No abstract

The proliferating cell nuclear antigen (PCNA) is now recognized as one of the key proteins in DNA metabolic events because of its direct interactions with many proteins involved in important cellular processes. We have determined the crystal structure of PCNA from a hyperthermophilic archaeon, Pyrococcus furiosus (PfuPCNA), at 2.1 Å resolution. PfuPCNA forms a toroidal, ring-shaped structure consisting of homotrimeric molecules, which is also observed in the PCNA crystals from human and yeast. The overall structure of PfuPCNA is highly conserved with other PCNA proteins, as well as with the bacterial ␤ clamp and the bacteriophage gp45. This result shows that the three-dimensional structure of the sliding clamp is conserved in the three domains of life. PfuPCNA has two remarkable features compared with the human and yeast PCNA molecules: it has more ion pairs and fewer intermolecular main chain hydrogen bonds. The former may contribute to the thermal stability of PfuPCNA, and the latter may be the cause of the stimulatory effect of PfuPCNA on the DNA synthesizing activity of P. furiosus DNA polymerases in the absence of the clamp loader replication factor C in vitro.

Background:  Proliferating cell nuclear antigen (PCNA), which is recognized as a DNA polymerase processivity factor, has direct interactions with various proteins involved in the important genetic information processes in Eukarya. We determined the crystal structure of PCNA from the hyperthermophilic archaeon, Pyrococcus furiosus (PfuPCNA) at 2.1 Å resolution, and found that the toroidal ring‐shaped structure, which consists of homotrimeric molecules, is highly conserved between the Eukarya and Archaea. This allowed us to examine its interaction with the loading factor at the atomic level. Results: The replication factor C (RFC) is known as the loading factor of PCNA on to the DNA strand. P. furiosus RFC (PfuRFC) has a PCNA binding motif (PIP‐box) at the C‐terminus of the large subunit (RFCL). An 11 residue‐peptide containing a PIP‐box sequence of RFCL inhibited the PCNA‐dependent primer extension ability of P. furiosus PolI in a concentration‐dependent manner. To understand the molecular interaction mechanism of PCNA with PCNA binding proteins, we solved the crystal structure of PfuPCNA complexed with the PIP‐box peptide. The interaction mode of the two molecules is remarkably similar to that of human PCNA and a peptide containing the PIP‐box of p21WAF1/CIP1. Moreover, the PIP‐box binding may have some effect on the stability of the ring structure of PfuPCNA by some domain shift. Conclusions: Our structural analysis on PfuPCNA suggests that the interaction mode of the PIP‐box with PCNA is generally conserved among the PCNA interacting proteins and that the functional meaning of the interaction via the PIP‐box possibly depends on each protein. A movement of the C‐terminal region of the PCNA monomer by PIP‐box binding may cause the PCNA ring to be more rigid, suitable for its functions.

Two methylated bis(ethylenedithio)tetrathiafulvalene (ET) derivatives, Me2ET and Me4ET were stereoselectively synthesized to examine the effect of methylation on conformations of dihydrodithiin rings and molecular arrangements in the crystalline state. Since the donating ability of Me2ET and Me4ET are similar to that of ET, the methylated ET derivatives are considered to be appropriate to investigate the “lattice pressure” effect on ET radical salts by changing the volume of donor molecules. The upper limit of an activation energy for the ring inversion of the dimethylated dihydrodithiin in solution was estimated to be 32 kJ mol−1 by 13C NMR spectroscopy. The X-ray structure analyses revealed that orientations of methyl groups are fixed to axial in Me2ET and to equatorial in Me4ET, accompanied by the change of molecular stacking. The “volume of a methyl group” was evaluated by comparing the molecular volumes of Me2ET and Me4ET with that of ET, and the effective volume for the axial methyl group turns out to be 15% larger than that of the equatorial. The solid state 13C NMR (CP/MAS) spectra of ET and its derivatives showed that the chemical shifts of resonance lines reflect the conformations of dihydrodithiin rings in crystals.

[structure: see text] UCS1025A and B, novel pentacyclic polyketides with an unprecedented furopyrrolizidine skeleton, were isolated from the fungus Acremonium sp. KY4917. The structures and stereochemistry were elucidated by a combination of two-dimensional NMR and X-ray crystallographic analysis. UCS1025A showed unique chemical equilibria involving three tautomeric isomers and exhibited antimicrobial activity and antiproliferative activity against human tumor cell lines.