Departments of Physiology and Biophysics and
Departments of Physiology and Biophysics andChemistry, University of Washington, Seattle, WA 98195
Proc Natl Acad Sci U S A. 2014 Jul 8;111(27):9816-21. doi: 10.1073/pnas.1405371111. Epub 2014 Jun 23.
Binding of 3',5'-cyclic adenosine monophosphate (cAMP) to hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels regulates their gating. cAMP binds to a conserved intracellular cyclic nucleotide-binding domain (CNBD) in the channel, increasing the rate and extent of activation of the channel and shifting activation to less hyperpolarized voltages. The structural mechanism underlying this regulation, however, is unknown. We used double electron-electron resonance (DEER) spectroscopy to directly map the conformational ensembles of the CNBD in the absence and presence of cAMP. Site-directed, double-cysteine mutants in a soluble CNBD fragment were spin-labeled, and interspin label distance distributions were determined using DEER. We found motions of up to 10 Å induced by the binding of cAMP. In addition, the distributions were narrower in the presence of cAMP. Continuous-wave electron paramagnetic resonance studies revealed changes in mobility associated with cAMP binding, indicating less conformational heterogeneity in the cAMP-bound state. From the measured DEER distributions, we constructed a coarse-grained elastic-network structural model of the cAMP-induced conformational transition. We find that binding of cAMP triggers a reorientation of several helices within the CNBD, including the C-helix closest to the cAMP-binding site. These results provide a basis for understanding how the binding of cAMP is coupled to channel opening in HCN and related channels.
3'，5'-环磷酸腺苷（cAMP）与超极化激活环核苷酸门控（HCN）离子通道的结合调节其门控。cAMP 与通道中保守的细胞内环核苷酸结合域（CNBD）结合，增加通道的激活速率和程度，并将激活转移到更去极化的电压。然而，这种调节的结构机制尚不清楚。我们使用双电子-电子共振（DEER）光谱技术直接绘制了 CNBD 在没有和存在 cAMP 时的构象总体。在可溶性 CNBD 片段中的定点双半胱氨酸突变体被自旋标记，并使用 DEER 确定了自旋标记之间的距离分布。我们发现 cAMP 结合诱导了高达 10 Å 的运动。此外，在存在 cAMP 的情况下，分布更窄。连续波电子顺磁共振研究揭示了与 cAMP 结合相关的迁移率变化，表明 cAMP 结合状态的构象异质性较小。根据测量的 DEER 分布，我们构建了 cAMP 诱导构象转变的粗粒度弹性网络结构模型。我们发现，cAMP 的结合引发了 CNBD 内几个螺旋的重新取向，包括最接近 cAMP 结合位点的 C 螺旋。这些结果为理解 cAMP 的结合如何与 HCN 和相关通道的通道开放偶联提供了基础。
