The structure of human SFPQ reveals a coiled-coil mediated polymer essential for functional aggregation in gene regulation.
Bottom Line: Small-angle X-ray scattering and transmission electron microscopy experiments show that polymerization is reversible in solution and can be templated by DNA.We demonstrate that the ability to polymerize is essential for the cellular functions of SFPQ: disruptive mutation of the coiled-coil interaction motif results in SFPQ mislocalization, reduced formation of nuclear bodies, abrogated molecular interactions and deficient transcriptional regulation.The coiled-coil interaction motif thus provides a molecular explanation for the functional aggregation of SFPQ that directs its role in regulating many aspects of cellular nucleic acid metabolism.
Affiliation: School of Chemistry and Biochemistry, University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia.Show MeSH
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Mentions: To examine the significance of the intact DBD and the polymerization domain for DNA binding, we next tested the ability of a number of the SFPQ constructs to bind to promoter DNA using an EMSA with the GAGE6 promoter—a reported SFPQ target gene (7)—as a probe (Figure 4). In order to limit extensive unworkable polymerization, we again used heteromeric SFPQ/NONO proteins, where NONO is unchanged (dimerization domain, 53–312) and SFPQ is varied: SFPQ-214–598 comprises DBD, dimerization domain and polymerization domain, SFPQ-214–535 comprises DBD and dimerization domain, and SFPQ-276–535 matches Crystal 3 (dimerization domain) (Figure 1b). To specifically address the role of the coiled-coil interaction motif in the polymerization domain, a mutated variant SFPQ-214–598mut was also generated, where the four key residues of the motif—L535, L539, L546 and M549 (Figure 2a)—were substituted to alanine. These four hydrophobic residues are the ‘knobs’ that stick into ‘holes’ that stabilize coiled-coil interactions. The alanine mutation was designed to maintain α-helical structure while disrupting the ability to form the coiled-coil interaction.
Affiliation: School of Chemistry and Biochemistry, University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia.