Abstract

Photoactivated adenylyl cyclase (PAC) is a unique protein that, upon blue light exposure, catalyzes cAMP production. The crystal structures of two PACs, from Oscillatoria acuminata (OaPAC) and Beggiatoa sp. (bPAC), have been solved, and they show a high degree of similarity. However, the photoactivity of OaPAC is much lower than that of bPAC, and the regulatory mechanism of PAC photoactivity, which induces the difference in activity between OaPAC and bPAC, has not yet been clarified. Here, we investigated the role of the C-terminal region in OaPAC, the length of which is the only notable difference from bPAC. We found that the photoactivity of OaPAC was inversely proportional to the C-terminal length. However, the deletion of more than nine amino acids did not further increase the activity, indicating that the nine amino acids at the C-terminal critically affect the photoactivity. Besides, absorption spectral features of light-sensing domains (BLUF domains) of the C-terminal deletion mutants showed similar light-dependent spectral shifts as in WT, indicating that the C-terminal region influences the activity without interacting with the BLUF domain. The study characterizes new PAC mutants with modified photoactivities, which could be useful as optogenetics tools.

Highlights

  • Photoactivated adenylyl cyclase (PAC) is a unique protein that, upon blue light exposure, catalyzes cAMP production

  • We aimed to investigate the mechanism of regulating adenylyl cyclase activity in OaPAC, focusing on the C-terminal region

  • A conserved residue closest to the C-terminus in OaPAC, bPAC, and PAC α-chains from Euglena gracilis (PACα) is a glutamic acid (E348 at OaPAC)

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Summary

Introduction

Photoactivated adenylyl cyclase (PAC) is a unique protein that, upon blue light exposure, catalyzes cAMP production. C-terminal deletion mutants showed similar light-dependent spectral shifts as in WT, indicating that the C-terminal region influences the activity without interacting with the BLUF domain. (bPAC)[5,6,7,8,9,10], which have expanded the use of PAC as an optogenetic tool and in the control of intracellular cAMP levels by blue light in hippocampal neurons, Caenorhabditis elegans, pancreatic β-cells, etc.[7,10,11,12,13,14]. The structures of OaPAC and bPAC are similar, both forming homo-dimers, and each subunit having the BLUF domain at the N-terminus and AC domain at the C-terminus (10,15, Fig. 1a). An active site is located in the interface between AC domains, where cAMP is produced from ATP

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