Crystal structure, nucleic-acid binding properties, and dimerization model of Pur-alpha

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

Of bits and bugs

Pur-α is a nucleic acid-binding protein involved in cell cycle control, transcription, and neuronal function. Initially no prediction of the three-dimensional structure of Pur-α was possible. However, recently we solved the X-ray structure of Pur-α from the fruitfly Drosophila melanogaster and showed that it contains a so-called PUR domain. Here we explain how we exploited bioinformatics tools in combination with X-ray structure determination of a bacterial homolog to obtain diffracting crystals and the high-resolution structure of Drosophila Pur-α. First, we used sensitive methods for remote-homology detection to find three repetitive regions in Pur-α. We realized that our lack of understanding how these repeats interact to form a globular domain was a major problem for crystallization and structure determination. With our information on the repeat motifs we then identified a distant bacterial homolog that contains only one repeat. We determined the bacterial crystal structure and found that two of the repeats interact to form a globular domain. Based on this bacterial structure, we calculated a computational model of the eukaryotic protein. The model allowed us to design a crystallizable fragment and to determine the structure of Drosophila Pur-α. Key for success was the fact that single repeats of the bacterial protein self-assembled into a globular domain, instructing us on the number and boundaries of repeats to be included for crystallization trials with the eukaryotic protein. This study demonstrates that the simpler structural domain arrangement of a distant prokaryotic protein can guide the design of eukaryotic crystallization constructs. Since many eukaryotic proteins contain multiple repeats or repeating domains, this approach might be instructive for structural studies of a range of proteins

Of Bits and Bugs — On the Use of Bioinformatics and a Bacterial Crystal Structure to Solve a Eukaryotic Repeat-Protein Structure

Pur-α is a nucleic acid-binding protein involved in cell cycle control, transcription, and neuronal function. Initially no prediction of the three-dimensional structure of Pur-α was possible. However, recently we solved the X-ray structure of Pur-α from the fruitfly Drosophila melanogaster and showed that it contains a so-called PUR domain. Here we explain how we exploited bioinformatics tools in combination with X-ray structure determination of a bacterial homolog to obtain diffracting crystals and the high-resolution structure of Drosophila Pur-α. First, we used sensitive methods for remote-homology detection to find three repetitive regions in Pur-α. We realized that our lack of understanding how these repeats interact to form a globular domain was a major problem for crystallization and structure determination. With our information on the repeat motifs we then identified a distant bacterial homolog that contains only one repeat. We determined the bacterial crystal structure and found that two of the repeats interact to form a globular domain. Based on this bacterial structure, we calculated a computational model of the eukaryotic protein. The model allowed us to design a crystallizable fragment and to determine the structure of Drosophila Pur-α. Key for success was the fact that single repeats of the bacterial protein self-assembled into a globular domain, instructing us on the number and boundaries of repeats to be included for crystallization trials with the eukaryotic protein. This study demonstrates that the simpler structural domain arrangement of a distant prokaryotic protein can guide the design of eukaryotic crystallization constructs. Since many eukaryotic proteins contain multiple repeats or repeating domains, this approach might be instructive for structural studies of a range of proteins