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.

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 (MR) 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.

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.

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.

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).

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.

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.

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.