Molecular mechanism of photoactivation of a light-regulated adenylate cyclase

Abstract

The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) detects light through a flavin chromophore within the N-terminal BLUF domain. BLUF domains have been found in a number of different light-activated proteins, but with different relative orientations. The two BLUF domains of OaPAC are found in close contact with each other, forming a coiled coil at their interface. Crystallization does not impede the activity switching of the enzyme, but flash cooling the crystals to cryogenic temperatures prevents the signature spectral changes that occur on photoactivation/deactivation. High-resolution crystallographic analysis of OaPAC in the fully activated state has been achieved by cryocooling the crystals immediately after light exposure. Comparison of the isomorphous light- and dark-state structures shows that the active site undergoes minimal changes, yet enzyme activity may increase up to 50-fold, depending on conditions. The OaPAC models will assist the development of simple, direct means to raise the cyclic AMP levels of living cells by light, and other tools for optogenetics.

Keywords: BLUF domain; allostery; cAMP; optogenetics; photoactivation.

Fig. 1.

Spectral properties of OaPAC in crystals and in solution. (A) Room-temperature absorption spectra of OaPAC in solution. Blue and red lines show the light- and dark-adapted states, respectively. The difference is indicated as a dotted black line. (B) Room-temperature absorption spectra of OaPAC in a single crystal. Spectra are colored as in A. (C) Absorption spectra of single cryocooled OaPAC crystals. (D) Formation and decay kinetics of the light-adapted state of OaPAC in a single crystal at room temperature. Activation was monitored by absorbance change at 492 nm. The blue line indicates photoactivation under irradiation with a 405-nm laser. The red line indicates relaxation after removing the stimulation. Inset shows the agreement between the absorption spectra of the crystal before (red) and after (blue) the kinetic measurement. (E) Fitting a single exponential to the photoexcitation kinetics. (F) Fitting a single exponential to the relaxation kinetics.

Fig. 2.

Structural changes on photoactivation of OaPAC. (A) The 2Fo–DFc electron density map, contoured at 1 σ, of the photoactivated OaPAC crystal after 20-s stimulation with blue light. Selected sidechains close to the flavin are shown as stick models. Carbon atoms are colored blue, oxygen atoms red. (B) The position of the photoactivated chromophore within the binding pocket. Hydrogen bonds between the FMN and protein are shown as red dotted lines. Distances are given in ångstroms. (C) A view of the chromophore in the dark-adapted resting state. Hydrogen bonds are shown as blue dotted lines, and carbon atoms are colored yellow. (D) An overlay of the resting and photoactivated structures, colored as in B and C, superposed by least-squares fitting Cα atoms of the BLUF domain. The view is expanded and rotated relative to A–C to show the movement of the surface Trp-90 residue, which contacts Arg-106B in the photoactive state. (E) The 2Fo–DFc electron density map contoured at 0.8 σ. The carbon atoms of one subunit are colored blue, and the other subunit marine blue. (F) An overlay of the dark-state (yellow) and photoactivated (blue) models, in the region around Trp-90 shown in E, but from a different angle, showing the absence of any major shift of secondary structure elements. (G) Ribbon diagram showing an overlay of the BLUF domain in the dark (yellow) and photoactivated (blue) states, looking along the dimer axis. (H) The same overlay shown in G, but looking along the dimer axis from the AC domains.

Fig. 3.

Domain motions on photoactivation. Difference distance map showing the relative motions of Cα atoms throughout the OaPAC dimer on exposure to light. Blue and red indicate movement nearer or farther, respectively, with color saturation indicating a difference of 0.5 Å or more. The BLUF and AC domains move very slightly as separate blocks, as shown by the regions highlighted with black dotted lines. Black dotted arrows connect these blocks to ribbon diagrams, where red and blue arrows indicate the relative motion of the highlighted region on photoactivation. The closer approach of the N termini of helix α3 in each subunit is highlighted by a black circle; the ribbon diagram (Lower Left) shows this region, with one subunit colored in blue and the other marine blue.

Fig. 4.

Enzyme activity in the crystal. (A) Schematic diagram showing the experimental setup. Crystals of similar size (∼80 × 30 × 20 μm) were soaked in the dark for 24 h. Some crystals were exposed to blue light for 20 min before being removed from the buffer (“light buffer”). Other crystals were kept in the dark before separation from the buffer. cAMP levels in the buffer were tested before (“dark-buffer D”) and after (“dark-buffer L”) light exposure. The protein crystals were dissolved to determine the amount of protein present. (B) cAMP level in each buffer sample. Only buffer that held an OaPAC crystal exposed to light showed significant cAMP. The overall protein concentrations (crystal plus buffer) were 87 μM and 91 μM, respectively, for the crystal exposed to light and the crystal held in the dark. Only buffer that held an OaPAC crystal exposed to light showed significant cAMP. Three independent experiments were performed. (C) Western blot of a redissolved crystal and each buffer sample, using antibody specific for OaPAC. 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.

Fig. 5.

Hydrogen bonding scheme at the chromophore. (A) Schematic diagram showing the hydrogen bonding pattern in the dark state of OaPAC. Transfer of an electron from Tyr-6 to the flavin on illumination yields an excited biradical form. A possible rearrangement of the hydrogen bonding is shown that would tautomerize the Gln-48 sidechain. (B) The tautomeric form of Gln-48 remains hydrogen bonded to Tyr-6. The biradical form of BLUF domains decays within nanoseconds to the signaling state.