Influence of pump laser fluence on ultrafast myoglobin structural dynamics.

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump-probe experiments for the investigation of electronic and nuclear changes during light-induced reactions. On timescales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer1,2. However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore3-17. As multiphoton absorption may force the protein response into non-physiological pathways, it is of great concern18,19 whether this experimental approach20 allows valid conclusions to be drawn vis-à-vis biologically relevant single-photon-induced reactions18,19. Here we describe ultrafast pump-probe SFX experiments on the photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics21) are seen to depend strongly on pump laser energy, in line with quantum chemical analysis. Our results confirm both the feasibility and necessity of performing ultrafast TR-SFX pump-probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing both the design and the interpretation of ultrafast TR-SFX pump-probe experiments20 such that mechanistically relevant insight emerges.

Redox driven B12-ligand switch drives CarH photoresponse.

CarH is a coenzyme B12-dependent photoreceptor involved in regulating carotenoid biosynthesis. How light-triggered cleavage of the B12 Co-C bond culminates in CarH tetramer dissociation to initiate transcription remains unclear. Here, a series of crystal structures of the CarH B12-binding domain after illumination suggest formation of unforeseen intermediate states prior to tetramer dissociation. Unexpectedly, in the absence of oxygen, Co-C bond cleavage is followed by reorientation of the corrin ring and a switch from a lower to upper histidine-Co ligation, corresponding to a pentacoordinate state. Under aerobic conditions, rapid flash-cooling of crystals prior to deterioration upon illumination confirm a similar B12-ligand switch occurs. Removal of the upper His-ligating residue prevents monomer formation upon illumination. Combined with detailed solution spectroscopy and computational studies, these data demonstrate the CarH photoresponse integrates B12 photo- and redox-chemistry to drive large-scale conformational changes through stepwise Co-ligation changes.

Is orange carotenoid protein photoactivation a single-photon process?

In all published photoactivation mechanisms of orange carotenoid protein (OCP), absorption of a single photon by the orange dark state starts a cascade of red-shifted OCP ground-state intermediates that subsequently decay within hundreds of milliseconds, resulting in the formation of the final red form OCPR, which is the biologically active form that plays a key role in cyanobacteria photoprotection. A major challenge in deducing the photoactivation mechanism is to create a uniform description explaining both single-pulse excitation experiments, involving single-photon absorption, and continuous light irradiation experiments, where the red-shifted OCP intermediate species may undergo re-excitation. We thus investigated photoactivation of Synechocystis OCP using stationary irradiation light with a biologically relevant photon flux density coupled with nanosecond laser pulse excitation. The kinetics of photoactivation upon continuous and nanosecond pulse irradiation light show that the OCPR formation quantum yield increases with photon flux density; thus, a simple single-photon model cannot describe the data recorded for OCP in vitro. The results strongly suggest a consecutive absorption of two photons involving a red intermediate with ≈100 millisecond lifetime. This intermediate is required in the photoactivation mechanism and formation of the red active form OCPR.

Time-resolved serial femtosecond crystallography on fatty-acid photodecarboxylase: lessons learned.

Upon absorption of a blue-light photon, fatty-acid photodecarboxylase catalyzes the decarboxylation of free fatty acids to form hydrocarbons (for example alkanes or alkenes). The major components of the catalytic mechanism have recently been elucidated by combining static and time-resolved serial femtosecond crystallography (TR-SFX), time-resolved vibrational and electronic spectroscopies, quantum-chemical calculations and site-directed mutagenesis [Sorigué et al. (2021), Science, 372, eabd5687]. The TR-SFX experiments, which were carried out at four different picosecond to microsecond pump-probe delays, yielded input for the calculation of Fourier difference maps that demonstrated light-induced decarboxylation. Here, some of the difficulties encountered during the experiment as well as during data processing are highlighted, in particular regarding space-group assignment, a pump-laser power titration is described and data analysis is extended by structure-factor extrapolation of the TR-SFX data. Structure refinement against extrapolated structure factors reveals a reorientation of the generated hydrocarbon and the formation of a photoproduct close to Cys432 and Arg451. Identification of its chemical nature, CO2 or bicarbonate, was not possible because of the limited data quality, which was assigned to specificities of the crystalline system. Further TR-SFX experiments on a different crystal form are required to identify the photoproducts and their movements during the catalytic cycle.

Rational Control of Off-State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement.

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

Oligomerization processes limit photoactivation and recovery of the orange carotenoid protein.

The orange carotenoid protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection by quenching of the excess of light-harvested energy. The photoactivation mechanism remains elusive, in part due to absence of data pertaining to the timescales over which protein structural changes take place. It also remains unclear whether or not oligomerization of the dark-adapted and light-adapted OCP could play a role in the regulation of its energy-quenching activity. Here, we probed photoinduced structural changes in OCP by a combination of static and time-resolved X-ray scattering and steady-state and transient optical spectroscopy in the visible range. Our results suggest that oligomerization partakes in regulation of the OCP photocycle, with different oligomers slowing down the overall thermal recovery of the dark-adapted state of OCP. They furthermore reveal that upon non-photoproductive excitation a numbed state forms, which remains in a non-photoexcitable structural state for at least ≈0.5 µs after absorption of a first photon.

Structural and functional characterization of DdrC, a novel DNA damage-induced nucleoid associated protein involved in DNA compaction.

Deinococcus radiodurans is a spherical bacterium well-known for its outstanding resistance to DNA-damaging agents. Exposure to such agents leads to drastic changes in the transcriptome of D. radiodurans. In particular, four Deinococcus-specific genes, known as DNA Damage Response genes, are strongly up-regulated and have been shown to contribute to the resistance phenotype of D. radiodurans. One of these, DdrC, is expressed shortly after exposure to γ-radiation and is rapidly recruited to the nucleoid. In vitro, DdrC has been shown to compact circular DNA, circularize linear DNA, anneal complementary DNA strands and protect DNA from nucleases. To shed light on the possible functions of DdrC in D. radiodurans, we determined the crystal structure of the domain-swapped DdrC dimer at a resolution of 2.5 Šand further characterized its DNA binding and compaction properties. Notably, we show that DdrC bears two asymmetric DNA binding sites located on either side of the dimer and can modulate the topology and level of compaction of circular DNA. These findings suggest that DdrC may be a DNA damage-induced nucleoid-associated protein that enhances nucleoid compaction to limit the dispersion of the fragmented genome and facilitate DNA repair after exposure to severe DNA damaging conditions.

Xtrapol8 enables automatic elucidation of low-occupancy intermediate-states in crystallographic studies.

Unstable states studied in kinetic, time-resolved and ligand-based crystallography are often characterized by a low occupancy, which hinders structure determination by conventional methods. To automatically extract structural information pertaining to these states, we developed Xtrapol8, a program which (i) applies various flavors of Bayesian-statistics weighting to generate the most informative Fourier difference maps; (ii) determines the occupancy of the intermediate states by use of methods hitherto not available; (iii) calculates extrapolated structure factors using the various proposed formalisms while handling the issue of negative structure factor amplitudes, and (iv) refines the corresponding structures in real and reciprocal-space. The use of Xtrapol8 could accelerate data processing in kinetic and time-resolved crystallographic studies, and as well foster the identification of drug-targetable states in ligand-based crystallography.

Structure-function-dynamics relationships in the peculiar Planktothrix PCC7805 OCP1: Impact of his-tagging and carotenoid type.

The orange carotenoid protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection. Here, we report on the functional, spectral and structural characteristics of the peculiar Planktothrix PCC7805 OCP (Plankto-OCP). We show that this OCP variant is characterized by higher photoactivation and recovery rates, and a stronger energy-quenching activity, compared to other OCP studied thus far. We characterize the effect of the functionalizing carotenoid and of his-tagging on these reactions, and identify the time scales on which these modifications affect photoactivation. The presence of a his-tag at the C-terminus has a large influence on photoactivation, thermal recovery and PBS-fluorescence quenching, and likewise for the nature of the carotenoid that additionally affects the yield and characteristics of excited states and the ns-s dynamics of photoactivated OCP. By solving the structures of Plankto-OCP in the ECN- and CAN-functionalized states, each in two closely-related crystal forms, we further unveil the molecular breathing motions that animate Plankto-OCP at the monomer and dimer levels. We finally discuss the structural changes that could explain the peculiar properties of Plankto-OCP.

Unifying Perspective of the Ultrafast Photodynamics of Orange Carotenoid Proteins from Synechocystis: Peril of High-Power Excitation, Existence of Different S* States, and Influence of Tagging.

A substantial number of Orange Carotenoid Protein (OCP) studies have aimed to describe the evolution of singlet excited states leading to the formation of a photoactivated form, OCPR. The most recent one suggests that 3 ps-lived excited states are formed after the sub-100 fs decay of the initial S2 state. The S* state, which has the longest reported lifetime of a few to tens of picoseconds, is considered to be the precursor of the first red photoproduct P1. Here, we report the ultrafast photodynamics of the OCP from Synechocystis PCC 6803 carried out using visible-near infrared femtosecond time-resolved absorption spectroscopy as a function of the excitation pulse power and wavelength. We found that a carotenoid radical cation can form even at relatively low excitation power, obscuring the determination of photoactivation yields for P1. Moreover, the comparison of green (540 nm) and blue (470 nm) excitations revealed the existence of an hitherto uncharacterized excited state, denoted as S∼, living a few tens of picoseconds and formed only upon 470 nm excitation. Because neither the P1 quantum yield nor the photoactivation speed over hundreds of seconds vary under green and blue continuous irradiation, this S∼ species is unlikely to be involved in the photoactivation mechanism leading to OCPR. We also addressed the effect of His-tagging at the N- or C-termini on the excited-state photophysical properties. Differences in spectral signatures and lifetimes of the different excited states were observed at a variance with the usual assumption that His-tagging hardly influences protein dynamics and function. Altogether our results advocate for the careful consideration of the excitation power and His-tag position when comparing the photoactivation of different OCP variants and beg to revisit the notion that S* is the precursor of photoactivated OCPR.

Socialization of Providencia stuartii Enables Resistance to Environmental Insults.

Providencia stuartii is a highly social pathogen responsible for nosocomial chronic urinary tract infections. The bacterium indeed forms floating communities of cells (FCC) besides and prior-to canonical surface-attached biofilms (SAB). Within P. stuartii FCC, cells are riveted one to another owing to by self-interactions between its porins, viz. Omp-Pst1 and Omp-Pst2. In pathophysiological conditions, P. stuartii is principally exposed to high concentrations of urea, ammonia, bicarbonate, creatinine and to large variations of pH, questioning how these environmental cues affect socialization, and whether formation of SAB and FCC protects cells against those. Results from our investigations indicate that FCC and SAB can both form in the urinary tract, endowing cells with increased resistance and fitness. They additionally show that while Omp-Pst1 is the main gateway allowing penetration of urea, bicarbonate and ammonia into the periplasm, expression of Omp-Pst2 enables resistance to them.

Structural and biochemical characterisation of the Providencia stuartii arginine decarboxylase shows distinct polymerisation and regulation.

Bacterial homologous lysine and arginine decarboxylases play major roles in the acid stress response, physiology, antibiotic resistance and virulence. The Escherichia coli enzymes are considered as their archetypes. Whereas acid stress triggers polymerisation of the E. coli lysine decarboxylase LdcI, such behaviour has not been observed for the arginine decarboxylase Adc. Here we show that the Adc from a multidrug-resistant human pathogen Providencia stuartii massively polymerises into filaments whose cryo-EM structure reveals pronounced differences between Adc and LdcI assembly mechanisms. While the structural determinants of Adc polymerisation are conserved only in certain Providencia and Burkholderia species, acid stress-induced polymerisation of LdcI appears general for enterobacteria. Analysis of the expression, activity and oligomerisation of the P. stuartii Adc further highlights the distinct properties of this unusual protein and lays a platform for future investigation of the role of supramolecular assembly in the superfamily or arginine and lysine decarboxylases.

Erratum: Tetreau et al. How Does Bacillus thuringiensis Crystallize Such a Large Diversity of Toxins? Toxins 2021, 13, 443.

The authors wish to make the following corrections to this paper [...].

Can (We Make) Bacillus thuringiensis Crystallize More Than Its Toxins?

The development of finely tuned and reliable crystallization processes to obtain crystalline formulations of proteins has received growing interest from different scientific fields, including toxinology and structural biology, as well as from industry, notably for biotechnological and medical applications. As a natural crystal-making bacterium, Bacillus thuringiensis (Bt) has evolved through millions of years to produce hundreds of highly structurally diverse pesticidal proteins as micrometer-sized crystals. The long-term stability of Bt protein crystals in aqueous environments and their specific and controlled dissolution are characteristics that are particularly sought after. In this article, I explore whether the crystallization machinery of Bt can be hijacked as a means to produce (micro)crystalline formulations of proteins for three different applications: (i) to develop new bioinsecticidal formulations based on rationally improved crystalline toxins, (ii) to functionalize crystals with specific characteristics for biotechnological and medical applications, and (iii) to produce microcrystals of custom proteins for structural biology. By developing the needs of these different fields to figure out if and how Bt could meet each specific requirement, I discuss the already published and/or patented attempts and provide guidelines for future investigations in some underexplored yet promising domains.

How Does Bacillus thuringiensis Crystallize Such a Large Diversity of Toxins?

Bacillus thuringiensis (Bt) is a natural crystal-making bacterium. Bt diversified into many subspecies that have evolved to produce crystals of hundreds of pesticidal proteins with radically different structures. Their crystalline form ensures stability and controlled release of these major virulence factors. They are responsible for the toxicity and host specificity of Bt, explaining its worldwide use as a biological insecticide. Most research has been devoted to understanding the mechanisms of toxicity of these toxins while the features driving their crystallization have long remained elusive, essentially due to technical limitations. The evolution of methods in structural biology, pushing back the limits of the resolution attainable, now allows access to be gained to structural information hidden within natural crystals of such toxins. In this review, I present the main parameters that have been identified as key drivers of toxin crystallization in Bt, notably in the light of recent discoveries driven by structural biology studies. Then, I develop how the future evolution of structural biology will hopefully unveil new mechanisms of Bt toxin crystallization, opening the door to their hijacking with the aim of developing a versatile in vivo crystallization platform of high academic and industrial interest.

Discovery of a Potent Dual Inhibitor of Acetylcholinesterase and Butyrylcholinesterase with Antioxidant Activity that Alleviates Alzheimer-like Pathology in Old APP/PS1 Mice.

The combination of the scaffolds of the cholinesterase inhibitor huprine Y and the antioxidant capsaicin results in compounds with nanomolar potencies toward human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) that retain or improve the antioxidant properties of capsaicin. Crystal structures of their complexes with AChE and BChE revealed the molecular basis for their high potency. Brain penetration was confirmed by biodistribution studies in C57BL6 mice, with one compound (5i) displaying better brain/plasma ratio than donepezil. Chronic treatment of 10 month-old APP/PS1 mice with 5i (2 mg/kg, i.p., 3 times per week, 4 weeks) rescued learning and memory impairments, as measured by three different behavioral tests, delayed the Alzheimer-like pathology progression, as suggested by a significantly reduced Aß42/Aß40 ratio in the hippocampus, improved basal synaptic efficacy, and significantly reduced hippocampal oxidative stress and neuroinflammation. Compound 5i emerges as an interesting anti-Alzheimer lead with beneficial effects on cognitive symptoms and on some underlying disease mechanisms.

The C. elegans GATA transcription factor elt-2 mediates distinct transcriptional responses and opposite infection outcomes towards different Bacillus thuringiensis strains.

The nematode Caenorhabditis elegans has been extensively used as a model for the study of innate immune responses against bacterial pathogens. While it is well established that the worm mounts distinct transcriptional responses to different bacterial species, it is still unclear in how far it can fine-tune its response to different strains of a single pathogen species, especially if the strains vary in virulence and infection dynamics. To rectify this knowledge gap, we systematically analyzed the C. elegans response to two strains of Bacillus thuringiensis (Bt), MYBt18247 (Bt247) and MYBt18679 (Bt679), which produce different pore forming toxins (PFTs) and vary in infection dynamics. We combined host transcriptomics with cytopathological characterizations and identified both a common and also a differentiated response to the two strains, the latter comprising almost 10% of the infection responsive genes. Functional genetic analyses revealed that the AP-1 component gene jun-1 mediates the common response to both Bt strains. In contrast, the strain-specific response is mediated by the C. elegans GATA transcription factor ELT-2, a homolog of Drosophila SERPENT and vertebrate GATA4-6, and a known master regulator of intestinal responses in the nematode. elt-2 RNAi knockdown decreased resistance to Bt679, but remarkably, increased survival on Bt247. The elt-2 silencing-mediated increase in survival was characterized by reduced intestinal tissue damage despite a high pathogen burden and might thus involve increased tolerance. Additional functional genetic analyses confirmed the involvement of distinct signaling pathways in the C. elegans defense response: the p38-MAPK pathway acts either directly with or in parallel to elt-2 in mediating resistance to Bt679 infection but is not required for protection against Bt247. Our results further suggest that the elt-2 silencing-mediated increase in survival on Bt247 is multifactorial, influenced by the nuclear hormone receptors NHR-99 and NHR-193, and may further involve lipid metabolism and detoxification. Our study highlights that the nematode C. elegans with its comparatively simple immune defense system is capable of generating a differentiated response to distinct strains of the same pathogen species. Importantly, our study provides a molecular insight into the diversity of biological processes that are influenced by a single master regulator and jointly determine host survival after pathogen infection.

Chiral Separation, X-ray Structure, and Biological Evaluation of a Potent and Reversible Dual Binding Site AChE Inhibitor.

Acetylcholinesterase (AChE) inhibitors (AChEIs) still remain the leading therapeutic options for the symptomatic treatment of cognitive deficits associated with mild-to-moderate Alzheimer's disease. The search for new AChEIs benefits from well-established knowledge of the molecular interactions of selective AChEIs, such as donepezil and related dual binding site inhibitors. Starting from a previously disclosed coumarin-based inhibitor (±)-cis-1, active as racemate in the nanomolar range toward AChE, we proceeded on a double track by (i) achieving chiral resolution of the enantiomers of 1 by HPLC and (ii) preparing two close achiral analogues of 1, i.e., compounds 4 and 6. An eudismic ratio as high as 20 was observed for the (-) enantiomer of cis-1. The X-ray crystal structure of the complex between the (-)-cis-1 eutomer (coded as MC1420) and T. californica AChE was determined at 2.8 Å, and docking calculation results suggested that the eutomer in (1R,3S) absolute configuration should be energetically more favored in binding the enzyme than the eutomer in (1S,3R) configuration. The achiral analogues 4 and 6 were less effective in inhibiting AChE compared to (±)-cis-1, but interestingly butylamide 4 emerged as a potent inhibitor of butyrylcholinesterase (BChE).

Serial femtosecond crystallography on in vivo-grown crystals drives elucidation of mosquitocidal Cyt1Aa bioactivation cascade.

Cyt1Aa is the one of four crystalline protoxins produced by mosquitocidal bacterium Bacillus thuringiensis israelensis (Bti) that has been shown to delay the evolution of insect resistance in the field. Limiting our understanding of Bti efficacy and the path to improved toxicity and spectrum has been ignorance of how Cyt1Aa crystallizes in vivo and of its mechanism of toxicity. Here, we use serial femtosecond crystallography to determine the Cyt1Aa protoxin structure from sub-micron-sized crystals produced in Bti. Structures determined under various pH/redox conditions illuminate the role played by previously uncharacterized disulfide-bridge and domain-swapped interfaces from crystal formation in Bti to dissolution in the larval mosquito midgut. Biochemical, toxicological and biophysical methods enable the deconvolution of key steps in the Cyt1Aa bioactivation cascade. We additionally show that the size, shape, production yield, pH sensitivity and toxicity of Cyt1Aa crystals grown in Bti can be controlled by single atom substitution.

Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy.

Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive. Here we describe the off-to-on photoswitching mechanism in the RSFP rsEGFP2 by using a combination of time-resolved serial crystallography at an X-ray free-electron laser and ns-resolved pump-probe UV-visible spectroscopy. Ten ns after photoexcitation, the crystal structure features a chromophore that isomerized from trans to cis but the surrounding pocket features conformational differences compared to the final on-state. Spectroscopy identifies the chromophore in this ground-state photo-intermediate as being protonated. Deprotonation then occurs on the µs timescale and correlates with a conformational change of the conserved neighbouring histidine. Together with a previous excited-state study, our data allow establishing a detailed mechanism of off-to-on photoswitching in rsEGFP2.