Bioinformatics Institute (A*STAR) , 30 Biopolis Street, 07-01 Matrix, Singapore 138671.
Biochemistry. 2013 Oct 15;52(41):7339-49. doi: 10.1021/bi400912e. Epub 2013 Oct 1.
ADP ribosylation factor nucleotide site opener (ARNO) as a guanine nucleotide exchange factor (GEF) activates small GTPases called ADP ribosylation factors (Arfs), which function as molecular switches and regulate a variety of cell biological events. ARNO directly interacts with the transmembrane a2-subunit isoform of the proton-pumping vacuolar ATPase in an acidification-dependent manner, and this interaction plays a crucial role in the regulation of the protein degradation pathway. A recent study reported specific interactions of a2N with the ARNO375-400 peptide corresponding to the polybasic (PB) domain of ARNO, which is a crucial regulatory element in the autoregulation and modulation of Arf-GEF activity. Interestingly, phosphorylation of Ser392 completely abolishes this interaction, and the experimental structure shows significant structural rearrangements. To investigate the effect of Ser392 phosphorylation on the structure and dynamics of the ARNO375-400 peptide, we employed all atom molecular dynamics (MD) simulations of the phosphorylated and unphosphorylated PB domain of the ARNO protein. A Hamiltonian-based replica exchange method called biasing potential replica exchange MD was used to enhance conformational sampling. Simulations predicted that the isolated PB domain is highly flexible, with the C-terminal region of the unphosphorylated state being unstable. In contrast, Ser392 phosphorylation increases the overall stability of the peptide. In agreement with experimental results, our simulations further support the hypothesis that phosphorylation induces disorder to order transitions and provide new insights into the structural dynamics of the PB domain. Phosphorylation of Ser392 appears to stabilize the C-terminal α-helix via formation of salt bridges between phospho-Ser392 and Arg390, Lys395, and Lys396.
ADP 核糖基化因子核苷酸位点 opener（ARNO）作为鸟嘌呤核苷酸交换因子（GEF），激活称为 ADP 核糖基化因子（Arfs）的小 GTP 酶，它们作为分子开关调节各种细胞生物学事件。ARNO 以酸化依赖的方式直接与质子泵 vacuolar ATPase 的跨膜 a2 亚基同工型相互作用，这种相互作用在调节蛋白降解途径中起着至关重要的作用。最近的一项研究报告了 a2N 与 ARNO375-400 肽的特定相互作用，该肽对应于 ARNO 的多碱性（PB）结构域，这是 Arf-GEF 活性的自身调节和调制的关键调节元件。有趣的是，丝氨酸 392 的磷酸化完全消除了这种相互作用，实验结构显示出明显的结构重排。为了研究丝氨酸 392 磷酸化对 ARNO375-400 肽的结构和动力学的影响，我们采用了全原子分子动力学（MD）模拟 ARNO 蛋白的磷酸化和非磷酸化 PB 结构域。采用基于哈密顿的复制交换方法——偏置势能复制交换 MD 来增强构象采样。模拟预测，孤立的 PB 结构域具有高度的灵活性，非磷酸化状态的 C 末端区域不稳定。相比之下，丝氨酸 392 的磷酸化增加了肽的整体稳定性。与实验结果一致，我们的模拟进一步支持了磷酸化诱导无序到有序转变的假设，并提供了对 PB 结构域结构动力学的新见解。丝氨酸 392 的磷酸化似乎通过形成磷酸化丝氨酸 392 与 Arg390、Lys395 和 Lys396 之间的盐桥，稳定 C 末端α-螺旋。
