X-ray structure of Pur-α reveals a Whirly-like fold and an unusual nucleic-acid binding surface

Abstract

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-␣, acts as a transcriptional regulator, as host factor for viral replication, and as cofactor for mRNP localization in dendrites. Pur-␣-deficient mice show severe neurologic defects and die after birth. Nucleic-acid binding by Pur-␣ 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-␣, 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 ␤-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 sizeexclusion chromatography indicated that PUR repeat III mediates dimerization of Pur-␣. Surface envelopes calculated from SAXS data show that the Pur-␣ 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-␣ has direct implications for ssDNA/ ssRNA and dsDNA binding.crystal structure ͉ DNA binding ͉ RNA binding ͉ fragile X-associated tremor ͉ ataxia syndrome

Drosophila Pur-α binds to trinucleotide-repeat containing cellular RNAs and translocates to the early oocyte

“…A previous study identified three so-called PUR-repeats in the Drosophila Pur-α sequence; crystallographic analysis revealed that two repeats form a nucleic acid-binding domain with a whirly-like fold. 12 Dimerization of Pur-α, which forms a third, intermolecular RNA binding domain, is necessary to achieve optimal transport into the oocyte. Assembly of multiple RNA binding sites is a common strategy among RNA transport factors that likely increases affinity, specificity or halflife of the mRNP complex, i.e., the chance for the cargo to remain bound throughout the entire transport time.…”

“…Considering the solution structure model calculated from SAXS-data, a 12-mer RNA binding substrate may be too short to make contacts with more than one pur-domain. 12 Furthermore, while the GST-Pur-α 40-185 truncation we employed dimerizes via the GST-tag, it lacks the third RNA binding domain that is normally formed between both subunits. It is therefore possible that longer stretches of CAG/CGG repeats, as found e.g., in the Notch opa-repeat, 20 are bound with higher affinity by wild-type Pur-α.…”

“…This was also true-though to a lesser extent-in the context of fulllength Pur-α. A second set of point mutations (R65A, R142A) diminishes RNA binding less than the previous one 12 and impaired association of Cup, Tral and Me31B to a lesser extent (Fig. 3).…”

“…Crystallographic analysis of Drosophila Pur-α has revealed the three dimensional structure of its nucleic acid binding domain, leading to the identification of amino acids that are essential for nucleic acid binding. 12 In the fruitfly Drosophila melanogaster, a number of genes that are important for the development of the nervous system also take part in key signaling events during oogenesis. 13 The extensive transport and localization of mRNAs prior to translation that characterizes Drosophila oogenesis is also reminiscent of the dendritic transport of mRNAs in neurons.…”

“…Previous in vitro studies using electrophoretic mobility shift assays (EMSA) demonstrated binding of full-length Pur-α as well as a truncated protein containing only PUR-repeats I and II to r(CGG) 4 with a moderate affinity of 1-2 μM. 12 We repeated the affinity measurements comparing EMSA and fluorescence anisotropy, a solution based assay that does not require separation of bound and free probe ( Fig. 4 and inserts; quantification and curve-fitting is shown in Figs.…”