF6α (transparent bar).
Fig. 5.
Fig. 5.
Emerging structural model for AcID–activator complex formation. (A and B) The NMR coordinates for Med25 AcID (PDB ID code 2XNF) (2) were used to construct the initial structure of Med25 in CHARMM using the Multiscale Modeling Tools for Structural Biology. For B, VP16 (438–454) G450C was constructed in CHARMM as a helical peptide, which was then patched in CHARMM to Med25 C506 through the formation of a disulfide bond at C506 (transparent blue helix). Using GBSW implicit solvent, temperature replica exchange was implemented using the CHARMM22 force field (32). The RMSFs were calculated for each Med25 AcID residue by overlaying Cα atoms for all of the coordinate files produced from the simulations. The coloring correlates with the degree of dynamical behavior of each region. (C) Structure of chemical cochaperone 22 obtained from a Tethering screen. The bar graph is a comparison of koff for VP16 (467–488) (blue bars) for Med25 AcID or Med25 AcID with 22 covalently Tethered; the red bars summarize data from analogous experiments with ATF6α. The values shown are the average of 2–3 independent experiments with the indicated errors (SD).
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