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Structure of PCNA from Drosophila melanogaster.

ABSTRACT: Proliferating cell nuclear antigen (PCNA) plays essential roles in DNA replication, DNA repair, cell-cycle regulation and chromatin metabolism. The PCNA from Drosophila melanogaster (DmPCNA) was purified and crystallized. The crystal of DmPCNA diffracted to 2.0 Å resolution and belonged to space group H3, with unit-cell parameters a = b = 151.16, c = 38.28 Å. The structure of DmPCNA was determined by molecular replacement. DmPCNA forms a symmetric homotrimer in a head-to-tail manner. An interdomain connector loop (IDCL) links the N- and C-terminal domains. Additionally, the N-terminal and C-terminal domains contact each other through hydrophobic associations. Compared with human PCNA, the IDCL of DmPCNA has conformational changes, which may explain their difference in function. This work provides a structural basis for further functional and evolutionary studies of PCNA.

SUBMITTER: Wang K

PROVIDER: S-EPMC3614162 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Structure of PCNA from Drosophila melanogaster.

Wang Ke K Shi Zhubing Z Zhang Min M Cheng Dianlin D

Acta crystallographica. Section F, Structural biology and crystallization communications 20130328 Pt 4

Proliferating cell nuclear antigen (PCNA) plays essential roles in DNA replication, DNA repair, cell-cycle regulation and chromatin metabolism. The PCNA from Drosophila melanogaster (DmPCNA) was purified and crystallized. The crystal of DmPCNA diffracted to 2.0 Å resolution and belonged to space group H3, with unit-cell parameters a = b = 151.16, c = 38.28 Å. The structure of DmPCNA was determined by molecular replacement. DmPCNA forms a symmetric homotrimer in a head-to-tail manner. An interdom ...[more]

PMID: 23545643

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Project description:BACKGROUND:The Drosophila melanogaster Serpin 42 Da gene (previously Serpin 4) encodes a serine protease inhibitor that is capable of remarkable functional diversity through the alternative splicing of four different reactive centre loop exons. Eight protein isoforms of Serpin 42 Da have been identified to date, targeting the protease inhibitor to both different proteases and cellular locations. Biochemical and genetic studies suggest that Serpin 42 Da inhibits target proteases through the classical serpin 'suicide' inhibition mechanism, however the crystal structure of a representative Serpin 42 Da isoform remains to be determined. RESULTS:We report two high-resolution crystal structures of Serpin 42 Da representing the A/B isoforms in the cleaved conformation, belonging to two different space-groups and diffracting to 1.7 Å and 1.8 Å. Structural analysis reveals the archetypal serpin fold, with the major elements of secondary structure displaying significant homology to the vertebrate serpin, neuroserpin. Key residues known to have central roles in the serpin inhibitory mechanism are conserved in both the hinge and shutter regions of Serpin 42 Da. Furthermore, these structures identify important conserved interactions that appear to be of crucial importance in allowing the Serpin 42 Da fold to act as a versatile template for multiple reactive centre loops that have different sequences and protease specificities. CONCLUSIONS:In combination with previous biochemical and genetic studies, these structures confirm for the first time that the Serpin 42 Da isoforms are typical inhibitory serpin family members with the conserved serpin fold and inhibitory mechanism. Additionally, these data reveal the remarkable structural plasticity of serpins, whereby the basic fold is harnessed as a template for inhibition of a large spectrum of proteases by reactive centre loop exon 'switching'. This is the first structure of a Drosophila serpin reported to date, and will provide a platform for future mutational studies in Drosophila to ascertain the functional role of each of the Serpin 42 Da isoforms.

Project description:Using a complete set of RING domains from Drosophila melanogaster, all the solved RING domains and cocrystal structures of RING-containing ubiquitin-ligases (RING-E3) and ubiquitin-conjugating enzyme (E2) pairs, we analyzed RING domains structures from their primary to quarternary structures. The results showed that: i) putative orthologs of RING domains between Drosophila melanogaster and the human largely occur (118/139, 84.9%); ii) of the 118 orthologous pairs from Drosophila melanogaster and the human, 117 pairs (117/118, 99.2%) were found to retain entirely uniform domain architectures, only Iap2/Diap2 experienced evolutionary expansion of domain architecture; iii) 4 evolutionary structurally conserved regions (SCRs) are responsible for homologous folding of RING domains at the superfamily level; iv) besides the conserved Cys/His chelating zinc ions, 6 equivalent residues (4 hydrophobic and 2 polar residues) in the SCRs possess good-consensus and conservation- these 4 SCRs function in the structural positioning of 6 equivalent residues as determinants for RING-E3 catalysis; v) members of these RING proteins located nucleus, multiple subcellular compartments, membrane protein and mitochondrion are respectively 42 (42/139, 30.2%), 71 (71/139, 51.1%), 22 (22/139, 15.8%) and 4 (4/139, 2.9%); vi) CG15104 (Topors) and CG1134 (Mul1) in C3HC4, and CG3929 (Deltex) in C3H2C3 seem to display broader E2s binding profiles than other RING-E3s; vii) analyzing intermolecular interfaces of E2/RING-E3 complexes indicate that residues directly interacting with E2s are all from the SCRs in RING domains. Of the 6 residues, 2 hydrophobic ones contribute to constructing the conserved hydrophobic core, while the 2 hydrophobic and 2 polar residues directly participate in E2/RING-E3 interactions. Based on sequence and structural data, SCRs, conserved equivalent residues and features of intermolecular interfaces were extracted, highlighting the presence of a nucleus for RING domain fold and formation of catalytic core in which related residues and regions exhibit preferential evolutionary conservation.

Dataset Information

Structure of PCNA from Drosophila melanogaster.

Publications

Structure of PCNA from Drosophila melanogaster.

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