Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium

Abstract

Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit.optogenetics | X-ray crystallography | blue light | allostery

Molecular mechanism of photoactivation of a light-regulated adenylate cyclase

“…The conserved Tyr-125 of OaPAC, at the junction between the α3 helix and the AC domains, forms an intersubunit hydrogen bond with Asn-256B (8). OaPAC(Y125F) and OaPAC(Y125C) show no activity, yet OaPAC(Y125S) behaves normally.…”

“…This change leads to different downstream effects in different proteins; for example in BlrP1, it affects the coordination of essential metal ions at the active site of the neighboring domain (7). The BLUF domain of OaPAC (residues 1-100) shows similarities to BlrP1, but the mechanism of signal transmission to the active site must be entirely different because it involves mutual interactions of the two BLUF domains (8). The crystal structure of OaPAC in the dark state (PDB 4YUS) revealed that the BLUF domains sit at one end of a coiled coil (residues 103-125), with the AC domains at the other end (residues 126-350).…”

“…The two active sites are found over 45 Å from the FMN chromophores, which are themselves about 35 Å apart. Site-directed mutagenesis further suggested that the BLUF domains are able to elicit enzyme activity via the central coiled coil with only minor conformational changes (8). However, the lack of a crystal structure of the photoactivated form restricted the conclusions that could be drawn regarding the molecular changes at the flavin chromophore and how these might be communicated to the active site.…”

“…In BlrP1, a light-regulated cyclic nucleotide phosphodiesterase from Klebsiella pneumonia (6, 7), the two BLUF domains are not in mutual contact, and photoactivation produces a fourfold increase in enzyme activity. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a homodimer, with each subunit of 366 residues carrying an N-terminal BLUF domain and a C-terminal class IIIb adenylate cyclase (AC) domain (8). OaPAC shows a much stronger activity increase on light exposure than BlrP1, and its structure suggests that the two BLUF domains can function more cooperatively through a mutual interface.…”

“…The known structures show roughly 20 residues are highly conserved over the ferredoxin-like fold, which is ≈100 residues long. In OaPAC, a helix on the opposite face of the β-sheet forms a coiled coil with its symmetry mate in the dimer, so that any communication between the two bound FMN molecules must occur through several layers of secondary structure (8). Conserved tyrosine, glutamine, and methionine residues are found at the flavin binding site of all BLUF domains, equivalent to Tyr-6, Gln-48, and Met-92 in OaPAC.…”