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A crystallographic study of human NONO (p54(nrb)): overcoming pathological problems with purification, data collection and noncrystallographic symmetry.

Knott GJ, Panjikar S, Thorn A, Fox AH, Conte MR, Lee M, Bond CS
Acta Crystallogr D Struct Biol 72 761-9 (2016)
Cited: 8 times
EuropePMC logo PMID: 27303796

Abstract

Non-POU domain-containing octamer-binding protein (NONO, a.k.a. p54(nrb)) is a central player in nuclear gene regulation with rapidly emerging medical significance. NONO is a member of the highly conserved Drosophila behaviour/human splicing (DBHS) protein family, a dynamic family of obligatory dimeric nuclear regulatory mediators. However, work with the NONO homodimer has been limited by rapid irreversible sample aggregation. Here, it is reported that L-proline stabilizes purified NONO homodimers, enabling good-quality solution small-angle X-ray structure determination and crystallization. NONO crystallized in the apparent space group P21 with a unique axis (b) of 408.9 Å and with evidence of twinning, as indicated by the cumulative intensity distribution L statistic, suggesting the possibility of space group P1. Structure solution by molecular replacement shows a superhelical arrangement of six NONO homodimers (or 12 in P1) oriented parallel to the long axis, resulting in extensive noncrystallographic symmetry. Further analysis revealed that the crystal was not twinned, but the collected data suffered from highly overlapping reflections that obscured the L-test. Optimized data collection on a new crystal using higher energy X-rays, a smaller beam width and an increased sample-to-detector distance produced non-overlapping reflections to 2.6 Å resolution. The steps taken to analyse and overcome this series of practical difficulties and to produce a biologically informative structure are discussed.

Articles - 5ifm mentioned but not cited (4)

  1. A crystallographic study of human NONO (p54(nrb)): overcoming pathological problems with purification, data collection and noncrystallographic symmetry. Knott GJ, Panjikar S, Thorn A, Fox AH, Conte MR, Lee M, Bond CS. Acta Crystallogr D Struct Biol 72 761-769 (2016)
  2. Structural basis of dimerization and nucleic acid binding of human DBHS proteins NONO and PSPC1. Knott GJ, Chong YS, Passon DM, Liang XH, Deplazes E, Conte MR, Marshall AC, Lee M, Fox AH, Bond CS. Nucleic Acids Res 50 522-535 (2022)
  3. Molecular Modelling of NONO and SFPQ Dimerization Process and RNA Recognition Mechanism. Laurenzi T, Palazzolo L, Taiana E, Saporiti S, Ben Mariem O, Guerrini U, Neri A, Eberini I. Int J Mol Sci 23 7626 (2022)
  4. Paraspeckle subnuclear bodies depend on dynamic heterodimerisation of DBHS RNA-binding proteins via their structured domains. Lee PW, Marshall AC, Knott GJ, Kobelke S, Martelotto L, Cho E, McMillan PJ, Lee M, Bond CS, Fox AH. J Biol Chem 298 102563 (2022)


Articles citing this publication (4)

  1. G-quadruplexes offer a conserved structural motif for NONO recruitment to NEAT1 architectural lncRNA. Simko EAJ, Liu H, Zhang T, Velasquez A, Teli S, Haeusler AR, Wang J. Nucleic Acids Res 48 7421-7438 (2020)
  2. Crystal structure of a SFPQ/PSPC1 heterodimer provides insights into preferential heterodimerization of human DBHS family proteins. Huang J, Casas Garcia GP, Perugini MA, Fox AH, Bond CS, Lee M. J Biol Chem 293 6593-6602 (2018)
  3. The regulation of NONO by USP11 via deubiquitination is linked to the proliferation of melanoma cells. Feng P, Li L, Dai J, Zhou L, Liu J, Zhao J, Li X, Ling N, Qiu S, Zhang L, Xie T, Chen Y, Donovan MJ, Peng T, Song J, Ye M. J Cell Mol Med 25 1507-1517 (2021)
  4. Drug repurposing screens identify compounds that inhibit α-synuclein oligomers' membrane disruption and block antibody interactions. Somavarapu AK, Kleijwegt G, Nagaraj M, Alam P, Nielsen J, Otzen DE. Chem Sci 14 3030-3047 (2023)


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