Noval María G, Esperante Sebastian A, Molina Ivana G, Chemes Lucía B, Prat-Gay Gonzalo de
Protein Structure-Function and Engineering Laboratory, Fundación Instituto Leloir and IIBBA-CONICET , Av. Patricias Argentinas 435, 1405 Buenos Aires, Argentina.
CNPq, Laboratório de Genômica Estrutural, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro , Rio de Janeiro, RJ, Brazil.
Biochemistry. 2016 Mar 15;55(10):1441-54. doi: 10.1021/acs.biochem.5b01332. Epub 2016 Mar 4.
Intrinsic disorder is at the center of biochemical regulation and is particularly overrepresented among the often multifunctional viral proteins. Replication and transcription of the respiratory syncytial virus (RSV) relies on a RNA polymerase complex with a phosphoprotein cofactor P as the structural scaffold, which consists of a four-helix bundle tetramerization domain flanked by two domains predicted to be intrinsically disordered. Because intrinsic disorder cannot be reduced to a defined atomic structure, we tackled the experimental dissection of the disorder-order transitions of P by a domain fragmentation approach. P remains as a tetramer above 70 °C but shows a pronounced reversible secondary structure transition between 10 and 60 °C. While the N-terminal module behaves as a random coil-like IDP in a manner independent of tetramerization, the isolated C-terminal module displays a cooperative and reversible metastable transition. When linked to the tetramerization domain, the C-terminal module becomes markedly more structured and stable, with strong ANS binding. Therefore, the tertiary structure in the C-terminal module is not compact, conferring "late" molten globule-like IDP properties, stabilized by interactions favored by tetramerization. The presence of a folded structure highly sensitive to temperature, reversibly and almost instantly formed and broken, suggests a temperature sensing activity. The marginal stability allows for exposure of protein binding sites, offering a thermodynamic and kinetic fine-tuning in order-disorder transitions, essential for the assembly and function of the RSV RNA polymerase complex.
内在无序在生化调节中处于核心地位，并且在通常具有多种功能的病毒蛋白中尤为突出。呼吸道合胞病毒（RSV）的复制和转录依赖于一种RNA聚合酶复合物，该复合物以磷蛋白辅因子P作为结构支架，它由一个四螺旋束四聚化结构域组成，两侧是两个预测为内在无序的结构域。由于内在无序无法简化为确定的原子结构，我们通过结构域片段化方法对P的无序-有序转变进行了实验剖析。P在70℃以上仍保持四聚体状态，但在10℃至60℃之间显示出明显的可逆二级结构转变。虽然N端模块表现为随机卷曲样的内在无序蛋白，其行为与四聚化无关，但分离的C端模块显示出协同且可逆的亚稳态转变。当与四聚化结构域相连时，C端模块变得明显更具结构且更稳定，具有强烈的ANS结合。因此，C端模块中的三级结构并不紧凑，赋予其“晚期”熔球样内在无序蛋白特性，通过四聚化所促进的相互作用得以稳定。存在对温度高度敏感、可逆且几乎瞬间形成和破坏的折叠结构，表明具有温度传感活性。这种边缘稳定性允许蛋白质结合位点暴露，在无序-有序转变中提供热力学和动力学的精细调节，这对RSV RNA聚合酶复合物的组装和功能至关重要。
