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PLoS One
2013 Jan 01;83:e57345. doi: 10.1371/journal.pone.0057345.
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Solution structure of the QUA1 dimerization domain of pXqua, the Xenopus ortholog of Quaking.
Ali M
,
Broadhurst RW
.
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The STAR protein family member Quaking is essential for early development in vertebrates. For example, in oligodendrocyte cells it regulates the splicing, localization, translation and lifetime of a set of mRNAs that code for crucial components of myelin. The Quaking protein contains three contiguous conserved regions: a QUA1 oligomerization element, followed by a single-stranded RNA binding motif comprising the KH and QUA2 domains. An embryonic lethal point mutation in the QUA1 domain, E48G, is known to affect both the aggregation state and RNA-binding properties of the murine Quaking ortholog (QKI). Here we report the NMR solution structure of the QUA1 domain from the Xenopus laevis Quaking ortholog (pXqua), which forms a dimer composed of two perpendicularly docked α-helical hairpin motifs. Size exclusion chromatography studies of a range of mutants demonstrate that the dimeric state of the pXqua QUA1 domain is stabilized by a network of interactions between side-chains, with significant roles played by an intra-molecular hydrogen bond between Y41 and E72 (the counterpart to QKI E48) and an inter-protomer salt bridge between E72 and R67. These results are compared with recent structural and mutagenesis studies of QUA1 domains from the STAR family members QKI, GLD-1 and Sam68.
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Figure 2. Size exclusion chromatography studies.Typical analytical size exclusion chromatography profiles, using a Superdex S200 PC 3.2/30 column and a 20 mM Tris-HCl and 150 mM NaCl running buffer at pH 7.9, for: (A) wild type pXqua MltBP-QUA1 (black) and MltBP-QUA1-C59S (grey); and (B) MltBP-QUA1-C59S/E72G (black) and MltBP-QUA1-C59S/R67A/E72G (grey).
Figure 3. NMR spectroscopy.[1H,15N]-HSQC spectrum of pXqua QUA1-C59S, showing residue assignments for backbone amide and selected side-chain sites.
Figure 4. 15N relaxation parameters.Underneath a schematic defining the boundaries of α-helices in the structure of pXqua QUA1-C59S, NMR parameters for backbone amide sites are plotted as a function of residue number for: (A) the 15N longitudinal relaxation rate, R1; (B) the 15N transverse relaxation rate, R2; (C) the {1H}-15N heteronuclear Overhauser effect ratio (I’/I0, where I’ is the intensity when the 1H spectrum has been saturated and I0 is the intensity in the reference spectrum); and (D) 1H-15N residual dipolar coupling measurements.
Figure 5. Solution structure of the of pXqua QUA1-C59S dimer.(A) backbone overlay for the final ensemble of 20 lowest energy structures, with protomer A coloured from blue (NT) to green (CT) and protomer B from yellow (NT) to red (CT); (B) ribbon representation of the dimer structure; (C) representation showing the surface of protomer A, coloured according to charge from blue (positive) to red (negative), and protomer B in ribbon form with selected residues displayed as sticks; (D) backbone overlay of QUA1 dimerization domain structures for pXqua C59S (blue), GLD-1 (3K6T; red) and Sam68 (2XA6; green).
Figure 6. Comparison of structures of QUA1 domains from pXqua and QKI.(A) backbone overlay of dimeric QUA1 domain structures, with pXqua C59S (2YMJ) subunits displayed in red and salmon, and QKI C35S (4DNN) subunits in green and lime; (B) side-chain orientations of pXqua S59 and QKI S35, using same colours as in part (A); (C) side-chain orientations of pXqua F58 and QKI F34, using same colours as in part (A).
Figure 1. Domain organization and sequence alignments.(A) Domain organization of pXqua [16], showing location of QUA1, KH and QUA2 regions in yellow blocks, and the boundaries of the QUA1 construct used in this work as a black line below. (B) Sequence alignment and residue numbering for QUA1 regions of different STAR family members. Above, secondary structure observed for the pXqua QUA1-C59S dimer. Below, residues that contribute to the protomer non-polar core, along with non-polar and polar residues that participate in the dimer interface: capital letters indicate involvement in all known structures; small case indicates involvement only in pXqua; M and m indicate involvement in the protomer core; D and d indicate involvement in the dimer interface.
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