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From crystallographic data to the solution structure of photoreceptors: the case of the AppA BLUF domain† † Electronic supplementary information (ESI) available. See DOI: 10.1039/d1sc03000k

ABSTRACT: Photoreceptor proteins bind a chromophore, which, upon light absorption, modifies its geometry or its interactions with the protein, finally inducing the structural change needed to switch the protein from an inactive to an active or signaling state. In the Blue Light-Using Flavin (BLUF) family of photoreceptors, the chromophore is a flavin and the changes have been connected with a rearrangement of the hydrogen bond network around it on the basis of spectroscopic changes measured for the dark-to-light conversion. However, the exact conformational change triggered by the photoexcitation is still elusive mainly because a clear consensus on the identity not only of the light activated state but also of the dark one has not been achieved. Here, we present an integrated investigation that combines microsecond MD simulations starting from the two conflicting crystal structures available for the AppA BLUF domain with calculations of NMR, IR and UV-Vis spectra using a polarizable QM/MM approach. Thanks to such a combined analysis of the three different spectroscopic responses, a robust characterization of the structure of the dark state in solution is given together with the uncovering of important flaws of the most popular molecular mechanisms present in the literature for the dark-to-light activation. With an integrated molecular dynamics and QM/MM strategy we characterize the dark-state structure of a BLUF photoreceptor and ration alize the discrepancy between published crystal structures.

SUBMITTER: Hashem S

PROVIDER: S-EPMC8528011 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Acinetobacter nosocomialis is a Gram-negative opportunistic pathogen, whose ability to cause disease in humans is well recognized. Blue light has been shown to modulate important physiological traits related to persistence and virulence in this microorganism. In this work, we characterized the three Blue Light sensing Using FAD (BLUF) domain-containing proteins encoded in the A. nosocomialis genome, which account for the only canonical light sensors present in this microorganism. By focusing on a light-modulated bacterial process such as motility, the temperature dependence of light regulation was studied, as well as the expression pattern and spectroscopic characteristics of the different A. nosocomialis BLUFs. Our results show that the BLUF-containing proteins AnBLUF65 and AnBLUF46 encode active photoreceptors in the light-regulatory temperature range when expressed recombinantly. In fact, AnBLUF65 is an active photoreceptor in the temperature range from 15°C to 37°C, while AnBLUF46 between 15°C to 32°C, in vitro. In vivo, only the Acinetobacter baumannii BlsA's ortholog AnBLUF65 was expressed in A. nosocomialis cells recovered from motility plates. Moreover, complementation assays showed that AnBLUF65 is able to mediate light regulation of motility in A. baumannii ΔblsA strain at 30°C, confirming its role as photoreceptor and in modulation of motility by light. Intra-protein interactions analyzed using 3D models built based on A. baumannii´s BlsA photoreceptor, show that hydrophobic/aromatic intra-protein interactions may contribute to the stability of dark/light- adapted states of the studied proteins, reinforcing the previous notion on the importance of these interactions in BLUF photoreceptors. Overall, the results presented here reveal the presence of BLUF photoreceptors in A. nosocomialis with idiosyncratic characteristics respect to the previously characterized A. baumannii's BlsA, both regarding the photoactivity temperature-dependency as well as expression patterns, contributing thus to broaden our knowledge on the BLUF family.

Dataset Information

From crystallographic data to the solution structure of photoreceptors: the case of the AppA BLUF domain† † Electronic supplementary information (ESI) available. See DOI: 10.1039/d1sc03000k

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