Structural dynamics of IDR interactions in human SFPQ and implications for liquid-liquid phase separation.

IF 2.6 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS
Heidar J Koning, Valerie Lai, Ashish Sethi, Shatabdi Chakraborty, Ching Seng Ang, Archa H Fox, Anthony P Duff, Andrew E Whitten, Andrew C Marshall, Charles S Bond
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引用次数: 0

Abstract

The proteins SFPQ (splicing factor proline- and glutamine-rich) and NONO (non-POU domain-containing octamer-binding protein) are members of the Drosophila behaviour/human splicing (DBHS) protein family, sharing 76% sequence identity in their conserved DBHS domain. These proteins are critical for elements of pre- and post-transcriptional regulation in mammals and are primarily located in paraspeckles: ribonucleoprotein bodies templated by NEAT1 long noncoding RNA. Regions that are structured and predicted to be disordered (IDRs) in DBHS proteins facilitate various interactions, including dimerization, polymerization, nucleic acid binding and liquid-liquid phase separation, all of which have consequences for cell health, the pathology of some neurological diseases and cancer. To date, very limited structural work has been carried out on characterizing the IDRs of the DBHS proteins, largely due to their predicted disordered nature and the fact that this is often a bottleneck for conventional structural techniques. This is a problem worth addressing, as the IDRs have been shown to be critical to the material state of the protein as well as its function. In this study, we used small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS), together with lysine cross-linking mass spectrometry (XL-MS), to investigate the regions of SFPQ flanking the structured DBHS domain and the possibility of dimer partner exchange of full-length proteins. Our results demonstrate experimentally that the N- and C-terminal regions on either side of the folded DBHS domain are long, disordered and flexible in solution. Realistic modelling of disordered chains to fit the scattering data and the compaction of the different protein variants suggests that it is physically possible for the IDRs to be close enough to interact. The mass-spectrometry data additionally indicate that the C-terminal IDR can potentially interact with the folded DBHS domain and also shares some conformational space with the N-terminal IDR. Our small-angle neutron scattering (SANS) experiments reveal that full-length SFPQ is capable of swapping dimer partners with itself, which has implications for our understanding of the combinatorial dimerization of DBHS proteins within cells. Our study provides insight into possible interactions between different IDRs either in cis or in trans and how these may relate to protein function, and the possible impact of mutations in these regions. The dynamic dimer partner exchange of a full-length protein inferred from this study is a phenomenon that is integral to the function of DBHS proteins, allowing changes in gene-regulatory activity by altering levels of the various heterodimers or homodimers.

人SFPQ中IDR相互作用的结构动力学及其对液-液相分离的意义。
SFPQ蛋白(剪接因子脯氨酸和谷氨酰胺丰富)和NONO蛋白(非pou结构域含八聚体结合蛋白)是果蝇行为/人类剪接(DBHS)蛋白家族的成员,在其保守的DBHS结构域具有76%的序列一致性。这些蛋白在哺乳动物的转录前和转录后调控中起着至关重要的作用,主要位于旁斑:由NEAT1长链非编码RNA模板化的核糖核蛋白小体。DBHS蛋白中的结构和预测紊乱(IDRs)区域促进各种相互作用,包括二聚化、聚合、核酸结合和液-液相分离,所有这些都对细胞健康、一些神经系统疾病和癌症的病理产生影响。迄今为止,在表征DBHS蛋白的idr方面进行的结构工作非常有限,这主要是由于它们预测的无序性质,而且这通常是传统结构技术的瓶颈。这是一个值得解决的问题,因为idr已被证明对蛋白质的物质状态及其功能至关重要。在这项研究中,我们利用小角x射线散射(SAXS)和小角中子散射(SANS),结合赖氨酸交联质谱(XL-MS),研究了SFPQ在DBHS结构域两侧的区域以及全长蛋白二聚体伴侣交换的可能性。实验结果表明,折叠DBHS结构域两侧的N端和c端在溶液中是长、无序和灵活的。对无序链进行现实建模以拟合散射数据和不同蛋白质变体的压实表明,idr在物理上可能足够接近以相互作用。质谱数据还表明,c端IDR可能与折叠的DBHS结构域相互作用,并与n端IDR共享一些构象空间。我们的小角中子散射(SANS)实验表明,全长SFPQ能够与自身交换二聚体伙伴,这对我们理解DBHS蛋白在细胞内的组合二聚化具有重要意义。我们的研究深入了解了顺式或反式中不同idr之间可能的相互作用,以及这些相互作用如何与蛋白质功能相关,以及这些区域突变可能产生的影响。从本研究推断,全长蛋白的动态二聚体伴侣交换是DBHS蛋白功能不可或缺的现象,允许通过改变各种异源二聚体或同型二聚体的水平来改变基因调控活性。
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来源期刊
Acta Crystallographica. Section D, Structural Biology
Acta Crystallographica. Section D, Structural Biology BIOCHEMICAL RESEARCH METHODSBIOCHEMISTRY &-BIOCHEMISTRY & MOLECULAR BIOLOGY
CiteScore
4.50
自引率
13.60%
发文量
216
期刊介绍: Acta Crystallographica Section D welcomes the submission of articles covering any aspect of structural biology, with a particular emphasis on the structures of biological macromolecules or the methods used to determine them. Reports on new structures of biological importance may address the smallest macromolecules to the largest complex molecular machines. These structures may have been determined using any structural biology technique including crystallography, NMR, cryoEM and/or other techniques. The key criterion is that such articles must present significant new insights into biological, chemical or medical sciences. The inclusion of complementary data that support the conclusions drawn from the structural studies (such as binding studies, mass spectrometry, enzyme assays, or analysis of mutants or other modified forms of biological macromolecule) is encouraged. Methods articles may include new approaches to any aspect of biological structure determination or structure analysis but will only be accepted where they focus on new methods that are demonstrated to be of general applicability and importance to structural biology. Articles describing particularly difficult problems in structural biology are also welcomed, if the analysis would provide useful insights to others facing similar problems.
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