X-ray radiolytic labeling reveals the molecular basis of orange carotenoid protein photoprotection and its interactions with fluorescence recovery protein

Gupta, Sayan; Sutter, Markus; Remesh, Soumya G.; Domínguez-Martín, María Agustina; Bao, Han; Feng, Xinyu A.; Chan, Leanne-Jade G.; Kim, Young-Mo; Kerfeld, Cheryl A.; Ralston, Corie Y.

doi:10.1074/jbc.ra119.007592

Cited by 31 publications

(52 citation statements)

References 39 publications

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“…The most recent experiments also support the fact that the translocation process precedes the dissociation of the NTD and CTD domains by orders of magnitude. 16,19,20 These findings are fully confirmed by our results. In fact, our wt-MetaD simulations show that the T 2 state has a remarkable preference to undergo dissociation with respect to OCP O .…”

Section: Discussionsupporting

confidence: 91%

“…In spite of these limitations, our wt-MetaD simulations clearly show that the dissociation of the two domains follows the translocation of the carotenoid, confirming the hypothesis of the most recent working model for the photo-activation process 16,19,20 . Furthermore, they suggest that the opening is facilitated by the loss of interactions between the carotenoid and the CTD, and not by the rearrangement in the protein structure taking place during the translocation.…”

Section: Dissociationsupporting

confidence: 84%

“…On the contrary, if the system were prepared in state T 1 and thermally equilibrated, considering the relative height of the barriers found in our simulations ( Figure 2C), we would have observed only a fall back to OCP O . To support this working hypothesis of a out-of-equilibrium pathway for the events following the carotenoid excited state relaxation, we note that different experimental results 15,20 indicate that the OCP R →OCP O thermal relaxation follows a different path with respect to the photoactivation.…”

Section: Discussionmentioning

confidence: 74%

“…Experimental techniques have probed the evolution of OCP after the photo-activation either with a high time [16][17][18][19] or space resolution 20 . Nonetheless, a clear and complete characterization of the molecular mechanisms through which the carotenoid translocates between the two domains and the protein dissociates is not available.…”

mentioning

confidence: 99%

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The Molecular Mechanisms of Photoactivation of Orange Carotenoid Protein Revealed by Molecular Dynamics

Bondanza¹,

Cupellini

Faccioli³

et al. 2020

Preprint

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Light-harvesting in photosynthesis is accompanied by photoprotective processes. In cyanobacteria, the photoprotective role is played by a specialized complex, the Orange Carotenoid Protein which is activated by strong blue-green light. This photoactivation involves a unique series of structural changes which terminate with an opening of the complex into two separated domains, one of which acts as a quencher for the light-harvesting complexes. Many experimental studies have tried to reveal the molecular mechanisms through which the energy absorbed by the carotenoid finally leads to the large conformational change of the complex. Here for the first time, these mechanisms are revealed by simulating at atomistic level the whole dynamics of the complex through an effective combination of enhanced sampling techniques. On the basis of our findings, we can conclude that the carotenoid does not act as a spring that, releasing its internal strain, induces the dissociation, as it was previously proposed but as a "latch" locking together the two domains. The photochemically triggered displacement of the carotenoid breaks this balance, allowing the complex to dissociate. <br>

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Section: Discussionsupporting

confidence: 91%

Section: Dissociationsupporting

confidence: 84%

Section: Discussionmentioning

confidence: 74%

mentioning

confidence: 99%

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The Molecular Mechanisms of Photoactivation of Orange Carotenoid Protein Revealed by Molecular Dynamics

Bondanza¹,

Cupellini

Faccioli³

et al. 2020

Preprint

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“…The use of fusion constructs simplified interpretation of the XFMS data by preventing solution heterogeneity that might otherwise dominate mixtures of weak-binding components. XFMS has been used previously to study protein global -local dynamics (38) and proteinprotein interactions (39). The computational and experimental results together provide a useful description of cooperative structural changes that accompany activation of CheY by phosphorylation and by binding of FliMN, with implications for allosteric phenomena employed by phospho-relay systems.…”

Section: Introductionmentioning

confidence: 99%

Allosteric priming ofE. coliCheY by the flagellar motor protein FliM

Wheatley

Pandini

Gupta

et al. 2019

Preprint

Self Cite

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Phosphorylation of Escherichia coli CheY couple's chemoreceptor output to flagellar motor response. The N-termini of FliM subunits (FliMN) in the flagellar rotor prime CheY to bind FliN thereby inducing cooperative switching, an essential feature of bacterial chemotaxis. We analyzed molecular dynamics {MD} trajectories to identify networks of residues involved in the long-range allosteric activation of CheY by FliMN. The CheY backbone was partitioned into four dynamically coordinated sectors, with activation tracked by changes in sector size and interactions. Bound FliMN closed the central α4-β4 loop hinge to strengthen correlations between sectors around its binding interface and D57 phosphorylation site. Inward W58 sidechain movements adjacent to the CheY D57 phosphorylation site were coupled to corresponding K91 and Y106 sidechain movements at the FliMN interface. Studies of the constitutively active CheY D13K-Y106W double mutant have related its structural changes with invivo signaling properties. The MD revealed that D13K-Y106W fused the phosphorylation site and FliMN binding sectors into a new surface-exposed sector and locked the 106W sidechain in the innermost rotamer configuration in CheY-FliMN complexes. X-ray foot-printing with mass spectroscopy exploited FliMN-CheY fusion proteins to validate the concerted sidechain internalization of W58, K91 and Y106 triggered by bound FliMN and increased by D13K-Y106W. Oxidation rate was correlated with the solvent accessible surface area, with K109, another central element of the allosteric relay, an outlier likely due to hydrogen bonding. The measurements indicated the fusion proteins were an effective mimic of the crystallized complexes used for the MD simulations. In absence of the D13K-Y106W mutations, CheY Y106 sampled multiple inward rotamer states, but their coupling to backbone dynamics required bound FliMN to prolongation inward state lifetimes by bound FliMN. Thus, as simulations have found for kinases, control of CheY activation by aromatic residue reorientation is more subtle than a binary ON-OFF switch. Statement of SignificanceThe chemotaxis phospho-protein CheY is activated at the flagellar motor by the Nterminus (FliMN) of FliM subunits. Crystal structures of FliMN.CheY complexes with and without the phosphomimic D13K-Y106W double-mutation both have the residue 106 sidechain in the same IN orientation at the FliMN interface. Additional factors that explain activation were identified by atomistic simulations based on the crystal structures. Free CheY samples both IN and OUT Y106 rotamer states, but bound FliMN increases multiple IN-state lifetimes to alter backbone dynamics. D13K-Y106W triggers further alterations to select the W106 state seen in the crystals and create novel binding surfaces. Observed changes in sidechain position along the allosteric relay reported by the crystals were predicted and validated by X-ray foot-printing with mass-spectroscopy.

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A favorable path to domain separation in the orange carotenoid protein

et al. 2022

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The Orange Carotenoid Protein (OCP) is responsible for nonphotochemical quenching (NPQ) in cyanobacteria, a defense mechanism against potentially damaging effects of excess light conditions. This soluble two-domain protein undergoes profound conformational changes upon photoactivation, involving translocation of the ketocarotenoid inside the cavity followed by domain separation. Domain separation is a critical step in the photocycle of OCP because it exposes the N-terminal domain (NTD) to perform quenching of the phycobilisomes. Many details regarding the mechanism and energetics of OCP domain separation remain unknown. In this work, we apply metadynamics to elucidate the protein rearrangements that lead to the active, domainseparated, form of OCP. We find that translocation of the ketocarotenoid canthaxanthin has a profound effect on the energetic landscape and that domain separation only becomes favorable following translocation. We further explore, characterize, and validate the free energy surface (FES) using equilibrium simulations initiated from different states on the FES. Through pathway optimization methods, we characterize the most probable path to domain separation and reveal the barriers along that pathway. We find that the free energy barriers are relatively small (<5 kcal/mol), but the overall estimated kinetic rate is consistent with experimental measurements (>1 ms).Overall, our results provide detailed information on the requirement for canthaxanthin translocation to precede domain separation and an energetically feasible pathway to dissociation.

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X-ray radiolytic labeling reveals the molecular basis of orange carotenoid protein photoprotection and its interactions with fluorescence recovery protein

Cited by 31 publications

References 39 publications

The Molecular Mechanisms of Photoactivation of Orange Carotenoid Protein Revealed by Molecular Dynamics

The Molecular Mechanisms of Photoactivation of Orange Carotenoid Protein Revealed by Molecular Dynamics

Allosteric priming ofE. coliCheY by the flagellar motor protein FliM

A favorable path to domain separation in the orange carotenoid protein

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