X-ray structure of Pur-alpha 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-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.

Fig. 1.

The structure of Drosophila Pur-α identifies the PUR domain as an MRP1/MRP2/P24-like nucleic-acid binding protein. (A) Schematic drawing of the distinct protein regions of Pur-α (Top) and the two protein fragments used in this study (Middle and Bottom). PUR repeats have been identified in this study by structure determination. (B) Ribbon backbone model of the globular domain formed by two PUR repeats, termed PUR domain. Similar to the schematic drawing in (A), PUR repeat I is shown in green, PUR repeat II in blue, and the linker connecting both repeats in gray. (C) View from (B) rotated by 180° around the vertical axis. (D) Superposition of the structure of Pur-α (gray) with the structure of MRP2 (red). Orientation is identical to (C). (E) Rotated and magnified superposition from (D) showing the orientation of the β-sheets from Pur-α and MRP2 with respect to each other.

Fig. 2.

Surface conservation of the PUR domain. (A) Surface representation of the sequence conservation of the solvent-accessible surface of the PUR domain. Surface plot is based on the interspecies alignment shown in Fig. S1. Dark green coloration indicates complete conservation, whereas light green shows partially conserved and gray unconserved residues. Orientation is identical to Fig. 1B. Dashed line indicates the unconserved rim that separates the two highly conserved surface regions around the β-sheets. (B) Representation as in (A) rotated by 180° around the vertical axis (identical to orientation in Fig. 1C). (Insets) Cartoon representation of a close-up of the β-sheet from PUR repeats I and PUR repeat II (Fig. 1B). Color-coding is identical to (Fig. 1 B and C). (C) Close-up of surface conservation from Pur repeat II with the view identical to inset above (C). (D) Close-up of surface charge of Pur repeat I with the view identical to inset above (D).

Fig. 3.

Surface charges of the PUR domain. (A) Representation of the electrostatic potentials of the solvent-accessible surface of the PUR domain. Red and blue coloration indicate negative and positive electrostatic potentials, respectively. Orientation is identical to Figs. 1B and 2A. Dashed line indicates the unconserved rim that separates the two highly conserved surface regions around the β-sheets. (B) Representation as in (A) rotated by 180° around the vertical axis (identical to orientation in Figs. 1C and 2B). (Insets) Cartoon representation of a close-up of the β-sheet from PUR repeats I (A) and PUR repeat II (Fig. 1B). (C) Close-up of surface charges of Pur repeat II with the view identical to inset above (C). (D) Close-up of surface charges of Pur repeat I with the view identical to inset above (D).

Fig. 4.

DNA EMSAs with different Pur-α fragments. (A) Full-length Pur-α binds with high affinity to MF0677 24mer ssDNA but not to control A₍₂₄₎-mer ssDNA. (B) Full-length Pur-α also binds to a (CGG)₍₁₂₎-mer ssRNA. Pur-α (I-II) binds to the ssDNA (C) and to the ssRNA (D) with affinities comparable to full-length Pur-α. The double-mutation R65A and R142A in Pur-α (I-II) does not affect its binding to ssDNA (C) but showed a moderate reduction in ssRNA binding (D). The double-mutant R80A and R158A abolished binding of Pur-α (I-II) to ssDNA (E) as well as to ssRNA (F).

Fig. 5.

Analysis by SAXS. (A) Table summarizing molecular-weight calculations by size-exclusion chromatography and by SAXS. (B) Schematic drawing depicting the role of individual PUR repeats in the formation of PUR domains and Pur-α dimerization. (C) Scattering curve of Pur-α (I–II) and representative surface envelope calculated from SAXS measurements. (D) A fit of the crystal structure into the surface envelope of Pur-α (I–II) confirms the presence of intramolecular PUR domains in solution. (E) Scattering curve of Pur-α (I–III) and representative surface envelope calculated from SAXS measurements. These envelopes adopt a Z-like shape. (F) A fit of three PUR domains into the Z-like envelope is consistent with the presence of two intramolecular PUR domains and one intermolecular PUR domain (B). Structural models of Pur-α (I-II) were positioned manually in the envelopes to demonstrate the compatibility of envelopes with the size of PUR domains.