Dynamic Nuclear Polarization Provides New Insights into Chromophore Structure in Phytochrome Photoreceptors

Stöppler, Daniel; Song, Chen; Rossum, Barth‐Jan van; Geiger, Michel-Andreas; Lang, Christina; Mroginski, Maria‐Andrea; Jagtap, Anil P.; Sigurdsson, Snorri Th.; Matysik, Jörg; Hughes, Jon; Oschkinat, Hartmut

doi:10.1002/anie.201608119

Cited by 22 publications

(23 citation statements)

References 45 publications

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“…The experimental δ N values were closely matched by those computed using a QM/MM approach ( δ N21-N24 = 165.3, 140.7, 154.1, and 138.0 ppm, respectively; see Supplementary Table S3 ). It should also be noted that the recent 15 N DNP enhanced MAS NMR characterization of the Pr state in Cph1 finalized the assignment of the Pr resonances ( Stöppler et al, 2016 ), in which the previous tentative assignments of N22 and N23 ( Rohmer et al, 2008 ) were interchanged. This thus forms a solid basis on calculating the state-induced δ N changes of the chromophore in oat phyA3 (see below).…”

Section: Resultsmentioning

confidence: 97%

“…We have proposed that the distinct δ N difference between these inner ring nitrogens arises from the asymmetry of the strong hydrogen-bonding interactions with the nearby carbonyl backbone of D272 (D207 in Cph1) and the pyrrole water ( Rohmer et al, 2010 ). Recent DNP-enhanced MAS NMR studies on Cph1 in Pr have suggested that the positive charge carried by the tetrapyrrole system is mainly localized at ring B and thus would account for a downfield shift of N22 relative to that of N23 ( Stöppler et al, 2016 ).…”

Section: Discussionmentioning

confidence: 99%

“…Although spectroscopic methods do not rival crystallography in solving 3D structures of complete proteins, MAS NMR continues to gain in value for protein structural studies, as it unveils outstandingly precise molecular details without the need to crystalize the protein. MAS NMR has contributed to phytochrome research, Cph1 auto-assembled with u -[ 13 C, 15 N]-PCB providing information on the little-known Pfr→Pr back-conversion route, confirming that the chromophore is fully protonated in both Pr and Pfr, and revealing the existence of at least two Pr sub-states, only one of which is represented by the crystal structure ( Rohmer et al, 2008 , 2010 ; Song et al, 2011a , b ; Stöppler et al, 2016 ). We also showed that Pr is intrinsically less rigid than Pfr, implying a mesoscopic phase transition associated with photoconversion ( Song et al, 2011a ).…”

Section: Introductionmentioning

confidence: 99%

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3D Structures of Plant Phytochrome A as Pr and Pfr From Solid-State NMR: Implications for Molecular Function

et al. 2018

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We present structural information for oat phyA3 in the far-red-light-absorbing (Pfr) signaling state, to our knowledge the first three-dimensional (3D) information for a plant phytochrome as Pfr. Solid-state magic-angle spinning (MAS) NMR was used to detect interatomic contacts in the complete photosensory module [residues 1–595, including the NTE (N-terminal extension), PAS (Per/Arnt/Sim), GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) and PHY (phytochrome-specific) domains but with the C-terminal PAS repeat and transmitter-like module deleted] auto-assembled in vitro with 13C- and 15N-labeled phycocyanobilin (PCB) chromophore. Thereafter, quantum mechanics/molecular mechanics (QM/MM) enabled us to refine 3D structural models constrained by the NMR data. We provide definitive atomic assignments for all carbon and nitrogen atoms of the chromophore, showing the Pfr chromophore geometry to be periplanar ZZEssa with the D-ring in a β-facial disposition incompatible with many earlier notions regarding photoconversion yet supporting circular dichroism (CD) data. The Y268 side chain is shifted radically relative to published Pfr crystal structures in order to accommodate the β-facial ring D. Our findings support a photoconversion sequence beginning with Pr photoactivation via an anticlockwise D-ring Za→Ea photoflip followed by significant shifts at the coupling of ring A to the protein, a B-ring propionate partner swap from R317 to R287, changes in the C-ring propionate hydrogen-bonding network, breakage of the D272–R552 salt bridge accompanied by sheet-to-helix refolding of the tongue region stabilized by Y326–D272–S554 hydrogen bonding, and binding of the NTE to the hydrophobic side of ring A. We discuss phyA photoconversion, including the possible roles of mesoscopic phase transitions and protonation dynamics in the chromophore pocket. We also discuss possible associations between structural changes and translocation and signaling processes within the cell.

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Section: Resultsmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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3D Structures of Plant Phytochrome A as Pr and Pfr From Solid-State NMR: Implications for Molecular Function

et al. 2018

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“…The stabilizing role of the tongue was further probed by site-directed mutations of amino acids comprising three highly conserved tongue motifs. For these NMR investigations on all2699g1 and all2699g1-2 proteins, we used the PSM of the canonical cyanobacterial phytochrome Cph1 (Cph1Δ2) as a reference for the NMR analysis [28,29,30,31,32,33] (see alignment of the sequences in Figure S1). NMR data provided information on the chromophore geometry, flexibility of the GAF1-bound chromophore and its interactions with nearby amino acids.…”

Section: Introductionmentioning

confidence: 99%

MAS NMR on a Red/Far-Red Photochromic Cyanobacteriochrome All2699 from Nostoc

Bielytskyi

Otis

et al. 2019

IJMS

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Unlike canonical phytochromes, the GAF domain of cyanobacteriochromes (CBCRs) can bind bilins autonomously and is sufficient for functional photocycles. Despite the astonishing spectral diversity of CBCRs, the GAF1 domain of the three-GAF-domain photoreceptor all2699 from the cyanobacterium Nostoc 7120 is the only CBCR-GAF known that converts from a red-absorbing (Pr) dark state to a far-red-absorbing (Pfr) photoproduct, analogous to the more conservative phytochromes. Here we report a solid-state NMR spectroscopic study of all2699g1 in its Pr state. Conclusive NMR evidence unveils a particular stereochemical heterogeneity at the tetrahedral C31 atom, whereas the crystal structure shows exclusively the R-stereochemistry at this chiral center. Additional NMR experiments were performed on a construct comprising the GAF1 and GAF2 domains of all2699, showing a greater precision in the chromophore–protein interactions in the GAF1-2 construct. A 3D Pr structural model of the all2699g1-2 construct predicts a tongue-like region extending from the GAF2 domain (akin to canonical phytochromes) in the direction of the chromophore, shielding it from the solvent. In addition, this stabilizing element allows exclusively the R-stereochemistry for the chromophore-protein linkage. Site-directed mutagenesis performed on three conserved motifs in the hairpin-like tip confirms the interaction of the tongue region with the GAF1-bound chromophore.

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“…For this purpose, classical MD simulations were performed by several groups [25,26,27,28]. In addition, Mroginski and coworkers also employed QM/MM simulations [29,30,31] and some recent publications based on this approach can be found in references [32,33,34,35,36,37]. Recently a QM/MM approach was also applied to investigate the covalent binding of the biliverdin (BV) chromophore to phytochrome domains [38] in the NIR FP miRFP670, which was also investigated experimentally [39,40,41].…”

Section: Introductionmentioning

confidence: 99%

QM/MM Benchmarking of Cyanobacteriochrome Slr1393g3 Absorption Spectra

Wiebeler

Schapiro

2019

Molecules

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Cyanobacteriochromes are compact and spectrally diverse photoreceptor proteins that are promising candidates for biotechnological applications. Computational studies can contribute to an understanding at a molecular level of their wide spectral tuning and diversity. In this contribution, we benchmark methods to model a 110 nm shift in the UV/Vis absorption spectrum from a red- to a green-absorbing form of the cyanobacteriochrome Slr1393g3. Based on an assessment of semiempirical methods to describe the chromophore geometries of both forms in vacuo, we find that DFTB2+D leads to structures that are the closest to the reference method. The benchmark of the excited state calculations is based on snapshots from quantum mechanics/molecular mechanics molecular dynamics simulations. In our case, the methods RI-ADC(2) and sTD-DFT based on CAM-B3LYP ground state calculations perform the best, whereas no functional can be recommended to simulate the absorption spectra of both forms with time-dependent density functional theory. Furthermore, the difference in absorption for the lowest energy absorption maxima of both forms can already be modelled with optimized structures, but sampling is required to improve the shape of the absorption bands of both forms, in particular for the second band. This benchmark study can guide further computational studies, as it assesses essential components of a protocol to model the spectral tuning of both cyanobacteriochromes and the related phytochromes.

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Dynamic Nuclear Polarization Provides New Insights into Chromophore Structure in Phytochrome Photoreceptors

Cited by 22 publications

References 45 publications

3D Structures of Plant Phytochrome A as Pr and Pfr From Solid-State NMR: Implications for Molecular Function

3D Structures of Plant Phytochrome A as Pr and Pfr From Solid-State NMR: Implications for Molecular Function

MAS NMR on a Red/Far-Red Photochromic Cyanobacteriochrome All2699 from Nostoc

QM/MM Benchmarking of Cyanobacteriochrome Slr1393g3 Absorption Spectra

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