Recognition of Nucleosomes by Chromatin Factors: Lessons from Data-Driven Docking-Based Structures of Nucleosome-Protein Complexes

The function of chromatin ultimately depends on the many chromatin-associated proteins and protein complexes that regulate all DNA-templated processes such as transcription, repair and replication. As the molecular docking platform for these proteins, the nucleosome is the essential gatekeeper to the genome. As such, the nucleosome-binding activity of a myriad of proteins is essential for a healthy cell. Here, we review the molecular basis of nucleosome-protein interactions and classify the different binding modes available. The structural data needed for such studies not only come from traditional sources such as X-Ray crystallography but also increasingly from other sources. In particular, we highlight how partial interaction data, derived from for example NMR or mutagenesis, are used in data-driven docking to drive the modeling of the complex into an atomistic structure. This approach has opened up detailed insights for several nucleosome-protein complexes that were intractable or recalcitrant to traditional methods. These structures guide the formation of new hypotheses and advance our understanding of chromatin function at the molecular level

Recognition of Nucleosomes by Chromatin Factors: Lessons from Data-Driven Docking-Based Structures of Nucleosome-Protein Complexes

The function of chromatin ultimately depends on the many chromatin-associated proteins and protein complexes that regulate all DNA-templated processes such as transcription, repair and replication. As the molecular docking platform for these proteins, the nucleosome is the essential gatekeeper to the genome. As such, the nucleosome-binding activity of a myriad of proteins is essential for a healthy cell. Here, we review the molecular basis of nucleosome-protein interactions and classify the different binding modes available. The structural data needed for such studies not only come from traditional sources such as X-Ray crystallography but also increasingly from other sources. In particular, we highlight how partial interaction data, derived from for example NMR or mutagenesis, are used in data-driven docking to drive the modeling of the complex into an atomistic structure. This approach has opened up detailed insights for several nucleosome-protein complexes that were intractable or recalcitrant to traditional methods. These structures guide the formation of new hypotheses and advance our understanding of chromatin function at the molecular level

The experimental power of FR900359 to study Gq-regulated biological processes.

Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq

A Cell-Permeable Inhibitor to Trap Gαq Proteins in the Empty Pocket Conformation

In spite of the crucial role of heterotrimeric G proteins as molecular switches transmitting signals from G protein-coupled receptors, their selective manipulation with small molecule, cell-permeable inhibitors still remains an unmet challenge. Here, we report that the small molecule BIM-46187, previously classified as pan-G protein inhibitor, preferentially silences Gαq signaling in a cellular context-dependent manner. Investigations into its mode of action reveal that BIM traps Gαq in the empty pocket conformation by permitting GDP exit but interdicting GTP entry, a molecular mechanism not yet assigned to any other small molecule Gα inhibitor to date. Our data show that Gα proteins may be “frozen” pharmacologically in an intermediate conformation along their activation pathway and propose a pharmacological strategy to specifically silence Gα subclasses with cell-permeable inhibitors

The b-wave of the dark adapted flash electroretinogram in patients with advanced asymmetrical glaucoma and normal subjects

AIMS—To evaluate whether the b-wave of the dark adapted flash electroretinogram (ERG) is affected by glaucomatous damage.
METHODS—ERGs were recorded in 35 patients aged 33-65 years with advanced asymmetrical glaucomas (interocular difference of perimetric defects (mean deviation) >2 dB between the two fellow eyes of the glaucoma patients, primary and secondary open angle and low tension glaucomas) and 17 normal subjects matched for age and sex using white flashes of a xenon discharge tube in a Ganzfeld stimulator. After 30 minutes of dark adaptation luminance response functions were obtained using flashes of increasing scotopic luminance (highest 9.4 cd/s/m(2), lowest 5.5 log units below it). The parameters V(max), n, and K of the Naka-Rushton equation were computed from the measurement values based on the usual fitting procedure. These parameters, together with b-wave amplitudes and implicit times for all flash intensities, were compared interocularly and between the normal subjects and those with glaucoma. Correlations were computed between interocular differences of the mean deviation and interocular differences of V(max), n, K, b-wave amplitudes, and implicit times between the two fellow eyes of the patients with asymmetrical glaucomatous damage.
RESULTS—Implicit times were significantly longer (p<0.005) in the glaucoma patients than in the normal group for flash intensities of 9.4, 5.3, 1.7, 0.53, and 0.17 cd/s/m(2). b-Wave amplitudes did not differ significantly between the two study groups. Comparing the two fellow eyes of each patient with glaucoma, V(max) was significantly higher in the less damaged eye than in the more damaged eye. The interocular differences in the mean deviation correlated significantly with the interocular differences in the b-wave amplitudes, implicit times, and V(max).
CONCLUSIONS—These results suggest that glaucomas can lead to electrophysiologically measurable damage of the inner nuclear layer.


The a-wave of the dark adapted electroretinogram in glaucomas: are photoreceptors affected?

AIMS—To evaluate whether the a-wave of the dark adapted flash electroretinogram (ERG) is affected by glaucomatous damage.
METHODS—ERGs were recorded in 20 patients (age 33-65 years) with advanced glaucomas (primary and secondary open angle and low tension glaucomas) and 20 normals using a ganzfeld stimulus. After 30( )minutes of dark adaptation and pupil dilatation to at least 7.5( )mm in diameter, luminance response functions were obtained presenting white flashes of increasing scotopic luminance (the highest flash intensity being 9.4 cd/s/m(2), the lowest being 5.75 log units below it) with an interflash interval of 5 seconds. For each scotopic luminance, the responses of four flashes were averaged. The a-wave's amplitude was measured at 10, 11, and 12 ms. Within the glaucoma group, correlations between the interocular differences of the a-wave's amplitude and the mean deviation of a static perimetry (Octopus 500 perimeter, program G1) were computed for all flash intensities. Between normals and glaucomas, the a-wave's amplitude was compared for all flash intensities (paired t test).
RESULTS—Within the glaucoma group, the interocular differences of the a-wave's amplitudes correlated significantly with the differences of the MD for flash intensities of 9.4, 5.3, 1.7, and 0.5 cd/s/m(2). The a-wave's amplitude was significantly lower in the glaucoma compared with the normal group (p <0.005) for flash intensities of 9.4 and 5.3 cd/s/m(2).
CONCLUSION—These electrophysiological results imply that also the outer retinal structures, especially the photoreceptors, may be affected by glaucomatous damage.


Mimicking the Nucleosomal Context in Peptide-Based Binders of a H3K36me Reader Increases Binding Affinity While Altering the Binding Mode

Targeting of proteins in the histone modification machinery has emerged as a promising new direction to fight disease. The search for compounds that inhibit proteins that readout histone modification has led to several new epigenetic drugs, mostly for proteins involved in recognition of acetylated lysines. However, this approach proved to be a challenging task for methyllysine readers, which typically feature shallow binding pockets. Moreover, reader proteins of trimethyllysine K36 on the histone H3 (H3K36me3) not only bind the methyllysine but also the nucleosomal DNA. Here, we sought to find peptide-based binders of H3K36me3 reader PSIP1, which relies on DNA interactions to tightly bind H3K36me3 modified nucleosomes. We designed several peptides that mimic the nucleosomal context of H3K36me3 recognition by including negatively charged Glu-rich regions. Using a detailed NMR analysis, we find that addition of negative charges boosts binding affinity up to 50-fold while decreasing binding to the trimethyllysine binding pocket. Since screening and selection of compounds for reader domains is typically based solely on affinity measurements due to their lack of enzymatic activity, our case highlights the need to carefully control for the binding mode, in particular for the challenging case of H3K36me3 readers