Chengliang Wang

Structure of the JmjC-domain-containing protein JMJD5

The post-translational modification of histone tails is the principal process controlling epigenetic regulation in eukaryotes. The lysine methylation of histones is dynamically regulated by two distinct classes of enzymes: methyltransferases and demethylases. JMJD5, which plays an important role in cell-cycle progression, circadian rhythms and embryonic cell proliferation, has been shown to be a JmjC-domain-containing histone demethylase with enzymatic activity towards H3K36me2. Here, the crystal structure of human JMJD5 lacking the N-terminal 175 amino-acid residues is reported. The structure showed that the Gln275, Trp310 and Trp414 side chains might block the insertion of methylated lysine into the active centre of JMJD5, suppressing the histone demethylase activity of the truncated JMJD5 construct. A comparison of the structure of JMJD5 with that of FIH, a well characterized protein hydroxylase, revealed that human JMJD5 might function as a protein hydroxylase. The interaction between JMJD5 and the core histone octamer proteins indicated that the histone proteins could be potential substrates for JMJD5.

Crystal structure of Sa239 reveals the structural basis for the activation of ribokinase by monovalent cations.

Ribokinase is responsible for catalyzing the reaction of d-ribose and ATP to produce ribose-5-phosphate and ADP, which can be activated by monovalent cations such as potassium, cesium and ammonium. However, the exact activation mechanism of ribokinase remains elusive. Here we report the crystal structure of Sa239, a ribokinase from Staphylococcus aureus, in the absence of monovalent ions. In addition to the dimer form similar to that observed in Escherichia coli ribokinase structure, the structure of Sa239 demonstrates that the C-terminal tail protrudes from the remaining part and interacts with the neighboring molecule, resulting in an unexpected dimerization form. By comparing the structure of Sa239 to E. coli ribokinase, we propose that binding of the monovalent cation triggers the conformational change of the large ATP loop to organize the formation of nucleotide binding pocket, thus enabling ATP binding and enhancing catalytic activity. Our study uncovers the detailed structural basis for the activation mechanism of ribokinase by monovalent cations.

Structural basis for the substrate selectivity of PvuRts1I, a 5-hydroxymethylcytosine DNA restriction endonuclease

The crystal structure of PvuRts1I was determined and a 5-hydroxymethylcytosine-binding pocket was identified in the SRA-like domain. Enzyme variants were engineered to assist in hydroxymethylome mapping based on the crystal structure of PvuRts1I.

Structural basis for role of ring finger protein RNF168 RING domain

Ubiquitin adducts surrounding DNA double-strand breaks (DSBs) have emerged as molecular platforms important for the assembly of DNA damage mediator and repair proteins. Central to these chromatin modifications lies the E2 UBC13, which has been implicated in a bipartite role in priming and amplifying lys63-linked ubiquitin chains on histone molecules through coupling with the E3 RNF8 and RNF168. However, unlike the RNF8-UBC13 holoenyzme, exactly how RNF168 work in concert with UBC13 remains obscure. To provide a structural perspective for the RNF168-UBC13 complex, we solved the crystal structure of the RNF168 RING domain. Interestingly, while the RNF168 RING adopts a typical RING finger fold with two zinc ions coordinated by several conserved cystine and histine residues arranged in a C3HC4 “cross-brace” manner, structural superimposition of RNF168 RING with other UBC13-binding E3 ubiquitin ligases revealed substantial differences at its corresponding UBC13-binding interface. Consistently, and in stark contrast to that between RNF8 and UBC13, RNF168 did not stably associate with UBC13 in vitro or in vivo. Moreover, domain-swapping experiments indicated that the RNF8 and RNF168 RING domains are not functionally interchangeable. We propose that RNF8 and RNF168 operate in different modes with their cognate E2 UBC13 at DSBs.

Octameric structure of Staphylococcus aureus enolase in complex with phosphoenolpyruvate

Non-ligand-bound and PEP-bound structures of S. aureus enolase (Sa_enolase) were solved, and catalytic loop 1 in the PEP-bound structure was found to show both ‘open’ and ‘closed’ conformations. Structural and biochemical results indicate that octamerization is required for substrate binding and catalysis by Sa_enolase.

Structure of the JmjC domain-containing protein NO66 complexed with ribosomal protein Rpl8

The structure of the complex of NO66 and Rpl8 was solved in the native state and NO66 recognizes the consensus motif NHXH . Tetramerization is required for efficient substrate binding and catalysis by NO66.

Structure insights into the molecular mechanism of the interaction between UHRF2 and PCNA.

UHRF2 (Ubiquitin-like with PHD and ring finger domains 2) is an E3 ubiquitin ligase that plays important roles in DNA methylation, histone modifications and cell cycle regulation by interacting with multiple epigenetic or cell-cycle related proteins. Previous studied have identified PCNA (Proliferating cell nuclear antigen) as an interacting partner of UHRF2 by using the antibody microarray. However, the molecular mechanism and the function of UHRF2-PCNA interaction remains unclear. Here, we report the complex structure of PCNA and the peptide (784NEILQTLLDLFFPGYSK800) derived from UHRF2 that contains a PIP box. Structural analysis combined with mutagenesis experiments provide the molecular basis for the recognition of UHRF2 by PCNA via PIP-box.

Phosphorylation of CENP-C by Aurora B facilitates kinetochore attachment error correction in mitosis

Significance Kinetochores are large protein networks located on centromeres that mediate chromosome segregation during mitosis and maintain genomic stability. Mis12 complex (Mis12C) functions as a scaffold that targets Ndc80 and Knl1 complexes to the centromere by associating with CENP-C. Here, we provide insights into the molecular mechanism underlying the CENP-C–dependent kinetochore recruitment of Mis12C, which is negatively regulated by Aurora B-dependent CENP-C phosphorylation. Replacement of Schizosaccharomyces pombe Cnp3 with a phosphorylation-mimicking mutant, Cnp3T28E, results in defective chromosome segregation caused by improper kinetochore assembly. These findings indicate that Aurora B-dependent phosphorylation of CENP-C plays a role in interrupting the connection between the inner and outer kinetochore and is thus involved in the error correction/spindle assembly checkpoint pathway to prevent chromosome missegregation during mitosis. Kinetochores are superprotein complexes that orchestrate chromosome segregation via a dynamic interaction with spindle microtubules. A physical connection between CENP-C and the Mis12–Ndc80–Knl1 (KMN) protein network is an important pathway that is used to assemble kinetochores on CENP-A nucleosomes. Multiple outer kinetochore components are phosphorylated by Aurora B kinase to activate the spindle assembly checkpoint (SAC) and to ensure accurate chromosome segregation. However, it is unknown whether Aurora B can phosphorylate inner kinetochore components to facilitate proper mitotic chromosome segregation. Here, we reported the structure of the fission yeast Schizosaccharomyces pombe Mis12–Nnf1 complex and showed that N-terminal residues 26–50 in Cnp3 (the CENP-C homolog of S. pombe) are responsible for interacting with the Mis12 complex. Interestingly, Thr28 of Cnp3 is a substrate of Ark1 (the Aurora B homolog of S. pombe), and phosphorylation impairs the interaction between the Cnp3 and Mis12 complex. The expression of a phosphorylation-mimicking Cnp3 mutant results in defective chromosome segregation due to improper kinetochore assembly. These results establish a previously uncharacterized regulatory mechanism involved in CENP-C–Mis12-facilitated kinetochore attachment error correction to ensure accurate chromosome segregation during mitosis.

Structural Insights into the Regulation of Staphylococcus aureus Phosphofructokinase by Tetramer–Dimer Conversion

Most reported bacterial phosphofructokinases (Pfks) are tetramers that exhibit activity allosterically regulated via conformational changes between the R and T states. We report that the Pfk from Staphylococcus aureus NCTC 8325 ( SaPfk) exists as both an active tetramer and an inactive dimer in solution. Multiple effectors, including pH, ADP, ATP, and adenylyl-imidodiphosphate (AMP-PNP), cause equilibrium shifts from the tetramer to dimer, whereas the substrate F6P stabilizes SaPfk tetrameric assembly. Crystal structures of SaPfk in complex with different ligands and biochemical analysis reveal that the flexibility of the Gly150-Leu151 motif in helix α7 plays a role in tetramer-dimer conversion. Thus, we propose a molecular mechanism for allosteric regulation of bacterial Pfk via conversion between the tetramer and dimer in addition to the well-characterized R-state/T-state mechanism.

Enolase binds to RnpA in competition with PNPase in Staphylococcus aureus

The RNA degradosome of the pathogen Staphylococcus aureus regulates the metabolism of RNA, the expression of virulence factors, and the formation of biofilms. It is composed of the RNases J1/J2, RNase Y, CshA, PNPase, Enolase, Pfk, and a newly identified component, RnpA. However, the function and new partners of RnpA in RNA degradosome remain unknown. Here, we identified PNPase and Enolase as two novel partners for RnpA. Further studies revealed that Enolase interacts with RnpA in competition with PNPase. Enzymatic assays showed that RnpA increases Enolase activity but has no effect on PNPase. These findings provide more information about the functional relationship between RnpA and RNA degradosome.