Changdong Liu

Characterization and Structure Determination of the Cdt1 Binding Domain of Human Minichromosome Maintenance (Mcm) 6

The minichromosome maintenance (Mcm) 2–7 complex is the replicative helicase in eukaryotic species, and it plays essential roles in the initiation and elongation phases of DNA replication. During late M and early G1, the Mcm2–7 complex is loaded onto chromatin to form prereplicative complex in a Cdt1-dependent manner. However, the detailed molecular mechanism of this loading process is still elusive. In this study, we demonstrate that the previously uncharacterized C-terminal domain of human Mcm6 is the Cdt1 binding domain (CBD) and present its high resolution NMR structure. The structure of CBD exhibits a typical “winged helix” fold that is generally involved in protein-nucleic acid interaction. Nevertheless, the CBD failed to interact with DNA in our studies, indicating that it is specific for protein-protein interaction. The CBD-Cdt1 interaction involves the helix-turn-helix motif of CBD. The results reported here provide insight into the molecular mechanism of Mcm2–7 chromatin loading and prereplicative complex assembly.

SET8 recognizes the sequence RHRK20VLRDN within the N terminus of histone H4 and mono-methylates lysine 20.

Methylation of lysine 20 in histone H4 has been proven to play important roles in chromatin structure and gene regulation. SET8 is one of the methyltransferases identified to be specific for this modification. In this study, the minimal active SET domain of SET8 has been mapped to the region of amino acids 195–352. This region completely retains the same methylation activity and substrate specificity as the full-length SET8. The SET domain recognizes a stretch of specific amino acid sequence around lysine 20 of H4 for its methylation activity. Methylation assays with N terminus mutants of H4 that contain deletions and single alanine or glutamine substitutions of charged residues revealed that SET8 requires the sequence RHRK20VLRDN for methylation at lysine 20. The individual mutation of any charged residue in this sequence to alanine or glutamine abolished or greatly decreased levels of methylation of lysine 20 of H4 by SET8. Interestingly, mutation of lysine 16 to alanine, arginine, glutamine, or methionine did not affect methylation of lysine 20 by the SET domain. Mass spectrometric analysis of synthesized H4 N-terminal peptides modified by SET8 showed that SET8 selectively mono-methylates lysine 20 of H4. Taken together, our results suggested that the coordination between the amino acid sequence RHRK20VLRDN and the SET domain of SET8 determines the substrate specificity and multiplicity of methylation of lysine 20 of H4.

Structural insights into the Cdt1-mediated MCM2–7 chromatin loading

Initiation of DNA replication in eukaryotes is exquisitely regulated to ensure that DNA replication occurs exactly once in each cell division. A conserved and essential step for the initiation of eukaryotic DNA replication is the loading of the mini-chromosome maintenance 2–7 (MCM2–7) helicase onto chromatin at replication origins by Cdt1. To elucidate the molecular mechanism of this event, we determined the structure of the human Cdt1-Mcm6 binding domains, the Cdt1(410–440)/MCM6(708–821) complex by NMR. Our structural and site-directed mutagenesis studies showed that charge complementarity is a key determinant for the specific interaction between Cdt1 and Mcm2–7. When this interaction was interrupted by alanine substitutions of the conserved interacting residues, the corresponding yeast Cdt1 and Mcm6 mutants were defective in DNA replication and the chromatin loading of Mcm2, resulting in cell death. Having shown that Cdt1 and Mcm6 interact through their C-termini, and knowing that Cdt1 is tethered to Orc6 during the loading of MCM2–7, our results suggest that the MCM2–7 hexamer is loaded with its C terminal end facing the ORC complex. These results provide a structural basis for the Cdt1-mediated MCM2–7 chromatin loading.

Topology of a G-quadruplex DNA formed by C9orf72 hexanucleotide repeats associated with ALS and FTD.

Abnormal expansions of an intronic hexanucleotide GGGGCC (G4C2) repeat of the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Previous studies suggested that the C9orf72 hexanucleotide repeat expansion (HRE), either as DNA or the transcribed RNA, can fold into G-quadruplexes with distinct structures. These structural polymorphisms lead to abortive transcripts and contribute to the pathogenesis of ALS and FTD. Using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, we analyzed the structures of C9orf72 HRE DNA with various G4C2 repeats. They exhibited diverse G-quadruplex folds in potassium ions. Furthermore, we determined the topology of a G-quadruplex formed by d(G4C2)4. It favors a monomeric fold and forms a chair-type G-quadruplex with a four-layer antiparallel G-tetra core and three edgewise loops, which is distinct from known structures of chair-type G-quadruplexes. Our findings highlight the conformational heterogeneity of C9orf72 HRE DNA, and may lay the necessary structural basis for designing small molecules for the modulation of ALS/FTD pathogenesis.

Structural basis for homeodomain recognition by the cell-cycle regulator Geminin

Homeodomain-containing transcription factors play a fundamental role in the regulation of numerous developmental and cellular processes. Their multiple regulatory functions are accomplished through context-dependent inputs of target DNA sequences and collaborating protein partners. Previous studies have well established the sequence-specific DNA binding to homeodomains; however, little is known about how protein partners regulate their functions through targeting homeodomains. Here we report the solution structure of the Hox homeodomain in complex with the cell-cycle regulator, Geminin, which inhibits Hox transcriptional activity and enrolls Hox in cell proliferative control. Side-chain carboxylates of glutamates and aspartates in the C terminus of Geminin generate an overall charge pattern resembling the DNA phosphate backbone. These residues provide electrostatic interactions with homeodomain, which combine with the van der Waals contacts to form the stereospecific complex. We further showed that the interaction with Geminin is homeodomain subclass-selective and Hox paralog-specific, which relies on the stapling role of residues R43 and M54 in helix III and the basic amino acid cluster in the N terminus. Interestingly, we found that the C-terminal residue Ser184 of Geminin could be phosphorylated by Casein kinase II, resulting in the enhanced binding to Hox and more potent inhibitory effect on Hox transcriptional activity, indicating an additional layer of regulation. This structure provides insight into the molecular mechanism underlying homeodomain-protein recognition and may serve as a paradigm for interactions between homeodomains and DNA-competitive peptide inhibitors.

Molecular Basis of the General Base Catalysis of an α/β-hydrolase Catalytic Triad

Background: The α/β-hydrolase MenH uses its Ser-His-Asp triad as a specific general basis with a poorly understood mechanism. Results: An open-closed conformational change is identified in the enzyme catalysis. Conclusion: The conformational change controls the formation and reactivity of the catalytic triad. Significance: The catalytic versatility of the Ser-His-Asp triad is expanded by the open-closed structural change. The serine-histidine-aspartate triad is well known for its covalent, nucleophilic catalysis in a diverse array of enzymatic transformations. Here we show that its nucleophilicity is shielded and its catalytic role is limited to being a specific general base by an open-closed conformational change in the catalysis of (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase (or MenH), a typical α/β-hydrolase fold enzyme in the vitamin K biosynthetic pathway. This enzyme is found to adopt an open conformation without a functional triad in its ligand-free form and a closed conformation with a fully functional catalytic triad in the presence of its reaction product. The open-to-closed conformational transition involves movement of half of the α-helical cap domain, which causes extensive structural changes in the α/β-domain and forces the side chain of the triad histidine to adopt an energetically disfavored gauche conformation to form the functional triad. NMR analysis shows that the inactive open conformation without a triad prevails in ligand-free solution and is converted to the closed conformation with a properly formed triad by the reaction product. Mutation of the residues crucial to this open-closed transition either greatly decreases or completely eliminates the enzyme activity, supporting an important catalytic role for the structural change. These findings suggest that the open-closed conformational change tightly couples formation of the catalytic triad to substrate binding to enhance the substrate specificities and simultaneously shield the nucleophilicity of the triad, thus allowing it to expand its catalytic power beyond the nucleophilic catalysis.

Solution Structure of the Phosphotyrosine Binding (PTB) Domain of Human Tensin2 Protein in Complex with Deleted in Liver Cancer 1 (DLC1) Peptide Reveals a Novel Peptide Binding Mode

Background: DLC1 interacts with tensin2 PTB, playing a role as a tumor suppressor in many human cancers. Results: We solved the tensin2 PTB-DLC1 complex structure and showed its importance for co-localization of DLC1 and tensin2 in cells. Conclusion: The novel PTB-peptide binding mode provides a molecular basis for understanding the tumor suppression of DLC1 and tensin2. Significance: A novel PTB-peptide binding mode was observed. The protein deleted in liver cancer 1 (DLC1) interacts with the tensin family of focal adhesion proteins to play a role as a tumor suppressor in a wide spectrum of human cancers. This interaction has been proven to be crucial to the oncogenic inhibitory capacity and focal adhesion localization of DLC1. The phosphotyrosine binding (PTB) domain of tensin2 predominantly interacts with a novel site on DLC1, not the canonical NPXY motif. In this study, we characterized this interaction biochemically and determined the complex structure of tensin2 PTB domain with DLC1 peptide by NMR spectroscopy. Our HADDOCK-derived complex structure model elucidates the molecular mechanism by which tensin2 PTB domain recognizes DLC1 peptide and reveals a PTB-peptide binding mode that is unique in that peptide occupies the binding site opposite to the canonical NPXY motif interaction site with the peptide utilizing a non-canonical binding motif to bind in an extended conformation and that the N-terminal helix, which is unique to some Shc- and Dab-like PTB domains, is required for binding. Mutations of crucial residues defined for the PTB-DLC1 interaction affected the co-localization of DLC1 and tensin2 in cells and abolished DLC1-mediated growth suppression of hepatocellular carcinoma cells. This tensin2 PTB-DLC1 peptide complex with a novel binding mode extends the versatile binding repertoire of the PTB domains in mediating diverse cellular signaling pathways as well as provides a molecular and structural basis for better understanding the tumor-suppressive activity of DLC1 and tensin2.

1H, 15N and 13C chemical shift assignments of the SH2 domain of human tensin2 (TENC1)

Tensin is an important cytoplasmic phosphoprotein localized to integrin-mediated focal adhesion. It links actin cytoskeleton to extracellular matrix through its N-terminal actin-binding domain and C-terminal phosphotyrosine-binding domain. Studies of knockout mice revealed the critical roles of tensin in skeletal muscle regeneration, renal function and regulation of cell migration. The SH2 domain of tensin interacts with various tyrosine-phosphorylated proteins thus functions as a platform for dis/assembly of signaling molecules. It has also been implicated in recruiting a tumor supperssor protein DLC1 (deleted in live cancer 1) to the focal adhesion, which is required for oncogenic inhibition effect of DLC1 in a phosphotyrosine-independent manner. Here, we report complete chemical shift assignments of the SH2 domain of human tensin2 determined by triple resonance experiments. The resonance assignments serve as a basis for our further functional studies and structure determination by NMR spectroscopy. (BMRB deposits with accession number 16472).

Development of stapled helical peptides to perturb the Cdt1–Mcm6 interaction†

Six all‐hydrocarbon‐stapled Cdt1 MBD‐derived peptides have been designed and synthesized to perturb the Cdt1–Mcm6 interaction, which is involved in DNA replication. Inconsistency between the helicity of the obtained peptidomimetics and their binding affinity has been observed. The helicity of 13‐amino acid stapled peptides increased, while their binding to Mcm6 was decreased. On the other hand, the 30‐amino acid stapled peptides exhibited decreased helicity but increased binding affinity. Copyright

Characterizations of distinct parallel and antiparallel G-quadruplexes formed by two-repeat ALS and FTD related GGGGCC sequence

The large expansion of GGGGCC (G4C2) repeats of the C9orf72 gene have been found to lead to the pathogenesis of devastating neurological diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The structural polymorphisms of C9orf72 HRE DNA and RNA may cause aberrant transcription and contribute to the development of ALS and FTD. Here we showed that the two-repeat G4C2 DNA, d(G4C2)2, simultaneously formed parallel and antiparallel G-quadruplex conformations in the potassium solution. We separated different folds of d(G4C2)2 by anion exchange chromatography, followed with characterizations by circular dichroism and nuclear magnetic resonance spectroscopy. The parallel d(G4C2)2 G-quadruplex folded as a symmetric tetramer, while the antiparallel d(G4C2)2 adopted the topology of an asymmetric dimer. These folds are distinct from the antiparallel chair-type conformation we previously identified for the d(G4C2)4 G-quadruplex. Our findings have demonstrated the conformational heterogeneity of the C9orf72 HRE DNA, and provided new insights into the d(G4C2)n folding. Meanwhile, the purified d(G4C2)2 G-quadruplex samples are suitable for further three-dimensional structure characterizations, which are required for the structure-based design of small molecules targeting ALS and FTD related C9orf72 HRE.