methods-for-quantitative-detection-antibody-induced-complement

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

University of Amsterdam

UV damage endonuclease is a DNA repair enzyme that can both recognize damage such as UV lesions and introduce a nick directly 5' to them. Recently, the crystal structure of the enzyme from Thermus thermophilus was solved. In the electron density map of this structure, unexplained density near the active site was observed at the tip of Lys229. Based on this finding, it was proposed that Lys229 is post-translationally modified. In this article, we give evidence that this modification is a carboxyl group. By combining activity assays and X-ray crystallography on several point mutants, we show that the carboxyl group assists in metal binding required for catalysis by donating negative charge to the metal-coordinating residue His231. Moreover, functional and structural analysis of the K229R mutant reveals that if His231 shifts away, an increased activity results on both damaged and undamaged DNA. Taken together, the results show that T. thermophilus ultraviolet damage endonuclease is carboxylated and the modified lysine is required for proper catalysis and preventing increased incision of undamaged DNA.

Involvement of a carboxylated lysine in UV damage endonuclease.

Cockayne syndrome protein A is one of the main components in mammalian transcription coupled repair. Here, the overproduction, purification and crystallization of human Cockayne syndrome protein A in complex with its interacting partner DNA damage binding protein 1 are reported. The complex was coproduced in insect cells, copurified and crystallized using sitting drops with PEG 3350 and sodium citrate as crystallizing agents. The crystals had unit-cell parameters a = b = 142.03, c = 250.19 Å and diffracted to 2.9 Å resolution on beamline ID14-1 at the European Synchrotron Radiation Facility.

Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of Cockayne syndrome protein A in complex with DNA damage-binding protein 1.

Potato serine protease inhibitor (PSPI) constitutes about 22% of the total amount of proteins in potato tubers (cv. Elkana), making it the most abundant protease inhibitor in the plant. PSPI is a heterodimeric double-headed Kunitz-type serine protease inhibitor that can tightly and simultaneously bind two serine proteases by mimicking the substrate of the enzyme with its reactive-site loops. Here, the crystal structure of PSPI is reported, representing the first heterodimeric double-headed Kunitz-type serine protease inhibitor structure to be determined. PSPI has a β-trefoil fold and, based on the structure, two reactive-site loops bearing residues Phe75 and Lys95 were identified.

Structure of a post-translationally processed heterodimeric double-headed Kunitz-type serine protease inhibitor from potato.

Repairing damaged DNA is essential for an organism's survival. UV damage endonuclease (UVDE) is a DNA-repair enzyme that can recognize and incise different types of damaged DNA. We present the structure of Sulfolobus acidocaldarius UVDE on its own and in a pre-catalytic complex with UV-damaged DNA containing a 6-4 photoproduct showing a novel 'dual dinucleotide flip' mechanism for recognition of damaged dipyrimidines: the two purines opposite to the damaged pyrimidine bases are flipped into a dipurine-specific pocket, while the damaged bases are also flipped into another cleft.

Article	Year
Involvement of a carboxylated lysine in UV damage endonuclease. Protein science : a publication of the Protein Society\| PubMed ID: 19241382	2009
Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of Cockayne syndrome protein A in complex with DNA damage-binding protein 1. Acta crystallographica. Section F, Structural biology and crystallization communications\| PubMed ID: 22232169	2012
Structure of a post-translationally processed heterodimeric double-headed Kunitz-type serine protease inhibitor from potato. Acta crystallographica. Section D, Biological crystallography\| PubMed ID: 22751664	2012
UV damage endonuclease employs a novel dual-dinucleotide flipping mechanism to recognize different DNA lesions. Nucleic acids research\| PubMed ID: 23221644	2013

Elisabeth M. Meulenbroek

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