X-ray structure of Pur-alpha reveals a Whirly-like fold and an unusual nucleic-acid binding surface.
The PUR protein family is a distinct and highly conserved class that is characterized by its sequence-specific RNA- and DNA-binding. Its best-studied family member, Pur-alpha, acts as a transcriptional regulator, as host factor for viral replication, and as cofactor for mRNP localization in dendrites. Pur-alpha-deficient mice show severe neurologic defects and die after birth. Nucleic-acid binding by Pur-alpha is mediated by its central core region, for which no structural information is available. We determined the x-ray structure of residues 40 to 185 from Drosophila melanogaster Pur-alpha, which constitutes a major part of the core region. We found that this region contains two almost identical structural motifs, termed "PUR repeats," which interact with each other to form a PUR domain. DNA- and RNA-binding studies confirmed that PUR domains are indeed functional nucleic-acid binding domains. Database analysis show that PUR domains share a fold with the Whirly class of nucleic-acid binding proteins. Structural analysis combined with mutational studies suggest that a PUR domain binds nucleic acids through two independent surface regions involving concave beta-sheets. Structure-based sequence alignment revealed that the core region harbors a third PUR repeat at its C terminus. Subsequent characterization by small-angle x-ray scattering (SAXS) and size-exclusion chromatography indicated that PUR repeat III mediates dimerization of Pur-alpha. Surface envelopes calculated from SAXS data show that the Pur-alpha dimer consisting of repeats I to III is arranged in a Z-like shape. This unexpected domain organization of the entire core domain of Pur-alpha has direct implications for ssDNA/ssRNA and dsDNA binding.
Institute of Structural Biology, Helmholtz Zentrum München, German Research Center for Environmental Health, Marchionini-Strasse 25, Munich 81377, Germany.
Almut
A
Graebsch
Stéphane
S
Roche
Dierk
D
Niessing
D000818:Animals; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry* / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D008958:Models, Molecular; D009696:Nucleic Acids / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D053838:Scattering, Small Angle; D012996:Solutions; D055672:Static Electricity; D013499:Surface Properties; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*
D029721:Drosophila Proteins; D009696:Nucleic Acids; C545199:Pur-alpha protein, Drosophila; D012996:Solutions; D014157:Transcription Factors; D004247:DNA