The inner mechanics of rhodopsin guanylyl cyclase during cGMP-formation revealed by real-time FTIR spectroscopy

Enzymerhodopsins represent a recently discovered class of rhodopsins which includes histidine kinase rhodopsin, rhodopsin phosphodiesterases and rhodopsin guanylyl cyclases (RGCs). The regulatory influence of the rhodopsin domain on the enzyme activity is only partially understood and holds the key for a deeper understanding of intra-molecular signaling pathways. Here we present a UV-Vis and FTIR study about the light-induced dynamics of a RGC from the fungus Catenaria anguillulae, which provides insights into the catalytic process. After the spectroscopic characterization of the late rhodopsin photoproducts, we analyzed truncated variants and revealed the involvement of the cytosolic N-terminus in the structural rearrangements upon photo-activation of the protein. We tracked the catalytic reaction of RGC and the free GC domain independently by UV-light induced release of GTP from the photolabile NPE-GTP substrate. Our results show substrate binding to the dark-adapted RGC and GC alike and reveal differences between the constructs attributable to the regulatory influence of the rhodopsin on the conformation of the binding pocket. By monitoring the phosphate rearrangement during cGMP and pyrophosphate formation in light-activated RGC, we were able to confirm the M state as the active state of the protein. The described setup and experimental design enable real-time monitoring of substrate turnover in light-activated enzymes on a molecular scale, thus opening the pathway to a deeper understanding of enzyme activity and protein-protein interactions.

Methods
Preparation

To prepare samples for FTIR, 500 µl of the initial protein solution (1 OD) was concentrated with an Amicon Ultra 10 kDa centrifugal filter to a final OD of ~33 at 540 nm. 10-15 µl of this solution was then placed on a BaF2 window and concentrated by evaporating solvent water under a stream of dry air. For the CaRGC protein, this step has to be performed with much care to prevent complete dehydration of the sample, since it induces irreversible denaturation of the protein. Samples were then sealed with a second BaF2 window. To ensure reproducible and constant sample thickness, a 6 µm PTFE spacer was placed between the windows.

For measurements between 900 and 1800 cm−1, an optical cutoff filter at 1850 cm−1 was placed in the beamline. The spectral resolution was 2 cm−1. Illumination was performed with a pulsed laser for uncaging experiments (330 nm, 6 ns, 10 Hz, 30 mJ per pulse) and time resolved measurements (532 nm). While for continuous illumination a 50 mW continuous-wave (CW), a laser with an output maximum of 532 nm was used (no. 37028, Edmund Optics, York, UK). LED illumination was performed with a set of 520 nm LEDs with a FWHM>20 nm. 

Acquired data was initially processed using OPUS 7.5 software, whereas further processing, including baseline correction with a linear function and pre-spline as well as SVD and global fit procedures, was performed by a customized software developed for Octave 5.1.0.0 initially conceived by Dr. Eglof Ritter.

Enzyme turnover

All samples were prepared under red light >640 nm. Caged compounds NPE-GTP and NPE-ATP were purchased from Jena Bioscience GmbH (Jena, Germany). For reference, measurements on GTP, cGMP and PP, as well as the caged ATP and GTP compounds the substrate to Mn2+ ratio was 1:2. For measurements of the catalytic activity, the caged compound (NPE-GTP/ATP 50 µl, 10 mM) and manganese (MnCl2·4H2O 10 µl, 100 mM) were added to the diluted protein solution (1 OD) to ensure sufficient diffusion. After an incubation period of 30 min in the dark, the sample was concentrated with an Amicon Ultra 10 kDa centrifugal filter as described by the manufacturer.

RGC homology model

The CaRGC-43 model was generated using CHARMM and PyMol 2.5 based on a homology model of its rhodopsin and linker domains. The crystal structures of the rhodopsin phosphodiesterase from Salpingoeca rosetta SrRhoPDE (PDB-IDs: 7CJ3, 7D7Q)34 served as templates for homology modelling using the online platforms for protein structure prediction of Swiss-Modell and Robetta. The crystal waters and the orientation of both protomers were adopted from the template structures.

Secondary structure prediction on the full-length CaRGC sequence in JPred4 helped to identify several additional N- and C-terminal features besides the 7-TM-rhodopsin or GC domains as the following: an additional TM-helix 0, an elongated TM-helix 7, short helices on both the N-terminus (helix -1) and the C-terminus (helix 8) and short N-terminal β-sheets. These structures were modelled using CHARMM and then oriented and linked in PyMol in which the cryo-EM maps of the NO-activated human soluble guanylate cyclase (sGC) served as a template (EMDB-ID: EMD-9885). The final 43-truncated rhodopsin domain was linked to the crystal structure of the guanylate cyclase domain of RhGC in complex with GTP (PDB-ID: 6SIR) using PyMol.

酶视紫红质（Enzymerhodopsins）是近年发现的一类视紫红质，涵盖组氨酸激酶视紫红质、视紫红质磷酸二酯酶以及视紫红质鸟苷酸环化酶（RGCs）。目前学界对该视紫红质结构域对酶活性的调控作用仅部分阐明，而这正是深入解析分子内信号转导通路的关键所在。本研究针对来自真菌美丽筒壶菌（Catenaria anguillulae）的RGC开展了紫外-可见（UV-Vis）与傅里叶变换红外（FTIR）光谱研究，揭示其光诱导动力学过程，为催化机制提供新见解。在对晚期视紫红质光产物完成光谱表征后，我们通过截短变体分析，发现胞质N端参与了蛋白光激活后的构象重排。我们借助紫外光诱导光不稳定底物NPE-GTP释放三磷酸鸟苷（GTP），分别追踪了RGC与游离GC结构域的催化反应。结果表明，暗适应状态下的RGC与GC均可结合底物，并揭示不同构建体间的差异源于视紫红质对结合口袋构象的调控作用。通过监测光激活RGC过程中环磷酸鸟苷（cGMP）与焦磷酸（PPi）形成时的磷酸重排，我们确认M态为该蛋白的活性构象。本研究采用的实验装置与设计可实现分子尺度下光激活酶底物周转的实时监测，为深入理解酶活性与蛋白质-蛋白质相互作用开辟了新路径。

实验方法

样品制备
针对傅里叶变换红外（FTIR）光谱测试，取500 μl初始蛋白溶液（吸光度1 OD），使用Amicon Ultra 10 kDa离心超滤管浓缩至540 nm波长下吸光度约为33。随后取10~15 μl该溶液滴加至氟化钡（BaF₂）窗片上，通过干燥空气流蒸发溶剂水进行浓缩。针对CaRGC蛋白，该步骤需格外小心以避免样品完全脱水，否则会导致蛋白不可逆变性。随后用第二块BaF₂窗片密封样品。为保证样品厚度可重复且恒定，在两块窗片之间放置6 μm聚四氟乙烯（PTFE）垫片。

若测量范围为900~1800 cm⁻¹，需在光束路径中放置1850 cm⁻¹的光学截止滤光片，光谱分辨率设为2 cm⁻¹。光照射实验分为两类：光笼解实验使用脉冲激光（330 nm，6 ns脉冲宽度，10 Hz重复频率，单脉冲能量30 mJ），时间分辨测量则使用532 nm激光。连续光照实验采用输出峰值为532 nm的50 mW连续波（CW）激光（货号37028，Edmund Optics，英国约克）。LED光照则使用一组半高宽>20 nm的520 nm LED光源。

采集的数据最初使用OPUS 7.5软件进行预处理，后续处理（包括线性函数基线校正、预样条拟合、奇异值分解（SVD）以及全局拟合流程）则由针对Octave 5.1.0.0开发的定制化软件完成，该软件最初由Eglof Ritter博士研发。

酶活性周转实验
所有样品均在波长>640 nm的红光下制备。光笼化合物NPE-三磷酸鸟苷（NPE-GTP）与NPE-三磷酸腺苷（NPE-ATP）购自德国耶拿的Jena Bioscience GmbH公司。作为对照，针对三磷酸鸟苷（GTP）、环磷酸鸟苷（cGMP）以及焦磷酸（PPi）的测量中，底物与锰离子（Mn²⁺）的摩尔比为1:2。针对催化活性测量，将光笼化合物（NPE-GTP/ATP 50 μl，浓度10 mM）与氯化锰（MnCl₂·4H₂O 10 μl，浓度100 mM）加入稀释后的蛋白溶液（1 OD）中，以保证底物充分扩散。将样品在黑暗中孵育30分钟后，按照制造商说明使用Amicon Ultra 10 kDa离心超滤管进行浓缩。

RGC同源建模
CaRGC-43模型通过CHARMM与PyMol 2.5基于其视紫红质结构域与连接结构域的同源模型构建。以玫瑰领鞭毛虫（Salpingoeca rosetta）视紫红质磷酸二酯酶的晶体结构（PDB编号：7CJ3、7D7Q）³⁴作为模板，通过Swiss-Model与Robetta在线蛋白质结构预测平台完成同源建模。模板结构中的结晶水与两个原聚体的取向均直接沿用。

通过JPred4对全长CaRGC序列进行二级结构预测，除7次跨膜（7-TM）视紫红质结构域与GC结构域外，还鉴定出多个额外的N端与C端结构特征：额外的TM螺旋0、延长的TM螺旋7、N端（螺旋-1）与C端（螺旋8）的短螺旋，以及N端的短β折叠片。这些结构通过CHARMM建模后，在PyMol中进行定向与连接，其中以NO激活的人可溶性鸟苷酸环化酶（sGC）的冷冻电镜图谱（EMDB编号：EMD-9885）作为参考模板。最终将截短43个氨基酸的视紫红质结构域，与结合GTP的RhGC鸟苷酸环化酶结构域晶体结构（PDB编号：6SIR）通过PyMol进行连接。