Bromodomain Interactions with Acetylated Histone 4 Peptides in the BRD4 Tandem Domain: Effects on Domain Dynamics and Internal Flexibility.

The bromodomain and extra-terminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Like other BET proteins, BRD4 contains two bromodomains, BD1 and BD2, that can interact cooperatively with target proteins and designed ligands, with important implications for drug discovery. Here, we used nuclear magnetic resonance (NMR) spectroscopy to study the dynamics and interactions of the isolated bromodomains, as well as the tandem construct including both domains and the intervening linker, and investigated the effects of binding a tetra-acetylated peptide corresponding to the tail of histone 4. The peptide affinity is lower for both domains in the tandem construct than for the isolated domains. Using 15N spin relaxation, we determined the global rotational correlation times and residue-specific order parameters for BD1 and BD2. Isolated BD1 is monomeric in the apo state but apparently dimerizes upon binding the tetra-acetylated peptide. Isolated BD2 partially dimerizes in both the apo and peptide-bound states. The backbone order parameters reveal marked differences between BD1 and BD2, primarily in the acetyl-lysine binding site where the ZA loop is more flexible in BD2. Peptide binding reduces the order parameters of the ZA loop in BD1 and the ZA and BC loops in BD2. The AB loop, located distally from the binding site, shows variable dynamics that reflect the different dimerization propensities of the domains. These results provide a basis for understanding target recognition by BRD4.

NMR Molecular Replacement Provides New Insights into Binding Modes to Bromodomains of BRD4 and TRIM24.

Structure-based drug discovery (SBDD) largely relies on structural information from X-ray crystallography because traditional NMR structure calculation methods are too time consuming to be aligned with typical drug discovery timelines. The recently developed NMR molecular replacement (NMR2) method dramatically reduces the time needed to generate ligand-protein complex structures using published structures (apo or holo) of the target protein and treating all observed NOEs as ambiguous restraints, bypassing the laborious process of obtaining sequence-specific resonance assignments for the protein target. We apply this method to two therapeutic targets, the bromodomain of TRIM24 and the second bromodomain of BRD4. We show that the NMR2 methodology can guide SBDD by rationalizing the observed SAR. We also demonstrate that new types of restraints and selective methyl labeling have the potential to dramatically reduce "time to structure" and extend the method to targets beyond the reach of traditional NMR structure elucidation.

Potent and selective bivalent inhibitors of BET bromodomains.

Proteins of the bromodomain and extraterminal (BET) family, in particular bromodomain-containing protein 4 (BRD4), are of great interest as biological targets. BET proteins contain two separate bromodomains, and existing inhibitors bind to them monovalently. Here we describe the discovery and characterization of probe compound biBET, capable of engaging both bromodomains simultaneously in a bivalent, in cis binding mode. The evidence provided here was obtained in a variety of biophysical and cellular experiments. The bivalent binding results in very high cellular potency for BRD4 binding and pharmacological responses such as disruption of BRD4-mediator complex subunit 1 foci with an EC50 of 100 pM. These compounds will be of considerable utility as BET/BRD4 chemical probes. This work illustrates a novel concept in ligand design-simultaneous targeting of two separate domains with a drug-like small molecule-providing precedent for a potentially more effective paradigm for developing ligands for other multi-domain proteins.

Backbone resonance assignments for the SET domain of the human methyltransferase NSD2.

Aberrant NSD2 methyltransferase activity is implicated as the oncogenic driver in multiple myeloma, suggesting opportunities for novel therapeutic intervention. The methyltransferase activity of NSD2 resides in its catalytic SET domain, which is conserved among most lysine methyltransferases. Here we report the backbone [Formula: see text], N, C[Formula: see text], [Formula: see text] and side-chain [Formula: see text] assignments of a 25 kDa NSD2 SET domain construct, spanning residues 991-1203. A chemical shift analysis of C[Formula: see text], [Formula: see text] and [Formula: see text] resonances predicts a secondary structural pattern that is in agreement with homology models.

Segmental Isotope Labelling of an Individual Bromodomain of a Tandem Domain BRD4 Using Sortase A.

Bromodomain and extra-terminal (BET) family of proteins are one of the major readers of epigenetic marks and an important target class in oncology and other disease areas. The importance of the BET family of proteins is manifested by the explosion in the number of inhibitors against these targets that have successfully entered clinical trials. One important BET family member is bromodomain containing protein 4 (BRD4). Structural and biophysical studies of BRD4 are complicated by its tertiary-structure consisting of two bromodomains connected by a flexible inter-domain linker of approximately 180 amino acids. A detailed understanding of the interplay of these bromodomains will be key to rational drug design in BRD4, yet there are no reported three-dimensional structures of the multi-domain BRD4 and NMR studies of the tandem domain are hampered by the size of the protein. Here, we present a method for rapid Sortase A-mediated segmental labelling of the individual bromodomains of BRD4 that provides a powerful strategy that will enable NMR studies of ligand-bromodomain interactions with atomic detail. In our labelling strategy, we have used U-[2H,15N]-isotope labelling on the C-terminal bromodomain with selective introduction of 13CH3 methyl groups on Ile (δ1), Val and Leu, whereas the N-terminal bromodomain remained unlabelled. This labelling scheme resulted in significantly simplified NMR spectra and will allow for high-resolution interaction, structure and dynamics studies in the presence of ligands.

Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence.

The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly (15)N-labeled Ras as well as [(13)C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions.

Functional implications of large backbone amplitude motions of the glycoprotein 130-binding epitope of interleukin-6.

Human interleukin (IL)-6 plays a pivotal role in the immune response, hematopoiesis, the acute-phase response, and inflammation. IL-6 has three distinct receptor epitopes, termed sites I, II, and III, that facilitate the formation of a signaling complex. IL-6 signals via a homodimer of glycoprotein 130 (gp130) after initially forming a heterodimer with the nonsignaling α-receptor [IL-6 α-receptor (IL-6R)] via site I. Here, we present the backbone dynamics of apo-IL-6 as determined by analysis of NMR relaxation data with the extended model-free formalism of Lipari and Szabo. To alleviate significant resonance overlap in the HSQC-type spectra, cell-free protein synthesis was used to selectively (15) N-label residues, thereby ensuring a complete set of residue-specific dynamics. The calculated order parameters [square of the generalized model-free order parameter (S(2))] showed significant conformational heterogeneity among clusters of residues in IL-6. In particular, the N-terminal region of the long AB-loop, which corresponds spatially to one of the gp130 receptor binding epitopes (i.e. site III), experiences substantial fluctuations along the conformation of the main chain (S(2) = 0.3-0.8) that are not observed at the other two epitopes or in other cytokines. Thus, we postulate that dynamic properties of the AB-loop are responsible for inhibiting the interaction of IL-6 with gp130 in the absence of the IL-6R, and that binding of IL-6R at site I shifts the dynamic equilibrium to favor interaction with gp130 at site III. In addition, molecular dynamics simulations corroborated the NMR-derived dynamics, and showed that the BC-loop adopts different substates that possibly play a role in facilitating receptor assembly.

Structure and dynamics of human Nedd4-1 WW3 in complex with the αENaC PY motif.

Nedd4-1 (neuronal precursor cell expressed developmentally downregulated gene 4-1) is an E3 ubiquitin ligase that interacts with and negatively regulates the epithelial Na(+) channel (ENaC). The WW domains of Nedd4-1 bind to the ENaC subunits via recognition of PY motifs. Human Nedd4-1 (hNedd4-1) contains four WW domains with the third domain (WW3*) showing the strongest affinity to the PY motif. To understand the mechanism underlying this binding affinity, we have carried out NMR structural and dynamics analyses of the hNedd4-1 WW3* domain in complex with a peptide comprising the C-terminal tail of the human ENaC α-subunit. The structure reveals that the peptide interacts in a similar manner to other WW domain-ENaC peptide structures. Crucial interactions that likely provide binding affinity are the broad XP groove facilitating additional contacts between the WW3* domain and the peptide, compared to similar complexes, and the large surface area buried (83Å(2)) between R430 (WW3*) and L647' (αENaC). This corroborates the model-free analysis of the (15)N backbone relaxation data, which showed that R430 is the most rigid residue in the domain (S(2)=0.90±0.01). Carr-Purcell-Meiboom-Gill relaxation dispersion analysis identified two different conformational exchange processes on the µs-ms time-scale. One of these processes involves residues located at the peptide binding interface, suggesting conformational exchange may play a role in peptide recognition. Thus, both structural and dynamic features of the complex appear to define the high binding affinity. The results should aid interpretation of biochemical data and modeling interfaces between Nedd4-1 and other interacting proteins.