Integrated Computational Study of the Light-Activated Structure of the AppA BLUF Domain and Its Spectral Signatures

Blue light-using flavin (BLUF) proteins are light-sensors that regulate responsive movement, gene expression and enzyme activity in diverse organisms. Their signaling times range from seconds to minutes, indicating a uniquely flexible dark-state recovery mechanism. Unlike other light-sensors, the flavin chromophore is non-covalently bonded to the protein. Hence, the switching occurs via a change in the protein-flavin hydrogen-bond network, involving conserved residues transferring protons, tautomerizing, rotating, and approaching or leaving the chromophore pocket; triggering secondary structure displacements. The specific deactivation steps and residue roles have remained controversial. The detailed process is difficult to probe experimentally, and although simulations can track it, the computational effort is daunting. We combine forefront techniques to simulate, for the first time, explicit dynamics of the deactivation. A hybrid quantum mechanics/molecular mechanics scheme focuses the computational resolution in the flavin's vicinity, while our path-based methods sample the mechanism of dark-state recovery with high efficiency. Our protocol delivers free-energy profiles for the deactivation of two BLUF proteins, BlrB and AppA; corroborating a proposed mechanism based on the rotation and tautomerization of a conserved Gln. We find that the conformation of a Trp and a Met near the flavin is crucial to modulate the rate-determining barrier, which differs significantly between the BlrB and AppA proteins. Our work evidences how specific variations of the deactivation mechanism control vast differences in signaling times.

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Crystal structure of a bacterial photoactivated adenylate cyclase determined by serial femtosecond and serial synchrotron crystallography

Kapetanaki,

Coquelle,

von Stetten

et al. 2024

IUCrJ

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OaPAC is a recently discovered blue-light-using flavin adenosine dinucleotide (BLUF) photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata that uses adenosine triphosphate and translates the light signal into the production of cyclic adenosine monophosphate. Here, we report crystal structures of the enzyme in the absence of its natural substrate determined from room-temperature serial crystallography data collected at both an X-ray free-electron laser and a synchrotron, and we compare these structures with cryo-macromolecular crystallography structures obtained at a synchrotron by us and others. These results reveal slight differences in the structure of the enzyme due to data collection at different temperatures and X-ray sources. We further investigate the effect of the Y6W mutation in the BLUF domain, a mutation which results in a rearrangement of the hydrogen-bond network around the flavin and a notable rotation of the side chain of the critical Gln48 residue. These studies pave the way for picosecond–millisecond time-resolved serial crystallography experiments at X-ray free-electron lasers and synchrotrons in order to determine the early structural intermediates and correlate them with the well studied picosecond–millisecond spectroscopic intermediates.

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Integrated Computational Study of the Light-Activated Structure of the AppA BLUF Domain and Its Spectral Signatures

Cited by 3 publications

References 58 publications

Deciphering Photoreceptors Through Atomistic Modeling from Light Absorption to Conformational Response

Deciphering Photoreceptors Through Atomistic Modeling from Light Absorption to Conformational Response

Free energies of the Gln tautomerization and rotation mechanism of dark-state recovery in blue light-using flavin proteins

Crystal structure of a bacterial photoactivated adenylate cyclase determined by serial femtosecond and serial synchrotron crystallography

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