Single-Molecule Analysis Reveals Linked Cycles of RSC Chromatin Remodeling and Ace1p Transcription Factor Binding in Yeast.

It is unknown how the dynamic binding of transcription factors (TFs) is molecularly linked to chromatin remodeling and transcription. Using single-molecule tracking (SMT), we show that the chromatin remodeler RSC speeds up the search process of the TF Ace1p for its response elements (REs) at the CUP1 promoter. We quantified smFISH mRNA data using a gene bursting model and demonstrated that RSC regulates transcription bursts of CUP1 only by modulating TF occupancy but does not affect initiation and elongation rates. We show by SMT that RSC binds to activated promoters transiently, and based on MNase-seq data, that RSC does not affect the nucleosomal occupancy at CUP1. Therefore, transient binding of Ace1p and rapid bursts of transcription at CUP1 may be dependent on short repetitive cycles of nucleosome mobilization. This type of regulation reduces the transcriptional noise and ensures a homogeneous response of the cell population to heavy metal stress.

Single-Molecule Orientation Imaging Reveals the Nano-Architecture of Amyloid Fibrils Undergoing Growth and Decay.

Amyloid-beta (Aß42) aggregates are characteristic Alzheimer's disease signatures, but probing how their nanoscale architectures influence their growth and decay remains challenging using current technologies. Here, we apply time-lapse single-molecule orientation-localization microscopy (SMOLM) to measure the orientations and rotational "wobble" of Nile blue (NB) molecules transiently binding to Aß42 fibrils. We correlate fibril architectures measured by SMOLM with their growth and decay over the course of 5 to 20 min visualized by single-molecule localization microscopy (SMLM). We discover that stable Aß42 fibrils tend to be well-ordered and signified by well-aligned NB orientations and small wobble. SMOLM also shows that increasing order and disorder are signatures of growing and decaying fibrils, respectively. We also observe SMLM-invisible fibril remodeling, including steady growth and decay patterns that conserve ß-sheet organization. SMOLM reveals that increased fibril architectural heterogeneity is correlated with dynamic remodeling and that large-scale fibril remodeling tends to originate from strongly heterogeneous local regions.

Versatile Near-Infrared Super-Resolution Imaging of Amyloid Fibrils with the Fluorogenic Probe CRANAD-2.

CRANAD-2 is a fluorogenic curcumin derivative used for near-infrared detection and imaging in vivo of amyloid aggregates, which are involved in neurodegenerative diseases. We explore the performance of CRANAD-2 in two super-resolution imaging techniques, namely stimulated emission depletion (STED) and single-molecule localization microscopy (SMLM), with markedly different fluorophore requirements. By conveniently adapting the concentration of CRANAD-2, which transiently binds to amyloid fibrils, we show that it performs well in both techniques, achieving a resolution in the range of 45-55 nm. Correlation of SMLM with atomic force microscopy (AFM) validates the resolution of fine features in the reconstructed super-resolved image. The good performance and versatility of CRANAD-2 provides a powerful tool for near-infrared nanoscopic imaging of amyloids in vitro and in vivo.

Peptide-PAINT Super-Resolution Imaging Using Transient Coiled Coil Interactions.

Super-resolution microscopy is transforming research in the life sciences by enabling the visualization of structures and interactions on the nanoscale. DNA-PAINT is a relatively easy-to-implement single-molecule-based technique, which uses the programmable and transient interaction of dye-labeled oligonucleotides with their complements for super-resolution imaging. However, similar to many imaging approaches, it is still hampered by the subpar performance of labeling probes in terms of their large size and limited labeling efficiency. To overcome this, we here translate the programmability and transient binding nature of DNA-PAINT to coiled coil interactions of short peptides and introduce Peptide-PAINT. We benchmark and optimize its binding kinetics in a single-molecule assay and demonstrate its super-resolution capability using self-assembled DNA origami structures. Peptide-PAINT outperforms classical DNA-PAINT in terms of imaging speed and efficiency. Finally, we prove the suitability of Peptide-PAINT for cellular super-resolution imaging by visualizing the microtubule and vimentin network in fixed cells.

Macrocyclic FKBP51 Ligands Define a Transient Binding Mode with Enhanced Selectivity.

Subtype selectivity represents a challenge in many drug discovery campaigns. A typical example is the FK506 binding protein 51 (FKBP51), which has emerged as an attractive drug target. The most advanced FKBP51 ligands of the SAFit class are highly selective vs. FKBP52 but poorly discriminate against the homologs and off-targets FKBP12 and FKBP12.6. During a macrocyclization pilot study, we observed that many of these macrocyclic analogs have unanticipated and unprecedented preference for FKBP51 over FKBP12 and FKBP12.6. Structural studies revealed that these macrocycles bind with a new binding mode featuring a transient conformation, which is disfavored for the small FKBPs. Using a conformation-sensitive assay we show that this binding mode occurs in solution and is characteristic for this new class of compounds. The discovered macrocycles are non-immunosuppressive, engage FKBP51 in cells, and block the cellular effect of FKBP51 on IKKα. Our findings provide a new chemical scaffold for improved FKBP51 ligands and the structural basis for enhanced selectivity.

Soil organic carbon, extracellular polymeric substances (EPS), and soil structural stability as affected by previous and current land-use.

While soil microbial ecology, soil organic carbon (SOC) and soil physical quality are widely understood to be interrelated - the underlying drivers of emergent properties, from land management to biochemistry, are hotly debated. Biological binding agents, microbial exudates, or 'extracellular polymeric substances' (EPS) in soil are now receiving increased attention due to several of the existing methodological challenges having been overcome. We applied a recently developed approach to quantify soil EPS, as extracellular protein and extracellular polysaccharide, on the well-characterised soils of the Highfield Experiment, Rothamsted Research, UK. Our aim was to investigate the links between agricultural land use, SOC, transient binding agents known as EPS, and their impacts on soil physical quality (given by mean weight diameter of water stable aggregates; MWD). We compared the legacy effects from long-term previous land-uses (unfertilised grassland, fertilised arable, and fallow) which were established > 50 years prior to investigation, crossed with the same current land-uses established for a duration of only 2.5 years prior to sampling. Continuously fallow and grassland soils represented the poorest and greatest states of structural integrity, respectively. Total SOC and N were found to be affected by both previous and current land-uses, while extractable EPS and MWD were driven primarily by the current land-use. Land-use change between these two extremes (fallow â†’ grass; grass â†’ fallow) resulted in smaller SOC differences (64% increase or 37% loss) compared to MWD (125% increase or 78% loss). SOC concentration correlated well to MWD (adjusted R 2 = 0.72) but the greater SOC content from previous grassland was not found to contribute directly to the current stability (p < 0.05). Our work thus supports the view that certain distinct components of SOC, rather than the total pool, have disproportionately important effects on a soil's structural stability. EPS-protein was more closely related to aggregate stability than EPS-polysaccharide (p values of 0.002 and 0.027, respectively), and ranking soils with the 5 greatest concentrations of EPS-protein to their corresponding orders of stability (MWD) resulted in a perfect match. We confirmed that both EPS-protein and EPS-polysaccharide were transient fractions: supporting the founding models for aggregate formation. We suggest that management of transient binding agents such as EPS -as opposed to simply increasing the total SOC content- may be a more feasible strategy to improve soil structural integrity and help achieve environmental objectives.

Kinetically selective and potent inhibitors of HDAC8.

Histone deacetylase 8 (HDAC8) is an established and validated target for T-cell lymphoma and childhood neuroblastoma. The active site binding pocket of HDAC8 is highly conserved among all zinc-containing representatives of the histone deacetylase (HDAC) family. This explains that most HDACs are unselectively recognized by similar inhibitors featuring a zinc binding group (ZBG), a hydrophobic linker and a head group. In the light of this difficulty, the creation of isoenzyme-selectivity is one of the major challenges in the development of HDAC inhibitors. In a series of trifluoromethylketone inhibitors of HDAC8 compound 10 shows a distinct binding mechanism and a dramatically increased residence time (RT) providing kinetic selectivity against HDAC4. Combining the binding kinetics results with computational docking and binding site flexibility analysis suggests that 10 occupies the conserved catalytic site as well as an adjacent transient sub-pocket of HDAC8.

Single-Molecule Orientation Imaging Reveals the Nano-Architecture of Amyloid Fibrils Undergoing Growth and Decay.

Amyloid-beta (Aß42) aggregates are characteristic signatures of Alzheimer's disease, but probing how their nanoscale architectures influence their growth and decay remains challenging using current technologies. Here, we apply time-lapse single-molecule orientation-localization microscopy (SMOLM) to measure the orientations and rotational "wobble" of Nile blue (NB) molecules transiently binding to Aß42 fibrils. We quantify correlations between fibril architectures, measured by SMOLM, and their growth and decay visualized by single-molecule localization microscopy (SMLM). We discover that stable Aß42 fibrils tend to be well-ordered, signified by well-aligned NB orientations and small wobble. SMOLM also shows that increasing order and disorder are signatures of growing and decaying Aß42 fibrils, respectively. We also observe SMLM-invisible fibril remodeling, including steady growth and decay patterns that conserve ß-sheet organization. SMOLM reveals that increased heterogeneity in fibril architectures is correlated with more dynamic remodeling and that large-scale fibril remodeling tends to originate from local regions that exhibit strong heterogeneity.

The catalytic domain of free or ligand bound histone deacetylase 4 occurs in solution predominantly in closed conformation.

Human histone deacetylase 4 (HDAC4) is a key epigenetic regulator involved in a number of important cellular processes. This makes HDAC4 a promising target for the treatment of several cancers and neurodegenerative diseases, in particular Huntington's disease. HDAC4 is highly regulated by phosphorylation and oxidation, which determine its nuclear or cytosolic localization, and exerts its function through multiple interactions with other proteins, forming multiprotein complexes of varying composition. The catalytic domain of HDAC4 is known to interact with the SMRT/NCOR corepressor complex when the structural zinc-binding domain (sZBD) is intact and forms a closed conformation. Crystal structures of the HDAC4 catalytic domain have been reported showing an open conformation of HDAC4 when bound to certain ligands. Here, we investigated the relevance of this HDAC4 conformation under physiological conditions in solution. We show that proper zinc chelation in the sZBD is essential for enzyme function. Loss of the structural zinc ion not only leads to a massive decrease in enzyme activity, but it also has serious consequences for the overall structural integrity and stability of the protein. However, the Zn2+ free HDAC4 structure in solution is incompatible with the open conformation. In solution, the open conformation of HDAC4 was also not observed in the presence of a variety of structurally divergent ligands. This suggests that the open conformation of HDAC4 cannot be induced in solution, and therefore cannot be exploited for the development of HDAC4-specific inhibitors.

Multiresolution molecular dynamics simulations reveal the interplay between conformational variability and functional interactions in membrane-bound cytochrome P450 2B4.

Cytochrome P450 2B4 (CYP 2B4) is one of the best-characterized CYPs and serves as a key model system for understanding the mechanisms of microsomal class II CYPs, which metabolize most known drugs. The highly flexible nature of CYP 2B4 is apparent from crystal structures that show the active site with either a wide open or a closed heme binding cavity. Here, we investigated the conformational ensemble of the full-length CYP 2B4 in a phospholipid bilayer, using multiresolution molecular dynamics (MD) simulations. Coarse-grained MD simulations revealed two predominant orientations of CYP 2B4's globular domain with respect to the bilayer. Their refinement by atomistic resolution MD showed adaptation of the enzyme's interaction with the lipid bilayer, leading to open configurations that facilitate ligand access to the heme binding cavity. CAVER analysis of enzyme tunnels, AquaDuct analysis of water routes, and Random Acceleration Molecular Dynamics simulations of ligand dissociation support the conformation-dependent passage of molecules between the active site and the protein surroundings. Furthermore, simulation of the re-entry of the inhibitor bifonazole into the open conformation of CYP 2B4 resulted in binding at a transient hydrophobic pocket within the active site cavity that may play a role in substrate binding or allosteric regulation. Together, these results show how the open conformation of CYP 2B4 facilitates the binding of substrates from and release of products to the membrane, whereas the closed conformation prolongs the residence time of substrates or inhibitors and selectively allows the passage of smaller reactants via the solvent and water channels.

Accessing Transient Binding Pockets by Protein Engineering and Yeast Surface Display Screening.

The binding pocket of some therapeutic targets can acquire multiple conformations that, to some extent, depend on the protein dynamics and the interaction with other molecules. The inability to reach the binding pocket can impose a substantial or even insurmountable barrier for the de novo identification or optimization of small-molecule ligands. Herein, we describe a protocol for the engineering of a target protein and a yeast display FACS sorting strategy to identify protein variants with a stable transient binding pocket with improved binding for a cryptic site-specific ligand. This strategy may facilitate drug discovery using the resulting protein variants with accessible binding pockets for ligand screening.

A protein engineering approach toward understanding FKBP51 conformational dynamics and mechanisms of ligand binding.

Most proteins are flexible molecules that coexist in an ensemble of several conformations. Point mutations in the amino acid sequence of a protein can trigger structural changes that drive the protein population to a conformation distinct from the native state. Here, we report a protein engineering approach to better understand protein dynamics and ligand binding of the FK506-binding protein 51 (FKBP51), a prospective target for stress-related diseases, metabolic disorders, some types of cancers and chronic pain. By randomizing selected regions of its ligand-binding domain and sorting yeast display libraries expressing these variants, mutants with high affinity to conformation-specific FKBP51 selective ligands were identified. These improved mutants are valuable tools for the discovery of novel selective ligands that preferentially and specifically bind the FKBP51 active site in its open conformation state. Moreover, they will help us understand the conformational dynamics and ligand binding mechanics of the FKBP51 binding pocket.

Binding pocket stabilization by high-throughput screening of yeast display libraries.

Protein dynamics have a great influence on the binding pockets of some therapeutic targets. Flexible protein binding sites can result in transient binding pocket formation which might have a negative impact on drug screening efforts. Here, we describe a protein engineering strategy with FK506-binding protein 51 (FKBP51) as a model protein, which is a promising target for stress-related disorders. High-throughput screening of yeast display libraries of FKBP51 resulted in the identification of variants exhibiting higher affinity binding of conformation-specific FKBP51 selective inhibitors. The gene libraries of a random mutagenesis and site saturation mutagenesis of the FK1 domain of FKBP51 encoding sequence were used to create a yeast surface display library. Fluorescence-activated cell sorting for FKBP51 variants that bind conformation-specific fluorescently labeled ligands with high affinity allowed for the identification of 15 different protein variants with improved binding to either, or both FKBP51-specific ligands used in the screening, with improved affinities up to 34-fold compared to the wild type. These variants will pave the way to a better understanding of the conformational flexibility of the FKBP51 binding pocket and may enable the isolation of new selective ligands that preferably and selectively bind the active site of the protein in its open conformation state.

Med Chem Remote: The Frontiers in Medicinal Chemistry 2021.

Digital, but delicious! The Frontiers in Medicinal Chemistry 2021 meeting, originally intended to take place in Darmstadt, carried on as an online event from March 8-10 this year. Even with pandemic restrictions, the event co-presented by the Medicinal Chemistry Division of the German Chemical Society (GDCh), the German Pharmaceutical Society (DPhG), and the Swiss Chemical Society (SCS) proved to be a success, showcasing excellent speakers and facilitating participant interaction in an ingenious virtual setting. Over 350 participants from more than 10 countries gathered to discuss the latest trends and directions in medicinal chemistry, with sessions on molecular glues, covalent fragments, transient binding pockets and more. This report presents a summary of the key lectures and activities at the event.

Which Properties Allow Ligands to Open and Bind to the Transient Binding Pocket of Human Aldose Reductase?

The transient specificity pocket of aldose reductase only opens in response to specific ligands. This pocket may offer an advantage for the development of novel, more selective ligands for proteins with similar topology that lack such an adaptive pocket. Our aim was to elucidate which properties allow an inhibitor to bind in the specificity pocket. A series of inhibitors that share the same parent scaffold but differ in their attached aromatic substituents were screened using ITC and X-ray crystallography for their ability to occupy the pocket. Additionally, we investigated the electrostatic potentials and charge distribution across the attached terminal aromatic groups with respect to their potential to bind to the transient pocket of the enzyme using ESP calculations. These methods allowed us to confirm the previously established hypothesis that an electron-deficient aromatic group is an important prerequisite for opening and occupying the specificity pocket. We also demonstrated from our crystal structures that a pH shift between 5 and 8 does not affect the binding position of the ligand in the specificity pocket. This allows for a comparison between thermodynamic and crystallographic data collected at different pH values.

NMR experiments on the transient interaction of the intrinsically disordered N-terminal peptide of cystathionine-ß-synthase with heme.

The N-terminal segment of human cystathionine-ß-synthase (CBS(1-40)) constitutes an intrinsically disordered protein stretch that transiently interacts with heme. We illustrate that the HCBCACON experimental protocol provides an efficient alternative approach for probing transient interactions of intrinsically disordered proteins with heme in situations where the applicability of the conventional [1H, 15N]-HSQC experiment may be limited. This experiment starting with the excitation of protein side chain protons delivers information about the proline residues and thereby makes it possible to use these residues in interaction mapping experiments. Employing this approach in conjunction with site-specific mutation we show that transient heme binding is mediated by the Cys15-Pro16 motif of CBS(1-40).

A method to trap transient and weak interacting protein complexes for structural studies.

Several key biological events adopt a "hit-and-run" strategy in their transient interactions between binding partners. In some instances, the disordered nature of one of the binding partners severely hampers the success of co-crystallization, often leading to the crystallization of just one of the partners. Here, we discuss a method to trap weak and transient protein interactions for crystallization. This approach requires the structural details of at least one of the interacting partners and binding knowledge to dock the known minimum binding region (peptide) of the protein onto the other using an optimal-sized linker. Prior to crystallization, the purified linked construct should be verified for its intact folding and stability. Following structure determination, structure-guided functional studies are performed with independent, full-length unlinked proteins to validate the findings of the linked complex. We designed this approach and then validated its efficacy using a 24 amino acid minimum binding region of the intrinsically disordered, neuron-specific substrates, Neurogranin and Neuromodulin, joined via a Gly-linker to their interacting partner, Calmodulin. Moreover, the reported functional studies with independent full-length proteins confirmed the findings of the linked peptide complexes. Based on our studies, and in combination with the supporting literature, we suggest that optimized linkers can provide an environment to mimic the natural interactions between binding partners, and offer a useful strategy for structural studies to trap weak and transient interactions involved in several biological processes.