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Graebsch, Almut (2010): Crystal structure, nucleic-acid binding properties, and dimerization model of Pur-alpha. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

This study characterizes Pur-α structurally and functionally. Pur-α is a highly conserved RNA- and DNA-binding protein involved in a multitude of cellular processes such as transcription, replication, cell cycle control, and mRNA transport. No homologous proteins with known structures are available. X-ray crystallography is often hampered by the lack of diffraction-quality protein crystals. This study demonstrates how this bottleneck was overcome by the combination of iterative use of sensitive bioinformatics tools and structure determination of a bacterial homolog. The identification of three repeat regions (PUR repeats) in eukaryotic Pur-α enabled the detection of a bacterial homolog, which corresponds to one PUR repeat. The crystal structure of Borrelia burgdorferi Pur-α was solved at 1.9 Å and was employed for precise domain boundary prediction for the Drosophila melanogaster ortholog. Therewith it became possible to obtain diffraction-quality crystals of eukaryotic Pur-α. The crystal structure of D. melanogaster Pur-α repeats I-II was solved at 2.1 Å and shares a highly conserved fold with B. burgdorferi Pur-α. One PUR repeat has an overall ββββα− topology, and two PUR repeats interact with each other to form a globular PUR domain. Small angle X-ray scattering (SAXS) analysis together with analytical size-exclusion chromatography provided evidence that dimerization of full length Pur-α requires PUR repeat III. PUR repeat III is proposed to form a PUR domain with a PUR repeat III from another Pur-α molecule. Surface envelopes calculated from SAXS data comply with this dimerization model. DNA- as well as RNA-binding properties of Pur-α were examined by filter binding assays and electrophoretic mobility shift assays. Structure-guided mutagenesis identified the β-sheets of the PUR domain as the nucleic-acid binding surface. To assess the protein-binding properties of D. melanogaster Pur-α, a yeast-two-hybrid screen was commissioned and evaluated. It confirmed the self-interaction of Pur-α and yielded Arrestin1, LaminC, Eye and Cka as putative previously unknown interaction partners.