Xiaoming Tu

Pup, a prokaryotic ubiquitin-like protein, is an intrinsically disordered protein.

Pup (prokaryotic ubiquitin-like protein) from Mycobacterium tuberculosis is the first ubiquitin-like protein identified in non-eukaryotic cells. Although different ubiquitin-like proteins from eukaryotes share low sequence similarity, their 3D (three-dimensional) structures exhibit highly conserved typical ubiquitin-like folds. Interestingly, our studies reveal that Pup not only shares low sequence similarity, but also presents a totally distinguished structure compared with other ubiquitin-like superfamily proteins. Diverse structure predictions combined with CD and NMR spectroscopic studies all demonstrate that Pup is an intrinsically disordered protein. Moreover, 1H-15N NOE (nuclear Overhauser effect) data and CSI (chemical shift index) analyses indicate that there is a residual secondary structure at the C-terminus of Pup. In M. tuberculosis, Mpa (mycobacterium proteasomal ATPase) is the regulatory cap ATPase of the proteasome that interacts with Pup and brings the substrates to the proteasome for degradation. In the present paper, SPR (surface plasmon resonance) and NMR perturbation studies imply that the C-terminus of Pup, ranging from residues 30 to 59, binds to Mpa probably through a hydrophobic interface. In addition, phylogenetic analysis clearly shows that the Pup family belongs to a unique and divergent evolutionary branch, suggesting that it is the most ancient and deeply branched family among ubiquitin-like proteins. This might explain the structural distinction between Pup and other ubiquitin-like superfamily proteins.

An aurora kinase homologue is involved in regulating both mitosis and cytokinesis in Trypanosoma brucei

The chromosomal passenger protein aurora kinases have been implicated in regulating chromosome segregation and cell division. Three aurora kinase homologues were identified (TbAUK1, -2 and -3) in the Trypanosome Genomic Data Base, and their expressions in the procyclic form of Trypanosoma brucei were knocked down individually by using the RNA interference technique. Only a knockdown of TbAUK1 arrested the cells in G2/M phase with each cell showing an extended posterior end, two kinetoplasts, and an enlarged nucleus, apparently the result of an inhibited kinetoplast multiplication and a failed mitosis. There is no mitotic spindle structure in the TbAUK1-depleted cell. The two kinetoplasts moved apart from each other but stopped just before cytokinesis, suggesting that cytokinesis was blocked in its early phase. Overexpression of TbAUK1 in the cells resulted in little change in cell growth. By immunofluorescence, TbAUK1 was primarily localized to the nucleus in interphase and to the mitotic spindle during apparent metaphase and anaphase. Thus, differing from other eukaryotes, TbAUK1 has an apparent triple function in coupling mitosis and kinetoplast replication with cytokinesis in T. brucei. T. brucei polo-like kinase, previously identified as the initiator of cytokinesis without apparent involvement in mitosis in the trypanosome, was either depleted or overexpressed in the TbAUK1-deficient cells. A dominant TbAUK1-depleted phenotype was demonstrated in both cases, suggesting that TbAUK1 plays an essential role in cytokinesis that cannot be affected by changes in the level of T. brucei polo-like kinase. To our knowledge, this is the first time that the function of an aurora B-like kinase is a prerequisite for polo-like kinase action in initiating cytokinesis. TbAUK1 is also the first identified protein that couples both mitosis and kinetoplast replication with cytokinesis in the trypanosome.

Crystal Structure of the Human SUV39H1 Chromodomain and Its Recognition of Histone H3K9me2/3.

SUV39H1, the first identified histone lysine methyltransferase in human, is involved in chromatin modification and gene regulation. SUV39H1 contains a chromodomain in its N-terminus, which potentially plays a role in methyl-lysine recognition and SUV39H1 targeting. In this study, the structure of the chromodomain of human SUV39H1 was determined by X-ray crystallography. The SUV39H1 chromodomain displays a generally conserved structure fold compared with other solved chromodomains. However, different from other chromodomains, the SUV39H1 chromodomain possesses a much longer helix at its C-terminus. Furthermore, the SUV39H1 chromodomain was shown to recognize histone H3K9me2/3 specifically.

Solution structure of the Pdp1 PWWP domain reveals its unique binding sites for methylated H4K20 and DNA

Methylation of H4K20 (Lys(20) of histone H4) plays an important role in the regulation of diverse cellular processes. In fission yeast, all three states of H4K20 methylation are catalysed by Set9. Pdp1 is a PWWP (proline-tryptophan-tryptophan-proline) domain-containing protein, which associates with Set9 to regulate its chromatin localization and methyltransferase activity towards H4K20. The structure of the Pdp1 PWWP domain, which is the first PWWP domain identified which binds to methyl-lysine at the H4K20 site, was determined in the present study by solution NMR. The Pdp1 PWWP domain adopts a classical PWWP fold, with a five-strand antiparallel β-barrel followed by three α-helices. However, it differs significantly from other PWWP domains in some structural aspects that account, in part, for its molecular recognition. Moreover, we revealed a unique binding pattern of the PWWP domain, in that the PWWP domain of Pdp1 bound not only to H4K20me3 (trimethylated Lys(20) of histone H4), but also to dsDNA (double-stranded DNA) via an aromatic cage and a positively charged area respectively. EMSAs (electrophoretic mobility-shift assays) illustrated the ability of the Pdp1 PWWP domain to bind to the nucleosome core particle, and further mutagenesis experiments indicated the crucial role of this binding activity in histone H4K20 di- and tri-methylation in yeast cells. The present study may shed light on a novel mechanism of histone methylation regulation by the PWWP domain.

The small ubiquitin-like modifier (SUMO) is essential in cell cycle regulation in Trypanosoma brucei.

SUMO, a reversible post-translational protein modifier, plays important roles in many processes of higher eukaryotic cell life. Although SUMO has been identified in many eukaryotes, SUMO and SUMO system are still unknown in some eukaryotic unicellular organisms, such as Trypanosoma brucei (T. brucei). In this study, only one SUMO homologue (TbSUMO) was identified in T. brucei. Expression of TbSUMO was knocked down by using RNA interference technique in procyclic-form T. brucei. The growth of TbSUMO-deficient cells was significantly inhibited. TbSUMO-deficient cells were arrested in G2/M phase accompanied with an obvious increase of 0N1K cells (zoids), and failed in chromosome segregation. These results indicate that TbSUMO is essential in cell cycle regulation, with one important role in mitosis. Meanwhile, the enrichment of zoids suggests the inhibition of mitosis does not prevent the cell division in procyclic-form T. brucei. HA-tagged TbSUMO was overexpressed in T. brucei and was shown to be localized to the nucleus through the whole cell cycle, further revealing its distinguished functions in nucleus. All these accumulated data imply that a SUMO system essential for regulating cell cycle progression might exist in the procyclic-form T. brucei.

Distinct cytoskeletal modulation and regulation of G1-S transition in the two life stages of Trypanosoma brucei

Procyclic-form Trypanosoma brucei is arrested in G1 phase with extended and/or branched posterior morphology when expression of its cdc2-related kinases 1 and 2 (CRK1 and CRK2) is knocked down by RNA interference. Transmission electron microscopy indicated that the mitochondrion in the cell is also extended and branched and associated with cortical microtubules in each elongated/branched posterior end. This posterior extension is apparently driven by the growing microtubule corset, as it can be blocked by rhizoxin, an inhibitor of microtubule assembly. In the bloodstream form of T. brucei, however, a knockdown of CRK1 and CRK2 resulted only in an enrichment of cells in G1 phase without cessation of DNA synthesis or elongated/branched posterior ends. A triple knockdown of CRK1, CRK2 and CycE1/CYC2 in the bloodstream form resulted in 15% of the cells arrested in G1 phase, but no cells had an abnormal posterior morphology. The double and triple knockdown bloodstream-form cells were differentiated in vitro into the procyclic form, and the latter thus generated bore the typical morphology of a procyclic form without an extended/branched posterior end, albeit arrested in the G1 phase as the bloodstream-form precursor. There is thus a major distinction in the mechanisms regulating G1-S transition and posterior morphogenesis between the two life stages of T. brucei.

Solution structure of the Taf14 YEATS domain and its roles in cell growth of Saccharomyces cerevisiae

Chromatin modifications play important roles in cellular biological process. A novel conserved domain family, YEATS, has been discovered in a variety of eukaryotic species ranging from yeasts to humans. Taf14, which is involved in a few protein complexes of chromatin remodelling and gene transcription, and is essential for keeping chromosome stability, regular cell growth and transcriptional regulation, contains a YEATS domain at its N-terminus. In the present study, we determined the solution structure of the Taf14 YEATS domain using NMR spectroscopy. The Taf14 YEATS domain adopts a global fold of an elongated β-sandwich, similar to the Yaf9 YEATS domain. However, the Taf14 YEATS domain differs significantly from the Yaf9 YEATS domain in some aspects, which might indicate different structural classes of the YEATS domain family. Functional studies indicate that the YEATS domain is critical for the function of Taf14 in inhibiting cell growth under stress conditions. In addition, our results show that the C-terminus of Taf14 is responsible for its interaction with Sth1, which is an essential component of the RSC complex. Taken together, this implies that Taf14 is involved in transcriptional activation of Saccharomyces cerevisiae and the YEATS domain of Taf14 might play a negative role in cell growth.

Role of the Helical Structure of the N-Terminal Region of Plasmodium falciparum Merozoite Surface Protein 2 in Fibril Formation and Membrane Interaction

Merozoite surface protein 2 (MSP2), an abundant glycosylphosphatidylinositol-anchored protein on the surface of Plasmodium falciparum merozoites, is a promising malaria vaccine candidate. MSP2 is intrinsically disordered and forms amyloid-like fibrils in solution under physiological conditions. The 25 N-terminal residues (MSP2(1-25)) play an important role in both fibril formation and membrane binding of the full-length protein. In this study, the fibril formation and solution structure of MSP2(1-25) in the membrane mimetic solvents sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), and trifluoroethanol (TFE) have been investigated by transmission electronic microscopy, turbidity, thioflavin T fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy. Turbidity data showed that the aggregation of MSP2(1-25) was suppressed in the presence of membrane mimetic solvents. CD spectra indicated that helical structure in MSP2(1-25) was stabilized in SDS and DPC micelles and in high concentrations of TFE. The structure of MSP2(1-25) in 50% aqueous TFE, determined using NMR, showed that the peptide formed an amphipathic helix encompassing residues 10-24. Low concentrations of TFE favored partially folded helical conformations, as demonstrated by CD and NMR, and promoted MSP2(1-25) fibril formation. Our data suggest that partially folded helical conformations of the N-terminal region of MSP2 are on the pathway to amyloid fibril formation, while higher degrees of helical structure stabilized by high concentrations of TFE or membrane mimetics suppress self-association and thus inhibit fibril formation. The roles of the induced helical conformations in membrane interactions are also discussed.

Solution structure of SUMO from Trypanosoma brucei and its interaction with Ubc9.

Post-translational modification by SUMO (small ubiquitin-related modifier) is an important mechanism that regulates a wide variety of cellular functions, such as nuclear transport, gene expression, stress response, cell cycle control, oncogenesis, and response to virus infection.1–3 SUMO proteins are conjugated to target proteins by an enzymatic cascade involving SUMO activating enzyme E1 (Aos1/Uba2, also named SAE1/SAE2),4–6 SUMO conjugating enzyme E2 (Ubc9),7,8 and E3 ligase. Trypanosoma brucei, the causative agent of African sleeping sickness, is a unicellular protozoan. The sequences of SUMO in Trypanosoma brucei (Tb-SUMO) are 37% and 33% identical with that of human SUMO-1 and yeast Smt3, respectively (Supporting Information Figure S1A). Here, the solution structure of truncated Tb-SUMO (residues 7–108) from T. brucei was determined by NMR spectroscopy. Its interaction surface with human Ubc9 was also determined by chemical shift perturbation using NMR spectroscopy. This is the first structure of SUMO determined in protist. The 3D structure of Tb-SUMO is highly conserved with that of SUMO-1 and Smt3. Tb-SUMO interacts with human Ubc9 through residues located on the b-sheet of TbSUMO, which is also similar to that of SUMO-1 and Smt3. All these results suggest the evolutionary conservation of SUMO and sumoylation in eukaryotes.

Solution Structure of Tensin2 SH2 Domain and Its Phosphotyrosine-Independent Interaction with DLC-1

Background Src homology 2 (SH2) domain is a conserved module involved in various biological processes. Tensin family member was reported to be involved in tumor suppression by interacting with DLC-1 (deleted-in-liver-cancer-1) via its SH2 domain. We explore here the important questions that what the structure of tensin2 SH2 domain is, and how it binds to DLC-1, which might reveal a novel binding mode. Principal Findings Tensin2 SH2 domain adopts a conserved SH2 fold that mainly consists of five β-strands flanked by two α-helices. Most SH2 domains recognize phosphorylated ligands specifically. However, tensin2 SH2 domain was identified to interact with nonphosphorylated ligand (DLC-1) as well as phosphorylated ligand. Conclusions We determined the solution structure of tensin2 SH2 domain using NMR spectroscopy, and revealed the interactions between tensin2 SH2 domain and its ligands in a phosphotyrosine-independent manner.