Small-Molecule Inhibitors of the MLL1 CXXC Domain, an Epigenetic Reader of DNA Methylation.

The CXXC domain is a reader of DNA methylation which preferentially binds to unmethylated CpG DNA motifs. Chromosomal translocations involving the MLL1 gene produce in-frame fusion proteins in which the N-terminal portion of the MLL1 protein harboring its CXXC domain is fused to the C-terminal portion of multiple partners. For the MLL-AF9 fusion, mutations which disrupt CXXC domain-DNA binding abrogate the ability to cause leukemia in mice. Based on this, we initiated an effort to develop small-molecule inhibitors of the MLL1 CXXC domain as a novel approach to therapy. We developed a fluorescence polarization-based assay for MLL CXXC domain-DNA binding and screened a library of Cys-reactive molecules. For the most potent hit from this screen, we have synthesized a library of analogs to explore the structure-activity relationship, defined the binding site using chemical shift perturbations in NMR spectra, and explored the selectivity of compounds across the CXXC domain family.

Observation of Arginine Side-Chain Motions Coupled to the Global Conformational Exchange Process in Deubiquitinase A.

Coupled motions have been demonstrated to be functionally important in a number of enzymes. Noncovalent side-chain interactions play essential roles in coordinating the motions across different structural elements in a protein. However, most of the dynamic studies of proteins are focused on backbone amides or methyl groups in the side chains and little is known about the polar and charged side chains. We have previously characterized the conformational dynamics of deubiquitinase A (DUBA), an isopeptidase, on the microsecond-to-millisecond (µs-ms) time scales with the amide 1H Carr-Purcell-Meiboom-Gill (CPMG) experiment. We detected a global conformational exchange process on a time scale of approximately 200 µs, which involves most of the structural elements in DUBA, including the active site and the substrate binding interface. Here, we extend our previous study on backbone amides to the arginine side-chain Nε-Hε groups using a modified 1H CPMG pulse sequence that can efficiently detect both backbone amide and arginine side-chain Nε-Hε signals in a single experiment. We found that the side chains of three arginines display motions on the same time scale as the backbone amides. Mutations of two of the three arginines to alanines result in a decrease in enzyme activity. One of these two arginines is located in a loop involved in substrate binding. This loop is not visible in the backbone amide-detected experiments due to excess line broadening induced by motions on the µs-ms time scales. These results clearly demonstrate that the motions of some arginine side chains are coupled to the global conformational exchange process and provide an additional probe for motions in a functionally important loop that did not yield visible backbone amide signals, suggesting the value of side-chain experiments on DUBA. The modified 1H CPMG pulse sequence allows the simultaneous characterization of backbone and arginine side-chain dynamics without any increase in data acquisition time and can be applied to the dynamic studies of any protein that displays measurable amide 1H relaxation dispersion.

KIX domain determines a selective tumor-promoting role for EP300 and its vulnerability in small cell lung cancer.

EP300, a transcription coactivator important in proliferation and differentiation, is frequently mutated in diverse cancer types, including small cell lung cancer (SCLC). While these mutations are thought to result in loss of EP300 function, the impact on tumorigenesis remains largely unknown. Here, we demonstrate that EP300 mutants lacking acetyltransferase domain accelerate tumor development in mouse models of SCLC. However, unexpectedly, complete Ep300 knockout suppresses SCLC development and proliferation. Dissection of EP300 domains identified kinase inducible domain-interacting (KIX) domain, specifically its interaction with transcription factors including MYB, as the determinant of protumorigenic activity. Ala627 in EP300 KIX results in a higher protein-binding affinity than Asp647 at the equivalent position in CREBBP KIX, underlying the selectivity of KIX-binding partners for EP300. Blockade of KIX-mediated interactions inhibits SCLC development in mice and cell growth. This study unravels domain-specific roles for EP300 in SCLC and unique vulnerability of the EP300 KIX domain for therapeutic intervention.

The Intrinsically Disordered Proteins MLLT3 (AF9) and MLLT1 (ENL) - Multimodal Transcriptional Switches With Roles in Normal Hematopoiesis, MLL Fusion Leukemia, and Kidney Cancer.

AF9 (MLLT3) and ENL (MLLT1) are members of the YEATS family (named after the five proteins first shown to contain this domain: Yaf9, ENL, AF9, Taf14, Sas5) defined by the presence of a YEATS domain. The YEATS domain is an epigenetic reader that binds to acetylated and crotonylated lysines, unlike the bromodomain which can only bind to acetylated lysines. All members of this family have been shown to be components of various complexes with roles in chromatin remodeling, histone modification, histone variant deposition, and transcriptional regulation. MLLT3 is a critical regulator of hematopoiesis with a role in maintaining the hematopoietic stem or progenitor cell (HSPC) population. Approximately 10% of acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) patients harbor a translocation involving MLL (mixed lineage leukemia). In the context of MLL fusion patients with AML and ALL, MLL-AF9 and MLL-ENL fusions are observed in 34 and 31% of the patients, respectively. The intrinsically disordered C-terminal domain of MLLT3 (AHD, ANC1 homology domain) undergoes coupled binding and folding upon interaction with partner proteins AF4, DOT1L, BCOR, and CBX8. Backbone dynamics studies of the complexes suggest a role for dynamics in function. Inhibitors of the interaction of the intrinsically disordered AHD with partner proteins have been described, highlighting the feasibility of targeting intrinsically disordered regions. MLLT1 undergoes phase separation to enhance recruitment of the super elongation complex (SEC) and drive transcription. Mutations in MLLT1 observed in Wilms tumor patients enhance phase separation and transcription to drive an aberrant gene expression program.

Conformational Dynamics of Deubiquitinase A and Functional Implications.

Deubiquitinase A (DUBA) belongs to the ovarian tumor family of deubiquitinating enzymes and was initially identified as a negative regulator of type I interferons, whose overproduction has been linked to autoimmune diseases. The deubiquitinating activity of DUBA is positively regulated by phosphorylation at a single serine residue, S177, which results in minimal structural changes. We have previously shown that phosphorylation induces a two-state conformational equilibrium observed only in the active form of DUBA, highlighting the functional importance of DUBA dynamics. Here, we report the conformational dynamics of DUBA on the microsecond-to-millisecond time scales characterized by nuclear magnetic resonance relaxation dispersion experiments. We found that motions on these time scales are highly synchronized in the phosphorylated and nonphosphorylated DUBA. Despite the overall similarity of these two forms, different dynamic properties were observed in helix α1 and the neighboring regions, including residue S177, which likely contribute to the activation of DUBA by phosphorylation. Moreover, our data suggest that transient unfolding of helix α6 drives the global conformational process and that mutations can be introduced to modulate this process, which provides a basis for future studies to define the exact functional roles of motions in DUBA activation and substrate specificity.

Modulation of conformational equilibrium by phosphorylation underlies the activation of deubiquitinase A.

Deubiquitinases deconjugate ubiquitin modifications from target proteins and are involved in many cellular processes in eukaryotes. The functions of deubiquitinases are regulated by post-translational modifications, mainly phosphorylation and ubiquitination. Post-translational modifications can result in subtle changes in structural and dynamic properties, which are difficult to identify but functionally important. In this work, we used NMR spectroscopy to characterize the conformational properties of the human deubiquitinase A (DUBA), a negative regulator of type I interferon. DUBA activity is regulated by phosphorylation at a single serine residue, Ser-177. We found that the catalytic rate constant of DUBA is enhanced by phosphorylation. By comparing NMR and enzyme kinetics data among different forms of DUBA with low and high activities, we concluded that a two-state equilibrium that was present only in phosphorylated DUBA is important for DUBA activity. Our results highlight the importance of defining conformational dynamics in understanding the mechanism of DUBA activation.

Backbone 1H, 13C, and 15N resonance assignments of deubiquitinase A in non-phosphorylated and phosphorylated forms.

Ubiquitination is one of the most prevalent forms of post-translational modifications that are important for regulating many cellular processes in eukaryotes. Deubiquitinases are proteases that hydrolyze the isopeptide or peptide bonds formed between ubiquitin and the target proteins or within a polyubiquitin chain. Deubiquitinase A (DUBA) is a deubiquitinase known to be a negative regulator of innate immune responses in humans by suppressing production of type I interferons (INF-I). Excess INF-I production has been associated with autoimmune diseases. Phosphorylation of a single serine residue at position 177 is essential for the protease activity of DUBA. The structural and mechanistic basis of DUBA activation by phosphorylation and substrate specificity is not well understood. Here, we report the backbone resonance assignments of the isoform 2 of DUBA in both non-phosphorylated and phosphorylated forms. The reported assignments form the basis for future NMR studies on the structural and dynamical properties of both active and inactive forms of DUBA.

Role of methionine 71 in substrate recognition and structural integrity of bacterial peptidyl-tRNA hydrolase.

BACKGROUND: Bacterial peptidyl-tRNA hydrolase (Pth) is an essential enzyme that alleviates tRNA starvation by recycling prematurely dissociated peptidyl-tRNAs. The specificity of Pth for N-blocked-aminoacyl-tRNA has been proposed to be contingent upon conserved residue N14 forming a hydrogen bond with the carbonyl of the first peptide bond in the substrate. M71 is involved in forming a conserved hydrogen bond with N14. Other interactions facilitating this recognition are not known. METHODS: The structure, dynamics, and stability of the M71A mutant of Pth from Vibrio cholerae (VcPth) were characterized by X-ray crystallography, NMR spectroscopy, MD simulations and DSC. RESULTS: Crystal structure of M71A mutant was determined. In the structure, the dimer interface is formed by the insertion of six C-terminal residues of one molecule into the active site of another molecule. The side-chain amide of N14 was hydrogen bonded to the carbonyl of the last peptide bond formed between residues A196 and E197, and also to A71. The CSP profile of mutation was similar to that observed for the N14D mutant. M71A mutation lowered the thermal stability of the protein. CONCLUSION: Our results indicate that the interactions of M71 with N14 and H24 play an important role in optimal positioning of their side-chains relative to the peptidyl-tRNA substrate. Overall, these interactions of M71 are important for the activity, stability, and compactness of the protein. SIGNIFICANCE: The work presented provides original and new structural and dynamics information that significantly enhances our understanding of the network of interactions that govern this enzyme's activity and selectivity.

Unraveling the stereochemical and dynamic aspects of the catalytic site of bacterial peptidyl-tRNA hydrolase.

Bacterial peptidyl-tRNA hydrolase (Pth; EC 3.1.1.29) hydrolyzes the peptidyl-tRNAs accumulated in the cytoplasm and thereby prevents cell death by alleviating tRNA starvation. X-ray and NMR studies of Vibrio cholerae Pth (VcPth) and mutants of its key residues involved in catalysis show that the activity and selectivity of the protein depends on the stereochemistry and dynamics of residues H24, D97, N118, and N14. D97-H24 interaction is critical for activity because it increases the nucleophilicity of H24. The N118 and N14 have orthogonally competing interactions with H24, both of which reduce the nucleophilicity of H24 and are likely to be offset by positioning of a peptidyl-tRNA substrate. The region proximal to H24 and the lid region exhibit slow motions that may assist in accommodating the substrate. Helix α3 exhibits a slow wobble with intermediate time scale motions of its N-cap residue N118, which may work as a flypaper to position the scissile ester bond of the substrate. Overall, the dynamics of interactions between the side chains of N14, H24, D97, and N118, control the catalysis of substrate by this enzyme.

Structural characterization of peptidyl-tRNA hydrolase from Mycobacterium smegmatis by NMR spectroscopy.

BACKGROUND: Accumulation of toxic peptidyl-tRNAs in the bacterial cytoplasm is averted by the action of peptidyl-tRNA hydrolase (Pth), which cleaves peptidyl-tRNA into free tRNA and peptide. NMR studies are needed for a protein homolog with a complete crystal structure, for comparison with the NMR structure of Mycobacterium tuberculosis Pth. METHODS: The structure and dynamics of Mycobacterium smegmatis Pth (MsPth) were characterized by NMR spectroscopy and MD simulations. The thermal stability of MsPth was characterized by DSC. RESULTS: MsPth NMR structure has a central mixed seven stranded ß-sheet that is enclosed by six α-helices. NMR relaxation and MD simulations studies show that most of the ordered regions are rigid. Of the substrate binding segments, the gate loop is rigid, the base loop displays slow motions, while the lid loop displays fast timescale motions. MsPth displays high thermal stability characterized by a melting temperature of 61.71°C. CONCLUSION: The NMR structure of MsPth shares the canonical Pth fold with the NMR structure of MtPth. The motional characteristics for the lid region, the tip of helix α3, and the gate region, as indicated by MD simulations and NMR data, are similar for MsPth and MtPth. However, MsPth has relatively less rigid base loop and more compactly packed helices α5 and α6. The packing and the dynamic differences appear to be an important contributing factor to the thermal stability of MsPth, which is significantly higher than that of MtPth. SIGNIFICANCE: MsPth structure consolidates our understanding of the structure and dynamics of bacterial Pth proteins.

Solution structures and dynamics of ADF/cofilins UNC-60A and UNC-60B from Caenorhabditis elegans.

The nematode Caenorhabditis elegans has two ADF (actin-depolymerizing factor)/cofilin isoforms, UNC-60A and UNC-60B, which are expressed by the unc60 gene by alternative splicing. UNC-60A has higher activity to cause net depolymerization, and to inhibit polymerization, than UNC-60B. UNC-60B, on the other hand, shows much stronger severing activity than UNC-60A. To understand the structural basis of their functional differences, we have determined the solution structures of UNC-60A and UNC-60B proteins and characterized their backbone dynamics. Both UNC-60A and UNC-60B show a conserved ADF/cofilin fold. The G-actin (globular actin)-binding regions of the two proteins are structurally and dynamically conserved. Accordingly, UNC-60A and UNC-60B individually bind to rabbit muscle ADP-G-actin with high affinities, with Kd values of 32.25 nM and 8.62 nM respectively. The primary differences between these strong and weak severing proteins were observed in the orientation and dynamics of the F-actin (filamentous actin)-binding loop (F-loop). In the strong severing activity isoform UNC-60B, the orientation of the F-loop was towards the recently identified F-loop-binding region on F-actin, and the F-loop was relatively more flexible with 14 residues showing motions on a nanosecond-picosecond timescale. In contrast, in the weak severing protein isoform UNC-60A, the orientation of the F-loop was away from the F-loop-binding region and inclined towards its own C-terminal and strand ß6. It was also relatively less flexible with only five residues showing motions on a nanosecond-picosecond timescale. These differences in structure and dynamics seem to directly correlate with the differential F-actin site-binding and severing properties of UNC-60A and UNC-60B, and other related ADF/cofilin proteins.

NMR assignments of actin depolymerizing factor (ADF) like UNC-60A and cofilin like UNC-60B proteins of Caenorhabditis elegans.

The actin filament dynamics in nematode, Caenorhabditis elegans, is regulated by differential activity of two proteins UNC-60A and UNC-60B. UNC-60A exhibits strong pointed end depolymerization on C. elegans actin (Ce-actin), strong inhibition of polymerization, strong monomer sequestering activity, weak severing activity, and low affinity for F-actin binding, while UNC-60B exhibits strong pointed end depolymerization on rabbit muscle actin, strong severing activity, and high affinity for F-actin binding. Structural characterization of these proteins will help to understand (1) molecular mechanism of actin dynamics regulation and (2) the differential activity of these proteins. Here, we report (1)H, (13)C, and (15)N chemical shift assignments of these two proteins as determined by heteronuclear NMR experiments (at pH 6.5 and temperature 298 K).

Structural and docking studies of Leucaena leucocephala Cinnamoyl CoA reductase.

Lignin, a major constituent of plant call wall, is a phenolic heteropolymer. It plays a major role in the development of plants and their defense mechanism against pathogens. Therefore Lignin biosynthesis is one of the critical metabolic pathways. In lignin biosynthesis, the Cinnamoyl CoA reductase is a key enzyme which catalyzes the first step in the pathway. Cinnamoyl CoA reductase provides the substrates which represent the main transitional molecules of lignin biosynthesis pathway, exhibits a high in vitro kinetic preference for feruloyl CoA. In present study, the three-dimensional model of cinnamoyl CoA reductase was constructed based on the crystal structure of Grape Dihydroflavonol 4-Reductase. Furthermore, the docking studies were performed to understand the substrate interactions to the active site of CCR. It showed that residues ARG51, ASN52, ASP54 and ASN58 were involved in substrate binding. We also suggest that residue ARG51 in CCR is the determinant residue in competitive inhibition of other substrates. This structural and docking information have prospective implications to understand the mechanism of CCR enzymatic reaction with feruloyl CoA, however the approach will be applicable in prediction of substrates and engineering 3D structures of other enzymes as well.