Ke Ruan

De novo determination of internuclear vector orientations from residual dipolar couplings measured in three independent alignment media

The straightforward interpretation of solution state residual dipolar couplings (RDCs) in terms of internuclear vector orientations generally requires prior knowledge of the alignment tensor, which in turn is normally estimated using a structural model. We have developed a protocol which allows the requirement for prior structural knowledge to be dispensed with as long as RDC measurements can be made in three independent alignment media. This approach, called Rigid Structure from Dipolar Couplings (RSDC), allows vector orientations and alignment tensors to be determined de novo from just three independent sets of RDCs. It is shown that complications arising from the existence of multiple solutions can be overcome by careful consideration of alignment tensor magnitudes in addition to the agreement between measured and calculated RDCs. Extensive simulations as well applications to the proteins ubiquitin and Staphylococcal protein GB1 demonstrate that this method can provide robust determinations of alignment tensors and amide N–H bond orientations often with better than 10° accuracy, even in the presence of modest levels of internal dynamics.

Structural and Functional Insights into the Human Börjeson-Forssman-Lehmann Syndrome-associated Protein PHF6

Background: PHF6 gene is mutated in BFLS and adult acute myeloid and T-cell acute lymphoblastic leukemias. Results: Crystal structure of the second extended PHD domain of PHF6 was solved. Conclusion: PHF6-ePHD2 is a novel structural module and binds dsDNA. Significance: PHF6 may function as a transcriptional repressor using its ePHD domains binding to DNA and recruiting NuRD complex through its NoLS region to regulate gene transcription. The plant homeodomain finger 6 (PHF6) was originally identified as the gene mutated in the X-linked mental retardation disorder Börjeson-Forssman-Lehmann syndrome. Mutations in the PHF6 gene have also been associated with T-cell acute lymphoblastic leukemia and acute myeloid leukemia. Approximately half of the disease-associated mutations are distributed in the second conserved extended plant homeodomain (ePHD2) of PHF6, indicating the functional importance of the ePHD2 domain. Here, we report the high resolution crystal structure of the ePHD2 domain of PHF6, which contains an N-terminal pre-PHD (C2HC zinc finger), a long linker, and an atypical PHD finger. PHF6-ePHD2 appears to fold as a novel integrated structural module. Structural analysis of PHF6-ePHD2 reveals pathological implication of PHF6 gene mutations in Börjeson-Forssman-Lehmann syndrome, T-cell acute lymphoblastic leukemia, and acute myeloid leukemia. The binding experiments show that PHF6-ePHD2 can bind dsDNA but not histones. We also demonstrate PHF6 protein directly interacts with the nucleosome remodeling and deacetylation complex component RBBP4. Via this interaction, PHF6 exerts its transcriptional repression activity. Taken together, these data support the hypothesis that PHF6 may function as a transcriptional repressor using its ePHD domains binding to the promoter region of its repressed gene, and this process was regulated by the nucleosome remodeling and deacetylation complex that was recruited to the genomic target site by NoLS region of PHF6.

Automated NMR Fragment Based Screening Identified a Novel Interface Blocker to the LARG/RhoA Complex

The small GTPase cycles between the inactive GDP form and the activated GTP form, catalyzed by the upstream guanine exchange factors. The modulation of such process by small molecules has been proven to be a fruitful route for therapeutic intervention to prevent the over-activation of the small GTPase. The fragment based approach emerging in the past decade has demonstrated its paramount potential in the discovery of inhibitors targeting such novel and challenging protein-protein interactions. The details regarding the procedure of NMR fragment screening from scratch have been rarely disclosed comprehensively, thus restricts its wider applications. To achieve a consistent screening applicable to a number of targets, we developed a highly automated protocol to cover every aspect of NMR fragment screening as possible, including the construction of small but diverse libray, determination of the aqueous solubility by NMR, grouping compounds with mutual dispersity to a cocktail, and the automated processing and visualization of the ligand based screening spectra. We exemplified our streamlined screening in RhoA alone and the complex of the small GTPase RhoA and its upstream guanine exchange factor LARG. Two hits were confirmed from the primary screening in cocktail and secondary screening over individual hits for LARG/RhoA complex, while one of them was also identified from the screening for RhoA alone. HSQC titration of the two hits over RhoA and LARG alone, respectively, identified one compound binding to RhoA.GDP at a 0.11 mM affinity, and perturbed the residues at the switch II region of RhoA. This hit blocked the formation of the LARG/RhoA complex, validated by the native gel electrophoresis, and the titration of RhoA to 15N labeled LARG in the absence and presence the compound, respectively. It therefore provides us a starting point toward a more potent inhibitor to RhoA activation catalyzed by LARG.

Multiplexed NMR: an automated CapNMR dual-sample probe.

A new generation of microscale, nuclear magnetic resonance (CapNMR) probe technology employs two independent detection elements to accommodate two samples simultaneously. Each detection element in the dual-sample CapNMR probe (DSP) delivers the same spectral resolution and S/N as in a CapNMR probe configured to accommodate one sample at a time. A high degree of electrical isolation allows the DSP to be used in a variety of data acquisition modes. Both samples are shimmed simultaneously to achieve high spectral resolution for simultaneous data acquisition, or alternatively, a flowcell-specific shim set is readily called via spectrometer subroutines to enable acquisition from one sample while the other is being loaded. An automation system accommodates loading of two samples via dual injection ports on an autosampler and two completely independent flowpaths leading to dedicated flowcells in the DSP probe.

Structural analysis of Stc1 provides insights into the coupling of RNAi and chromatin modification

Noncoding RNAs can modulate gene expression by directing modifications to histones that alter chromatin structure. In fission yeast, siRNAs produced via the RNAi pathway direct modifications associated with heterochromatin formation. siRNAs associate with the RNAi effector protein Argonaute 1 (Ago1), targeting the Ago1-containing RNA-induced transcriptional silencing (RITS) complex to homologous nascent transcripts. This promotes recruitment of the Clr4 complex (CLRC), which mediates methylation of histone H3 on lysine 9 (H3K9me) in cognate chromatin. A key question is how the RNAi and chromatin modification machineries are connected. Stc1 is a small protein recently shown to associate with both Ago1 and CLRC and to play a pivotal role in mediating the RNAi-dependent recruitment of CLRC to chromatin. To understand its mode of action, we have performed a detailed structural and functional analysis of the Stc1 protein. Our analyses reveal that the conserved N-terminal region of Stc1 represents an unusual tandem zinc finger domain, with similarities to common LIM domains but distinguished by a lack of preferred relative orientation of the two zinc fingers. We demonstrate that this tandem zinc finger domain is involved in binding Ago1, whereas the nonconserved C-terminal region mediates association with CLRC. These findings elucidate the molecular basis for the coupling of RNAi to chromatin modification in fission yeast.

Process of Fragment-Based Lead Discovery—A Perspective from NMR

Fragment-based lead discovery (FBLD) has proven fruitful during the past two decades for a variety of targets, even challenging protein–protein interaction (PPI) systems. Nuclear magnetic resonance (NMR) spectroscopy plays a vital role, from initial fragment-based screening to lead generation, because of its power to probe the intrinsically weak interactions between targets and low-molecular-weight fragments. Here, we review the NMR FBLD process from initial library construction to lead generation. We describe technical aspects regarding fragment library design, ligand- and protein-observed screening, and protein–ligand structure model generation. For weak binders, the initial hit-to-lead evolution can be guided by structural information retrieved from NMR spectroscopy, including chemical shift perturbation, transferred pseudocontact shifts, and paramagnetic relaxation enhancement. This perspective examines structure-guided optimization from weak fragment screening hits to potent leads for challenging PPI targets.

NMR characterization of weak interactions between RhoGDI2 and fragment screening hits.

BACKGROUND The delineation of intrinsically weak interactions between novel targets and fragment screening hits has long limited the pace of hit-to-lead evolution. Rho guanine-nucleotide dissociation inhibitor 2 (RhoGDI2) is a novel target that lacks any chemical probes for the treatment of tumor metastasis. METHODS Protein-observed and ligand-observed NMR spectroscopy was used to characterize the weak interactions between RhoGDI2 and fragment screening hits. RESULTS We identified three hits of RhoGDI2 using streamlined NMR fragment-based screening. The binding site residues were assigned using non-uniformly sampled Cα- and Hα-based three dimensional NMR spectra. The molecular docking to the proposed geranylgeranyl binding pocket of RhoGDI2 was guided by NMR restraints of chemical shift perturbations and ligand-observed transferred paramagnetic relaxation enhancement. We further validated the weak RhoGDI2-hit interactions using mutagenesis and structure-affinity analysis. CONCLUSIONS Weak interactions between RhoGDI2 and fragment screening hits were delineated using an integrated NMR approach. GENERAL INTERESTS Binders to RhoGDI2 as a potential anti-cancer target have been first reported, and their weak interactions were depicted using NMR spectroscopy. Our work highlights the powerfulness and the versatility of the integrative NMR techniques to provide valuable structural insight into the intrinsically weak interactions between RhoGDI2 and the fragment screening hits, which could hardly be conceived using other biochemical techniques.

Interfacial tension of the aqueous two-phase systems of cationic-anionic surfactant mixtures

Abstract Interfacial tensions of the aqueous two-phase systems formed by cationic-anionic surfactant mixtures were measured using spinning drop method. The effects of surfactant structure, molar ratio of cationic to anionic surfactants, surfactant concentration, salt, and temperature on the interfacial tensions were investigated. It was shown that the values of the interfacial tensions of the aqueous two-phase were in the scale of ultra-low interfacial tensions at certain molar ratios of cationic to anionic surfactants. Three types of interfacial tension curves were observed. The first curve comprised two curves that were located on either side of 1:1 molar ratio, and the interfacial tension decreased with the increase of excessive surfactant components. The second one was a saddle-shaped curve that strode over the 1:1 molar ratio. The third type was a saddle-shaped curve that was located beside the 1:1 molar ratio. The types of interfacial tensions depended on the molecular structure of the surfactants such as the hydrophilic groups and the lengths and symmetry of hydrophobic chains.

NMR Fragment Screening Hit Induces Plasticity of BRD7/9 Bromodomains

The complex biology associated with inhibition of bromodomain and extra‐terminal (BET) domains by chemical probes has attracted increasing attention, and there is a need to identify non‐BET bromodomain (BD) inhibitors. Several potent inhibitors of the BRD9 BD have recently been discovered, with anticancer and anti‐inflammation activity. However, its paralogue, BRD7 BD, remains unexploited. Here, we identified new chemotypes targeting BRD7 BD by using NMR fragment‐based screening. BRD7/9 BDs exhibit similar patterns of chemical‐shift perturbation upon the titration of hit compound 1. The crystal structure revealed that 1 repels the Y222 group of BRD9 BD in a similar way to that for butyryllysine, but not acetyllysine and known inhibitors. Hit 1 induced less rearrangement of residue F161 of BRD9 BD than acetyllysine, butyryllysine, and crotonyllysine. Our study provides structural insight into a new generation of butyryllysine mimics for probing the function of BRD7/9 BD.

Interfacial Tension of Aqueous Three‐Phase Systems Formed by Triton X‐100/PEG/Dextran

A novel aqueous three‐phase system was formed spontaneously when a nonionic surfactant (Triton X‐100) and two polymers (PEG and dextran) were mixed. The interfacial tension between the phases was measured by the spinning drop method. It was shown that the values of interfacial tension were extremely small. The interfacial tensions of the top/middle phases were much lower than those of the middle/bottom phases. The interfacial tension was affected by component concentrations, temperature, added salts, and the density difference between two phases. Temperature exhibited a special effect on interfacial tension: with the increase of temperature, interfacial tension increases significantly.