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

Abstract

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.

Diversity of rhodopsin cyclases in zoospore-forming fungi

“…In RgRGC1, the late L-intermediate likely corresponds to the active state of the enzyme. We also note that the homodimeric RGCs from Rhizophydiales identified here have short N-termini, whereas the long cytoplasmic N terminus of CaRGC has been shown to contribute to light activation of the enzyme ( 20 ). Thus, a detailed mechanism of how light-triggered enzyme activation occurs in various RGCs needs to be further analyzed in future studies.…”

“…This raises the question of what prevents the full-length RgRGC1 to function as a homodimer. Notably, the photocycle of RgRGC1 reveals no M-like state with deprotonated RSB, which has been proposed to be the enzyme-activating state in homodimeric CaRGC ( 3 , 19 , 20 ). However, after fusion of the RgRGC1 cyclase (RGC(a) heterodimer, Fig.…”

“…Furthermore, the primary signal-propagating pathway that couples the light-induced retinal isomerization to enzyme activation is obviously conserved in both RGB(b) and RGC(a). In homodimeric CaRGC (RGC(b)), light-activation of the enzyme has been attributed to the M-like state of the retinal photocycle ( 3 , 20 ), which is known to be a consequence of larger conformational changes that cause deprotonation of the chromophore in many microbial rhodopsins. Notably, RgRGC1 (RGC(a)) has a prolonged L-like state like CaRGC but lacks the M-like photo intermediate.…”

Far-Red Absorbing Rhodopsins, Insights From Heterodimeric Rhodopsin-Cyclases

“…Enzymerhodopsins, particularly RGC sequences, have a prolonged N-terminus preceding the additional transmembrane α-helix (Figures 2A, 6D). For homodimeric RGCs, the cytoplasmic N-terminus is involved in enzyme activation (Fischer et al, 2021) and impaired light-regulation with high-dark activity was observed upon its truncation (Gao et al, 2015). Remarkably, all three RGCs of R. globosum have significantly shorter N-termini than homodimeric RGCs, and future experiments may explore the impact of this part on functional heterodimerization.…”