Box jellyfish and vertebrates are separated by >500 million years of evolution yet have structurally analogous lens eyes that employ rhodopsin photopigments for vision. All opsins possess a negatively charged residue—the counterion—to maintain visible-light sensitivity and facilitate photoisomerization of their retinaldehyde chromophore. In vertebrate rhodopsins, the molecular evolution of the counterion position—from a highly conserved distal location in the second extracellular loop (E181) to a proximal location in the third transmembrane helix (E113)—is established as a key driver of higher fidelity photoreception. Here, we use computational biology and heterologous action spectroscopy to determine whether the appearance of the advanced visual apparatus in box jellyfish was also accompanied by changes in the opsin tertiary structure. We found that the counterion in an opsin from the lens eye of the box jellyfish Carybdea rastonii (JellyOp) has also moved to a unique proximal location within the transmembrane bundle—E94 in TM2. Furthermore, we reveal that this Schiff base/counterion system includes an additional positive charge—R186—that has coevolved with E94 to functionally separate E94 and E181 in the chromophore-binding pocket of JellyOp. By engineering this pocket—neutralizing R186 and E94, or swapping E94 with the vertebrate counterion E113—we can recreate versions of the invertebrate and vertebrate counterion systems, respectively, supporting a relatively similar overall architecture in this region of animal opsins. In summary, our data establish the third only counterion site in animal opsins and reveal convergent evolution of tertiary structure in opsins from distantly related species with advanced visual systems.

箱形水母和脊椎动物相隔超过 5 亿年的进化，但具有结构相似的晶状体眼睛，它们利用视紫红质光色素进行视觉。所有视蛋白都具有带负电荷的残基 - 抗衡离子 - 以保持可见光敏感性并促进其视黄醛发色团的光异构化。在脊椎动物视紫红质中，反离子位置的分子进化——从第二个细胞外环 (E181) 中高度保守的远端位置到第三跨膜螺旋 (E113) 中的近端位置——被确定为更高保真度光感受的关键驱动因素。在这里，我们使用计算生物学和异源作用光谱来确定箱形水母中高级视觉装置的出现是否也伴随着视蛋白三级结构的变化。Carybdea rastonii (JellyOp) 也移动到跨膜束内的一个独特的近端位置——TM2 中的 E94。此外，我们揭示了这种席夫碱/反离子系统包括一个额外的正电荷——R186——它与 E94 共同进化以在 JellyOp 的发色团结合口袋中功能分离 E94 和 E181。通过设计这个口袋——中和 R186 和 E94，或将 E94 与脊椎动物抗衡离子 E113 交换——我们可以分别重建无脊椎动物和脊椎动物抗衡离子系统的版本，从而支持该动物视蛋白区域中相对相似的整体架构。总之，我们的数据在动物视蛋白中建立了第三个唯一的反离子位点，并揭示了具有先进视觉系统的远亲物种视蛋白三级结构的趋同进化。