Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche (Alicante), Spain; Instituto de Biocomputación y Física de Sistemas Complejos (BIFI), Joint Units IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Universidad de Zaragoza, Zaragoza, Spain.
Centro de Investigación Príncipe Felipe, 41930, Valencia, Spain.
Biochim Biophys Acta Proteins Proteom. 2019 Nov;1867(11):140252. doi: 10.1016/j.bbapap.2019.07.005. Epub 2019 Jul 17.
Intrinsically disordered proteins (IDPs) explore diverse conformations in their free states and, a few of them, also in their molecular complexes. This functional plasticity is essential for the function of IDPs, although their dynamics in both free and bound states is poorly understood. NUPR1 is a protumoral multifunctional IDP, activated during the acute phases of pancreatitis. It interacts with DNA and other IDPs, such as prothymosin α (ProTα), with dissociation constants of ~0.5 μM, and a 1:1 stoichiometry. We studied the structure and picosecond-to-nanosecond (ps-ns) dynamics by using both NMR and SAXS in: (i) isolated NUPR1; (ii) the NUPR1/ProTα complex; and (iii) the NUPR1/double stranded (ds) GGGCGCGCCC complex. Our SAXS findings show that NUPR1 remained disordered when bound to either partner, adopting a worm-like conformation; the fuzziness of bound NUPR1 was also pinpointed by NMR. Residues with the largest values of the relaxation rates (R, R, R and η), in the free and bound species, were mainly clustered around the 30s region of the sequence, which agree with one of the protein hot-spots already identified by site-directed mutagenesis. Not only residues in this region had larger relaxation rates, but they also moved slower than the rest of the molecule, as indicated by the reduced spectral density approach (RSDA). Upon binding, the energy landscape of NUPR1 was not funneled down to a specific, well-folded conformation, but rather its backbone flexibility was kept, with distinct motions occurring at the hot-spot region.
无规卷曲蛋白（IDPs）在其自由状态下探索多种构象，其中一些在其分子复合物中也是如此。这种功能灵活性对于 IDPs 的功能至关重要，尽管它们在自由和结合状态下的动力学仍了解甚少。NUPR1 是一种促肿瘤多功能 IDP，在胰腺炎的急性阶段被激活。它与 DNA 和其他 IDPs 相互作用，如胸腺素前体 α（ProTα），解离常数约为 0.5μM，且为 1:1 比例。我们使用 NMR 和 SAXS 研究了结构和皮秒到纳秒（ps-ns）动力学：（i）分离的 NUPR1；（ii）NUPR1/ProTα 复合物；和（iii）NUPR1/双链（ds）GGGCGCGCCC 复合物。我们的 SAXS 研究结果表明，NUPR1 与任一伴侣结合时仍然保持无序状态，采用蠕虫样构象；NMR 也指出了结合 NUPR1 的模糊性。在游离和结合物种中，具有最大弛豫率（R、R、R 和 η）的残基主要聚集在序列的 30s 区域周围，这与通过定点突变已鉴定的蛋白质热点之一一致。不仅该区域的残基具有更大的弛豫率，而且它们的运动速度也比分子的其余部分慢，如减少的光谱密度方法（RSDA）所示。结合后，NUPR1 的能量景观并没有被引导到特定的、完全折叠的构象，而是保持其构象柔性，在热点区域发生明显的运动。
