Rongsheng Ma

Crystal structure of tRNA m1G9 methyltransferase Trm10: insight into the catalytic mechanism and recognition of tRNA substrate

Transfer RNA (tRNA) methylation is necessary for the proper biological function of tRNA. The N1 methylation of guanine at Position 9 (m1G9) of tRNA, which is widely identified in eukaryotes and archaea, was found to be catalyzed by the Trm10 family of methyltransferases (MTases). Here, we report the first crystal structures of the tRNA MTase spTrm10 from Schizosaccharomyces pombe in the presence and absence of its methyl donor product S-adenosyl-homocysteine (SAH) and its ortholog scTrm10 from Saccharomyces cerevisiae in complex with SAH. Our crystal structures indicated that the MTase domain (the catalytic domain) of the Trm10 family displays a typical SpoU-TrmD (SPOUT) fold. Furthermore, small angle X-ray scattering analysis reveals that Trm10 behaves as a monomer in solution, whereas other members of the SPOUT superfamily all function as homodimers. We also performed tRNA MTase assays and isothermal titration calorimetry experiments to investigate the catalytic mechanism of Trm10 in vitro. In combination with mutational analysis and electrophoretic mobility shift assays, our results provide insights into the substrate tRNA recognition mechanism of Trm10 family MTases.

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.

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.

The polar warhead of a TRIM24 bromodomain inhibitor rearranges a water-mediated interaction network

Tripartite motif‐containing protein 24 (TRIM24) is closely correlated with multiple cancers, and a recent study demonstrated that the bromodomain of TRIM24 is essential for the proliferation of lethal castration‐resistant prostate cancer. Here, we identify three new inhibitors of the TRIM24 bromodomain using NMR fragment‐based screening. The crystal structures of two new inhibitors in complex with the TRIM24 bromodomain reveal that the water‐bridged interaction network is conserved in the same fashion as those for known benzoimidazolone inhibitors. Interestingly, the polar substitution on the warhead of one new inhibitor pulls the whole ligand approximately 2 Å into the inner side pocket of the TRIM24 bromodomain, and thus exhibits a binding mode significantly different from other known bromodomain ligands. This mode provides a useful handle for further hit‐to‐lead evolution toward novel inhibitors of the TRIM24 bromodomain.

Fluorine Pseudocontact Shifts Used for Characterizing the Protein–Ligand Interaction Mode in the Limit of NMR Intermediate Exchange

The characterization of protein-ligand interaction modes becomes recalcitrant in the NMR intermediate exchange regime as the interface resonances are broadened beyond detection. Here, we determined the 19 F low-populated bound-state pseudocontact shifts (PCSs) of mono- and di-fluorinated inhibitors of the BRM bromodomain using a highly skewed protein/ligand ratio. The bound-state 19 F PCSs were retrieved from 19 F chemical exchange saturation transfer (CEST) in the presence of the lanthanide-labeled protein, which was termed the 19 F PCS-CEST approach. These PCSs enriched in spatial information enabled the identification of best-fitting poses, which agree well with the crystal structure of a more soluble analog in complex with the BRM bromodomain. This approach fills the gap of the NMR structural characterization of lead-like inhibitors with moderate affinities to target proteins, which are essential for structure-guided hit-to-lead evolution.

Dynamic Nature of CTCF Tandem 11 Zinc Fingers in Multivalent Recognition of DNA As Revealed by NMR Spectroscopy

The 11 zinc fingers (ZFs) of the transcription factor CTCF play a versatile role in the regulation of gene expression. CTCF binds to numerous genomic sites to form chromatin loops and topologically associated domains and thus mediates the 3D architecture of chromatin. Although CTCF inter-ZF plasticity is essential for the recognition of multiple genomic sites, the dynamic nature of its 11 ZFs remains unknown. We assigned the chemical shifts of the CTCF ZFs 1-11 and solved the solution structures of each ZF. NMR backbone dynamics, residual dipolar couplings, and small-angle X-ray scattering experiments suggest a high inter-ZF plasticity of the free-form ZFs 1-11. As exemplified by two different protocadherin DNA sequences, the titration of DNAs to 15N-labeled CTCF ZFs 1-11 enabled systematic mapping of binding of CTCF ZFs to various chromatin sites. Our work paves the way for illustrating the molecular basis of the versatile DNA recognized by CTCF and has interesting implications for its conformational transition during DNA binding.

Ligand Proton Pseudocontact Shifts Determined from Paramagnetic Relaxation Dispersion in the Limit of NMR Intermediate Exchange

Delineation of protein-ligand interaction modes is key for rational drug discovery. The availability of complex crystal structures is often limited by the aqueous solubility of the compounds, while lead-like compounds with micromolar affinities normally fall into the NMR intermediate exchange regime, in which severe line broadening to beyond the detection of interfacial resonances limits NMR applications. Here, we developed a new method to retrieve low-populated bound-state 1H pseudocontact shifts (PCSs) using paramagnetic relaxation dispersion (RD). We evaluated using a 1H PCS-RD approach in a BRM bromodomain lead-like inhibitor to filter molecular docking poses using multiple intermolecular structural restraints. Considering the universal presence of proton atoms in druglike compounds, our work will have wide application in structure-guided drug discovery even under an extreme condition of NMR intermediate exchange and low aqueous solubility of ligands.

Structural plasticity of the TDRD3 Tudor domain probed by a fragment screening hit

As a reader of di‐methylated arginine on various proteins, such as histone, RNA polymerase II, PIWI and Fragile X mental retardation protein, the Tudor domain of Tudor domain‐containing protein 3 (TDRD3) mediates transcriptional activation in nucleus and formation of stress granules in the cytoplasm. Despite the TDRD3 implication in cancer cell proliferation and invasion, warheads to block the di‐methylated arginine recognition pocket of the TDRD3 Tudor domain have not yet been uncovered. Here we identified 14 small molecule hits against the TDRD3 Tudor domain through NMR fragment‐based screening. These hits were further cross‐validated by using competitive fluorescence polarization and isothermal titration calorimetry experiments. The crystal structure of the TDRD3 Tudor domain in complex with hit 1 reveals a distinct binding mode from the nature substrate. Hit 1 protrudes into the aromatic cage of the TDRD3 Tudor domain, where the aromatic residues are tilted to accommodate a sandwich‐like π–π interaction. The side chain of the conserved residue N596 swings away 3.1 Å to form a direct hydrogen bond with hit 1. Moreover, this compound shows a decreased affinity against the single Tudor domain of survival motor neuron protein, but no detectable binding to neither the tandem Tudor domain of TP53‐binding protein 1 nor the extended Tudor domain of staphylococcal nuclease domain‐containing protein 1. Our work depicts the structural plasticity of the TDRD3 Tudor domain and paves the way for the subsequent structure‐guided discovery of selective inhibitors targeting Tudor domains.