Hursey Matthew S, Reitz Abigail D, Kihn Kyle C, Deredge Daniel J, Michel Sarah L J
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201-1180, United States.
Biochemistry. 2025 Jan 7;64(1):156-169. doi: 10.1021/acs.biochem.4c00470. Epub 2024 Dec 16.
Ran-binding domain-containing protein 2 (ZRANB2) is a zinc finger (ZF) protein that plays a key role in alternative splicing. ZRANB2 is composed of two ZF domains that contain four invariant cysteine residues per domain. ZRANB2 binds RNA targets that contain AGGUAA sequence motifs. Three constructs of ZRANB2, ZRANB2-ZF1 (first ZF domain), ZRANB2-ZF2 (second ZF domain), and ZRANB2-2D (both ZF domains), were isolated in the apo form and shown to bind Zn(II) via UV-visible-monitored competitive titrations with Co(II) as a spectroscopic probe. Zn binding to each construct led to the adoption of a limited secondary structure of each domain, as measured by circular dichroism (CD). Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) of the two-domain construct, ZRANB2-2D, revealed that both ZF domains adopt a more rigid structure upon Zn binding. Zn binding to the first ZF domain resulted in a greater decrease in the conformational dynamics than Zn binding to the second ZF domain. RNA binding to TRA2B pre-mRNA, a physiological splicing target, was measured by fluorescence anisotropy (FA), and high-affinity RNA binding was found to require Zn coordination to both domains. HDX-MS of ZRANB2-2D with TRA2B RNA as well as two optimized RNA sequences that contain a single and double AGGUAA hexamer revealed additional protection from H/D exchange for ZRANB2 in the presence of RNA. Here, greater protection was observed for the second ZF of ZRANB2-2D, suggesting a larger effect on conformational dynamics. A model for zinc-mediated RNA binding of ZRANB2 is proposed.
含Ran结合结构域蛋白2（ZRANB2）是一种锌指（ZF）蛋白，在可变剪接中起关键作用。ZRANB2由两个ZF结构域组成，每个结构域含有四个不变的半胱氨酸残基。ZRANB2结合含有AGGUAA序列基序的RNA靶标。以脱辅基形式分离出ZRANB2的三种构建体，即ZRANB2-ZF1（第一个ZF结构域）、ZRANB2-ZF2（第二个ZF结构域）和ZRANB2-2D（两个ZF结构域），并通过以Co(II)作为光谱探针的紫外可见监测竞争滴定法显示它们能结合Zn(II)。通过圆二色性（CD）测量，锌与每个构建体的结合导致每个结构域采用有限的二级结构。两结构域构建体ZRANB2-2D的氢氘交换联用质谱（HDX-MS）表明，两个ZF结构域在结合锌后都采用了更刚性的结构。锌与第一个ZF结构域的结合导致构象动力学的下降比锌与第二个ZF结构域的结合更大。通过荧光各向异性（FA）测量了RNA与生理剪接靶标TRA2B前体mRNA的结合，发现高亲和力RNA结合需要两个结构域都与锌配位。以TRA2B RNA以及包含单链和双链AGGUAA六聚体的两个优化RNA序列对ZRANB2-2D进行HDX-MS，结果显示在存在RNA的情况下，ZRANB2对H/D交换有额外的保护作用。在此，观察到ZRANB2-2D的第二个ZF有更大的保护作用，表明对构象动力学有更大的影响。本文提出了一个锌介导的ZRANB2 RNA结合模型。
