1H, 13C and 15N resonance assignment of the YTH domain of YTHDC2.

In humans, YTH (YT521-B homology) domain containing protein 2 (YTHDC2) plays a crucial role in the phase-shift from mitosis to meiosis. YTH domains bind to methylated adenosine nucleotides such as m6A. In a phylogenic tree, the YTH domain of YTHDC2 (YTH2) and that of the YTH containing protein YTHDC1 (YTH1) belong to the same sub-group. However, the binding affinity of m6A differs between these proteins. Here, we report 1H, 13C and 15N resonance assignment of YTH2 and its solution structure to examine the difference of the structural architecture and the dynamic properties of YTH1 and YTH2. YTH2 adopts a ß1-α1-ß2-α2-ß3-ß4-ß5-α3-ß6-α4 topology, which was also observed in YTH1. However, the ß4-ß5 loops of YTH1 and YTH2 are distinct in length and amino acid composition. Our data revealed that, unlike in YTH1, the structure of m6A-binding pocket of YTH2 formed by the ß4-ß5 loop is stabilized by electrostatic interaction. This assignment and the structural information for YTH2 will provide the insight on the further functional research of YTHDC2.

Chemical and structural studies provide a mechanistic basis for recognition of the MYC G-quadruplex.

G-quadruplexes (G4s) are noncanonical DNA structures that frequently occur in the promoter regions of oncogenes, such as MYC, and regulate gene expression. Although G4s are attractive therapeutic targets, ligands capable of discriminating between different G4 structures are rare. Here, we describe DC-34, a small molecule that potently downregulates MYC transcription in cancer cells by a G4-dependent mechanism. Inhibition by DC-34 is significantly greater for MYC than other G4-driven genes. We use chemical, biophysical, biological, and structural studies to demonstrate a molecular rationale for the recognition of the MYC G4. We solve the structure of the MYC G4 in complex with DC-34 by NMR spectroscopy and illustrate specific contacts responsible for affinity and selectivity. Modification of DC-34 reveals features required for G4 affinity, biological activity, and validates the derived NMR structure. This work advances the design of quadruplex-interacting small molecules to control gene expression in therapeutic areas such as cancer.

Myosin VI Contains a Compact Structural Motif that Binds to Ubiquitin Chains.

Myosin VI is critical for cargo trafficking and sorting during early endocytosis and autophagosome maturation, and abnormalities in these processes are linked to cancers, neurodegeneration, deafness, and hypertropic cardiomyopathy. We identify a structured domain in myosin VI, myosin VI ubiquitin-binding domain (MyUb), that binds to ubiquitin chains, especially those linked via K63, K11, and K29. Herein, we solve the solution structure of MyUb and MyUb:K63-linked diubiquitin. MyUb folds as a compact helix-turn-helix-like motif and nestles between the ubiquitins of K63-linked diubiquitin, interacting with distinct surfaces of each. A nine-amino-acid extension at the C-terminal helix (Helix2) of MyUb is required for myosin VI interaction with endocytic and autophagic adaptors. Structure-guided mutations revealed that a functional MyUb is necessary for optineurin interaction. In addition, we found that an isoform-specific helix restricts MyUb binding to ubiquitin chains. This work provides fundamental insights into myosin VI interaction with ubiquitinated cargo and functional adaptors.

Diverse functions of myosin VI elucidated by an isoform-specific α-helix domain.

Myosin VI functions in endocytosis and cell motility. Alternative splicing of myosin VI mRNA generates two distinct isoform types, myosin VI(short) and myosin VI(long), which differ in the C-terminal region. Their physiological and pathological roles remain unknown. Here we identified an isoform-specific regulatory helix, named the α2-linker, that defines specific conformations and hence determines the target selectivity of human myosin VI. The presence of the α2-linker structurally defines a new clathrin-binding domain that is unique to myosin VI(long) and masks the known RRL interaction motif. This finding is relevant to ovarian cancer, in which alternative myosin VI splicing is aberrantly regulated, and exon skipping dictates cell addiction to myosin VI(short) in tumor-cell migration. The RRL interactor optineurin contributes to this process by selectively binding myosin VI(short). Thus, the α2-linker acts like a molecular switch that assigns myosin VI to distinct endocytic (myosin VI(long)) or migratory (myosin VI(short)) functional roles.

Dual-site interactions of p53 protein transactivation domain with anti-apoptotic Bcl-2 family proteins reveal a highly convergent mechanism of divergent p53 pathways.

Molecular interactions between the tumor suppressor p53 and the anti-apoptotic Bcl-2 family proteins play an important role in the transcription-independent apoptosis of p53. The p53 transactivation domain (p53TAD) contains two conserved ΦXXΦΦ motifs (Φ indicates a bulky hydrophobic residue and X is any other residue) referred to as p53TAD1 (residues 15-29) and p53TAD2 (residues 39-57). We previously showed that p53TAD1 can act as a binding motif for anti-apoptotic Bcl-2 family proteins. In this study, we have identified p53TAD2 as a binding motif for anti-apoptotic Bcl-2 family proteins by using NMR spectroscopy, and we calculated the structures of Bcl-X(L)/Bcl-2 in complex with the p53TAD2 peptide. NMR chemical shift perturbation data showed that p53TAD2 peptide binds to diverse members of the anti-apoptotic Bcl-2 family independently of p53TAD1, and the binding between p53TAD2 and p53TAD1 to Bcl-X(L) is competitive. Refined structural models of the Bcl-X(L)·p53TAD2 and Bcl-2·p53TAD2 complexes showed that the binding sites occupied by p53TAD2 in Bcl-X(L) and Bcl-2 overlap well with those occupied by pro-apoptotic BH3 peptides. Taken together with the mutagenesis, isothermal titration calorimetry, and paramagnetic relaxation enhancement data, our structural comparisons provided the structural basis of p53TAD2-mediated interaction with the anti-apoptotic proteins, revealing that Bcl-X(L)/Bcl-2, MDM2, and cAMP-response element-binding protein-binding protein/p300 share highly similar modes of binding to the dual p53TAD motifs, p53TAD1 and p53TAD2. In conclusion, our results suggest that the dual-site interaction of p53TAD is a highly conserved mechanism underlying target protein binding in the transcription-dependent and transcription-independent apoptotic pathways of p53.

Structural insight into the interaction of ADP-ribose with the PARP WWE domains.

The WWE domain is often identified in proteins associated with ubiquitination or poly-ADP-ribosylation. Structural information about WWE domains has been obtained for the ubiquitination-related proteins, such as Deltex and RNF146, but not yet for the poly-ADP-ribose polymerases (PARPs). Here we determined the solution structures of the WWE domains from PARP11 and PARP14, and compared them with that of the RNF146 WWE domain. NMR perturbation experiments revealed the specific differences in their ADP-ribose recognition modes that correlated with their individual biological activities. The present structural information sheds light on the ADP-ribose recognition modes by the PARP WWE domains.

Structural insights into the dual-targeting mechanism of Nutlin-3.

Multi-targeting therapy is an emerging strategy of drug discovery to improve therapeutic efficacy, safety and resistance profiles. In this study, we monitored the binding of a potent MDM2 inhibitor Nutlin-3 with anti-apoptotic Bcl-2 family proteins using NMR spectroscopy. Our results showed the universal binding of Nutlin-3 with diverse anti-apoptotic Bcl-2 family proteins. Taken together with the binding data for Nutlin-3 analogs, the structural model of the Bcl-X(L)/Nutlin-3 complex showed that the binding mode of Nutlin-3 resembles that of the Bcl-X(L)/Bcl-2 inhibitors, suggesting the molecular mechanism of transcription-independent mitochondrial apoptosis by Nutlin-3. Finally, our structural comparison provides structural insights into the dual-targeting mechanism of how Nutlin-3 can bind to two different target proteins, MDM2 and anti-apoptotic Bcl-2 family proteins in a similar manner.

Solution structure of the cysteine-rich domain in Fn14, a member of the tumor necrosis factor receptor superfamily.

Fn14 is the smallest member of the tumor necrosis factor (TNF) receptor superfamily, and specifically binds to its ligand, TWEAK (TNF-like weak inducer of apoptosis), which is a member of the TNF superfamily. The receptor-ligand recognition between Fn14 and TWEAK induces a variety of cellular processes for tissue remodeling and is also involved in the pathogenesis of some human diseases, such as cancer, chronic autoimmune diseases, and acute ischaemic stroke. The extracellular ligand-binding region of Fn14 is composed of 53 amino acid residues and forms a single, cysteine-rich domain (CRD). In this study, we determined the solution structure of the Fn14 CRD (Glu28-Ala70) by heteronuclear NMR, with a (13)C-/(15)N-labeled sample. The tertiary structure of the CRD comprises a beta-sheet with two strands, followed by a 3(10) helix and a C-terminal alpha-helix, and is stabilized by three disulfide bonds connecting Cys36-Cys49, Cys52-Cys67, and Cys55-Cys64. Comparison of the disulfide bond connectivities and the tertiary structures with those of other CRDs revealed that the Fn14 CRD is similar to the fourth CRD of TNF receptor 1 (A1-C2 module type), but not to the CRD of B-cell maturation antigen and the second CRD of transmembrane activator and CAML (calcium modulator and cyclophilin ligand) interactor (A1-D2 module type). This is the first structural report about the A1-C2 type CRD that could bind to the known target.

Synaptic PDZ domain-mediated protein interactions are disrupted by inhalational anesthetics.

Anesthetics exert multiple effects on the central nervous system through altering synaptic transmission, but the mechanisms for this process are poorly understood. PDZ domain-mediated protein interactions play a central role in organizing signaling complexes around synaptic receptors for efficient signal transduction. We report here that clinically relevant concentrations of inhalational anesthetics dose-dependently and specifically inhibit the PDZ domain-mediated protein interaction between PSD-95 or PSD-93 and the N-methyl-d-aspartate receptor or neuronal nitric-oxide synthase. These inhibitory effects are immediate, potent, and reversible and occur at a hydrophobic peptide-binding groove on the surface of the second PDZ domain of PSD-95 in a manner relevant to anesthetic action. These findings reveal the PDZ domain as a new molecular target for inhalational anesthetics.