The Nedd4-1 WW Domain Recognizes the PY Motif Peptide through Coupled Folding and Binding Equilibria.
The Nedd4-1 WW Domain Recognizes the PY Motif Peptide through Coupled Folding and Binding Equilibria.
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DOI：
10.1021/acs.biochem.5b01028
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发表时间：
2016-01
期刊：
影响因子：
2.9
通讯作者：
Vineet Panwalkar;Philipp Neudecker;M. Schmitz;Justin Lecher;Marianne Schulte;Karima Medini;M. Stoldt
中科院分区：
文献类型：
--
作者：
Vineet Panwalkar;Philipp Neudecker;M. Schmitz;Justin Lecher;Marianne Schulte;Karima Medini;M. Stoldt
人Nedd4-1（神经元前细胞表达发育下调基因4-1）的四个WW结构域与人上皮Na（+）通道（hENaC）亚基的PPxY （PY）基相互作用，其中第三个WW结构域（WW3*）的亲和力最高。我们之前已经证明，WW3*的α-hENaC PY基序结合界面在毫秒时间尺度上发生了构象交换，表明构象采样在肽识别中起作用。为了进一步了解这一作用，我们研究了hNedd4-1 WW3*的结构和动力学。apo-WW3*的核Overhauser效应衍生的结构类似于α-hENaC肽的结构域，尽管特定的侧链构象在肽结合时发生了变化，这一点通过分子动力学模拟得到了进一步的研究。对（15）N核磁共振自旋弛豫数据的无模型分析表明，WW3*的载子和肽结合态具有相似的皮秒和纳秒骨干时间尺度动力学。然而，apo-WW3*在毫秒时间尺度上表现出明显的化学交换，这种交换在肽结合时被淬灭。(1)不同温度下的HN和（15）N carr - purcell - meiboomm - gill （CPMG）弛豫色散实验表明，apo-WW3*存在于天然折叠肽结合态和随机线圈状变性态之间的平衡状态。通过拟合圆二色光谱监测的热变性曲线并结合CPMG数据，确定了折叠平衡的热力学，得出在37°C时未折叠态填充到~ 20%的结论。这些结果表明，hNedd4-1 WW3*结构域与α-hENaC的结合与折叠平衡是耦合的。
The four WW domains of human Nedd4-1 (neuronal precursor cell expressed developmentally downregulated gene 4-1) interact with the PPxY (PY) motifs of the human epithelial Na(+) channel (hENaC) subunits, with the third WW domain (WW3*) showing the highest affinity. We have shown previously that the α-hENaC PY motif binding interface of WW3* undergoes conformational exchange on the millisecond time scale, indicating that conformational sampling plays a role in peptide recognition. To further understand this role, the structure and dynamics of hNedd4-1 WW3* were investigated. The nuclear Overhauser effect-derived structure of apo-WW3* resembles the domain in complex with the α-hENaC peptide, although particular side chain conformations change upon peptide binding, which was further investigated by molecular dynamics simulations. Model-free analysis of the (15)N nuclear magnetic resonance spin relaxation data showed that the apo and peptide-bound states of WW3* have similar backbone picosecond to nanosecond time scale dynamics. However, apo-WW3* exhibits pronounced chemical exchange on the millisecond time scale that is quenched upon peptide binding. (1)HN and (15)N Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments at various temperatures revealed that apo-WW3* exists in an equilibrium between the natively folded peptide binding-competent state and a random coil-like denatured state. The thermodynamics of the folding equilibrium was determined by fitting a thermal denaturation profile monitored by circular dichroism spectroscopy in combination with the CPMG data, leading to the conclusion that the unfolded state is populated to ∼ 20% at 37 °C. These results show that the binding of the hNedd4-1 WW3* domain to α-hENaC is coupled to the folding equilibrium.