Key Residues for the Light Regulation of the Blue Light-Activated Adenylyl Cyclase from Beggiatoa sp.

Abstract

Photoactivated adenylyl cyclases are powerful tools for optogenetics and for investigating signal transduction mechanisms in biological photoreceptors. Because of its large increase in enzyme activity in the light, the BLUF (blue light sensor using flavin adenine dinucleotide)-activated adenylyl cyclase (bPAC) from Beggiatoa sp. is a highly attractive model system for studying BLUF domain signaling. In this report, we studied the influence of site-directed mutations within the BLUF domain on the light regulation of the cyclase domain and determined key elements for signal transduction and color tuning. Photoactivation of the cyclase domain is accomplished via strand β5 of the BLUF domain and involves the formation of helical structures in the cyclase domain as assigned by vibrational spectroscopy. In agreement with earlier studies, we observed severely impaired signaling in mutations directly on strand β5 as well as in mutations affecting the hydrogen bond network around the flavin. Moreover, we identified a bPAC mutant with red-shifted absorbance and a decreased dark activity that is highly valuable for long-term optogenetic experiments. Additionally, we discovered a mutant that forms a stable neutral flavin semiquinone radical in the BLUF domain and surprisingly exhibits an inversion of light activation.

Molecular mechanism of photoactivation of a light-regulated adenylate cyclase

“…No protein was detected in any buffer sample from which the crystal had been removed, showing all enzyme activity must come from protein within the crystal. FTIR spectra of bPAC, which they interpreted as indicating a major transformation from random coil to helical, and suggested this possibly created a functioning active site (38). Our results appear to rule out any such marked rearrangement, in OaPAC at least.…”

“…It remains unclear how the imidic tautomer is maintained for any length of time, given the roughly 40 kJ/mol higher energy (36), but the stronger hydrogen bonds formed by Gln-48 with Tyr-6 and the flavin must offset the energy cost to some extent, as will the stronger Asn-30 hydrogen bonds to the chromophore. Different BLUF proteins show different behavior if the conserved tryptophan is replaced with phenylalanine or alanine, with recovery of the dark state being accelerated in some cases and retarded in others, but dark-state activity is increased (23,37,38). A similar pseudolit state is found if the key methionine is replaced with alanine, which can also be explained by a change in the relative stability of different orientations of the tryptophan sidechain at the protein surface (16,38).…”

“…Different BLUF proteins show different behavior if the conserved tryptophan is replaced with phenylalanine or alanine, with recovery of the dark state being accelerated in some cases and retarded in others, but dark-state activity is increased (23,37,38). A similar pseudolit state is found if the key methionine is replaced with alanine, which can also be explained by a change in the relative stability of different orientations of the tryptophan sidechain at the protein surface (16,38). Some BLUF domains show alternative conformations with either the conserved methionine or tryptophan close to the flavin.…”

Seeing the light with BLUF proteins

“…However, the coiled-coil appears to be rigid in the crystal structure, and it is hard to imagine any substantial mechanical mechanism like that observed in haemoglobin. Light-minus-dark differences in the FTIR spectra of bPAC have been interpreted to indicate a significant tertiary change from random coil to helical (Stierl et al 2014). On the basis of the dark-state OaPAC model, the FTIR spectra of bPAC can be interpreted as a stiffening of the coiled-coil at the N-terminal end, which is sensed at the AC domain.…”

“…BlrP1 shows that tryptophan is not absolutely required for this process and that BLUF domains can operate without a shared interface. Different BLUF proteins show different behaviour if the conserved tryptophan is replaced with phenylalanine or alanine, with recovery of the dark state being accelerated in some cases and retarded in others, but dark-state activity is increased (Bonetti et al 2009;Laan et al 2006;Stierl et al 2014). A similar pseudo-lit state is found if the key methionine is replaced with alanine, which can also be explained by a change in the relative stability of different orientations of the tryptophan side-chain at the protein surface (Fudim et al 2015;Stierl et al 2014).…”