Structural analysis of the regulatory GAF domains of cGMP phosphodiesterase elucidates the allosteric communication pathway

Regulation of photoreceptor phosphodiesterase (PDE6) activity is responsible for the speed, sensitivity, and recovery of the photoresponse during visual signaling in vertebrate photoreceptor cells. It is hypothesized that the physiological differences in the light responsiveness of rods and cones may result in part from differences in the structure and regulation of the distinct isoforms of rod and cone PDE6. Although rod and cone PDE6 catalytic subunits share a similar domain organization consisting of tandem GAF domains (GAFa and GAFb) and a catalytic domain, cone PDE6 is a homodimer whereas rod PDE6 consists of two homologous catalytic subunits. Here we provide the x-ray crystal structure of cone GAFab regulatory domain solved at 3.3 Å resolution, in conjunction with chemical cross-linking and mass spectrometric analysis of conformational changes to GAFab induced upon binding of cGMP and the PDE6 inhibitory γ-subunit (Pγ). Ligand-induced changes in cross-linked residues implicate multiple conformational changes in the GAFa and GAFb domains in forming an allosteric communication network that communicates with the PDE6 catalytic domains. Molecular dynamics (MD) simulations of cone GAFab revealed asymmetry in the two GAFab subunits forming the homodimer and allosteric perturbations on cGMP binding. Cross-linking of Pγ to GAFab in conjunction with solution NMR spectroscopy of isotopically labeled Pγ identified the central polycationic region of Pγ interacting with the GAFb domain. These results provide a mechanistic basis for developing allosteric activators of PDE6 with therapeutic implications for halting the progression of several retinal degenerative diseases.

脊椎动物感光细胞的视觉信号传导过程中，感光磷酸二酯酶（photoreceptor phosphodiesterase, PDE6）的活性调控决定了光响应的速度、灵敏度与恢复能力。有假说认为，视杆细胞与视锥细胞的光响应生理差异，部分源于二者各自PDE6同工酶在结构与调控机制上的区别。尽管视杆与视锥PDE6的催化亚基拥有相似的结构域排布：包含串联的GAF结构域（GAFa与GAFb）以及一个催化结构域，但视锥PDE6为同源二聚体，而视杆PDE6则由两个同源催化亚基组成。本研究解析了分辨率为3.3埃的视锥GAFab调控结构域的X射线晶体结构，并结合化学交联与质谱分析，探究了环磷酸鸟苷（cyclic guanosine monophosphate, cGMP）与PDE6抑制性γ亚基（Pγ）结合后，GAFab发生的构象变化。配体诱导的交联残基变化表明，GAFa与GAFb结构域发生的多重构象变化，共同构成了与PDE6催化结构域进行信号传递的变构通讯网络。视锥GAFab的分子动力学（molecular dynamics, MD）模拟结果显示，构成同源二聚体的两个GAFab亚基存在不对称性，且cGMP结合会引发变构扰动。通过Pγ与GAFab的交联实验，结合同位素标记Pγ的溶液核磁共振（nuclear magnetic resonance, NMR）光谱分析，研究人员确定了Pγ的中央多阳离子区域与GAFb结构域存在相互作用。本研究结果为开发PDE6变构激活剂提供了机制层面的理论基础，这对阻断多种视网膜退行性疾病的进展具有潜在治疗价值。