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. 2023 Nov 21;120(47):e2313835120.
doi: 10.1073/pnas.2313835120. Epub 2023 Nov 14.

Glutamine-rich regions of the disordered CREB transactivation domain mediate dynamic intra- and intermolecular interactions

Maria A Martinez-Yamout  1 , Irem Nasir  1 , Sergey Shnitkind  1 , Jamie P Ellis  1 , Rebecca B Berlow  1 , Gerard Kroon  1 , Ashok A Deniz  1 , H Jane Dyson  1 , Peter E Wright  1
Affiliations

Glutamine-rich regions of the disordered CREB transactivation domain mediate dynamic intra- and intermolecular interactions

Maria A Martinez-Yamout et al. Proc Natl Acad Sci U S A. .

Abstract

The cyclic AMP response element (CRE) binding protein (CREB) is a transcription factor that contains a 280-residue N-terminal transactivation domain and a basic leucine zipper that mediates interaction with DNA. The transactivation domain comprises three subdomains, the glutamine-rich domains Q1 and Q2 and the kinase inducible activation domain (KID). NMR chemical shifts show that the isolated subdomains are intrinsically disordered but have a propensity to populate local elements of secondary structure. The Q1 and Q2 domains exhibit a propensity for formation of short β-hairpin motifs that function as binding sites for glutamine-rich sequences. These motifs mediate intramolecular interactions between the CREB Q1 and Q2 domains as well as intermolecular interactions with the glutamine-rich Q1 domain of the TATA-box binding protein associated factor 4 (TAF4) subunit of transcription factor IID (TFIID). Using small-angle X-ray scattering, NMR, and single-molecule Förster resonance energy transfer, we show that the Q1, Q2, and KID regions remain dynamically disordered in a full-length CREB transactivation domain (CREBTAD) construct. The CREBTAD polypeptide chain is largely extended although some compaction is evident in the KID and Q2 domains. Paramagnetic relaxation enhancement reveals transient long-range contacts both within and between the Q1 and Q2 domains while the intervening KID domain is largely devoid of intramolecular interactions. Phosphorylation results in expansion of the KID domain, presumably making it more accessible for binding the CBP/p300 transcriptional coactivators. Our study reveals the complex nature of the interactions within the intrinsically disordered transactivation domain of CREB and provides molecular-level insights into dynamic and transient interactions mediated by the glutamine-rich domains.

Keywords: NMR; TFIID; intrinsically disordered protein; single-molecule FRET; transcriptional activation.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Schematic diagram showing the domains of CREB, the two glutamine-rich domains Q1 and Q2, the kinase-inducible domain KID, and the bZip. All domains are intrinsically disordered in isolation. Phosphorylated KID (pKID) folds into two helices, αA and αB, upon binding to the KIX domain of CBP (27). The structure of the complex (1KDX) is shown at the Bottom Left. The bZip domain dimerizes and binds to DNA (25), and the structure 1DH3 is shown at the Bottom Right.
Fig. 2.
Fig. 2.
Dimensionless Kratky plot of small-angle scattering data for CREBTAD (black) and pCREBTAD (red), using merged data. An equivalent plot using raw data is shown in SI Appendix, Fig. S1, together with a plot of log I(q) vs. q for CREBTAD and pCREBTAD.
Fig. 3.
Fig. 3.
Plot of secondary structure propensities calculated from the secondary chemical shifts of Cα, Cβ, and CO for the three isolated domains, using the program SSP (50). The boundaries of each construct are indicated by colored rectangles at the top of the figure. Red arrows within the graph show the location of putative β-turns identified using the MICS server (http://spin.niddk.nih.gov/bax/nmrserver/mics) (51).
Fig. 4.
Fig. 4.
Region of the 1H-15N HSQC spectrum of CREBTAD (residues 1 to 280) of CREB, processed with iterative soft thresholding (IST) (black), showing assignments mapped from the spectra of the individual domains Q1 (green), KID (red), and Q2 (blue). Overlays of the complete CREBTAD spectrum and those of the isolated domains are shown in SI Appendix, Fig. S4. Resonances that could not be sequentially assigned in the spectra of the individual domains are shown with a star.
Fig. 5.
Fig. 5.
Region of an overlay of the 1H-15N HSQC spectra of CREBTAD in the diamagnetic (reduced) form (black) and the paramagnetic (active spin label) form (red). (A) CREBTAD mutant A50C/C90A with spin label at C50. (B) Wild-type CREBTAD with spin label at C90. (C) CREBTAD mutant C90A/T172C with spin label at C172. (D) 15N-labeled CREBTAD mutant C90A in the presence of unlabeled CREBTAD mutant C90A/T172C with spin label at C172.
Fig. 6.
Fig. 6.
Plots of the signal intensity ratio (Ipara/Idia) for each of the spin-labeled constructs of CREBTAD, as a function of residue number. (A) CREBTAD mutant A50C/C90A with spin label at C50. (B) Wild-type CREBTAD with spin label at C90. (C) CREBTAD mutant C90A/T172C with spin label at C172. The intensity ratio for the intramolecular PRE (spin label incorporated into the 15N-labeled protein) is shown in black, and the ratio for the intermolecular PRE (spin label incorporated into an unlabeled C90A/T172C protein, mixed with 15N-labeled C90A without spin label) is shown in blue. Small bars below each panel indicate missing data points. Green bars represent prolines, red bars show residues with cross-peaks in the spectra of the individual domains but not in that of CREBTAD, and purple bars show residues whose cross-peaks are missing from both the isolated domain and CREBTAD spectra. Blue bars denote residues with resonance overlaps that preclude evaluation of the cross-peak intensity.
Fig. 7.
Fig. 7.
Plots of the 1H-15N cross-peak intensity ratio (intensity of resonance with maximum titrant concentration/intensity of free protein resonance) (red bars) and average chemical shift difference for 1H-15N cross-peaks {Δδ = √ [(δH, titrant – δH, free)2 + ((δN, titrant – δN, free)2)/5]} (blue) for (A) CREB Q2 (residues 160 to 280) with the addition of a 10-fold molar excess of CREB Q1. (B) CREB Q2 (residues 160 to 280) with the addition of a fourfold molar excess of TAF Q1. (C) CREB Q1 (residues 1 to 98) with the addition of a fourfold molar excess of TAF Q1. Green bars represent prolines, purple bars show residues whose cross-peaks are missing from the spectra, and blue bars denote residues with resonance overlaps that precluded evaluation of the cross-peak intensity. It should be noted that several residues for which the Δδ (blue) is highest also correspond to cross-peaks of extremely low intensity, which may register as zero in the intensity ratio plot (red).
Fig. 8.
Fig. 8.
FRET histograms for the four constructs of CREB in sm-FRET buffer (20 mM TRIS, and 2 mM DTT, pH 7.5). Locations of fluorescent tags are indicated by stars on the schematic diagrams of the sequence, shown at the Top of the figure.
Fig. 9.
Fig. 9.
Effect of phosphorylation and increasing salt concentration on FRET efficiency. (A) Effect of increasing salt concentration on the FRET efficiency for the four fluorescently labeled CREBTAD samples: T2C/C90 (Q1) (red), C90/T160C (KID) (green), C90A/T172C/T259C (Q2) (blue), and T2C/C90A/T198C (Q1Q2) (black). (B) Effect of increasing salt concentration on the FRET efficiency for C90/T160C (KID) (green) and for C90/T160C phosphorylated at S98 and S133 by PKA (ppKID) (purple). (C) Effect of increasing salt concentration on the FRET efficiency for T2C/C90A/T198C (Q1Q2) (green) and for T2C/C90A/T198C phosphorylated at S98 and S133 by PKA (ppQ1Q2) (purple).
Fig. 10.
Fig. 10.
Cartoon representation of the predicted β-hairpin structures in the Q1 and Q2 domains of the CREBTAD. Transient population of these structures is supported by analysis of chemical shifts and PRE intensities and AlphaFold structure predictions. Regions with a propensity for β-structure (green arrows) and β-turns (black brackets) are indicated.
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