Biology subjects

Toul, M.

Publications and source records attributed to Toul, M..

8 recordsLinked to original sources

bioRxiv2026

Structural mechanism governing radiationless energy transfer in Renilla bioluminescence

The nonradiative transport of electronic excitation from one chromophore to another, known as resonance energy transfer, lies at the root of photochemical processes in biology. Unlike photosynthesis, bioluminescence converts chemical energy into light through an enzymatic oxygenation of an energy-rich luciferin. In glowing cnidarians, the energy is relocated from an excited oxyluciferin to a fluorescent protein, shifting the colour and enhancing the quantum yield of a photogenic reaction. How protein-chromophore complexes assemble during this interplay in real space, and what this association entails for function, are unknown. Here, we report co-crystal structures of a 120-kilodalton energy-transfer complex from the luminescent soft coral Renilla reniformis. We find a heterotetrameric 2:2 assembly composed of two coelenteramide-loaded luciferases (RrLuc) docked at opposite sides of a head-to-tail dimer of green fluorescent protein (RrGFP). The edge-to-edge distance between donor and acceptor chromophores is below 3 nm, favouring the Forster-type radiationless energy transfer. Furthermore, RrGFP serves not only as a colour-switchable antenna and luminescence amplifier but also tunes the efficiency of luciferase catalysis by controlling its inherent dynamics. Our results provide detailed spatial information about intermolecular dipole-dipole coupling in Renilla bioluminescence, including the arrangement of donor-acceptor pairs that secure excited-state energy transfer with exquisite precision.

biochemistry↗

bioRxiv2026

Fibrin Selective Alteplase with Improved Thrombolysis and Inhibition Resistance Engineered by Rational Design

Thrombolytic enzymes represent an important class of proteolytic biocatalysts for medical applications, yet currently used FDA-approved variants, including alteplase and tenecteplase, remain limited by suboptimal catalytic efficiency, off-target activity, and susceptibility to inhibition. These limitations reflect the complexity of enzyme function in physiological environments, where therapeutic performance depends on the simultaneous optimization of multiple catalytic and biophysical properties. Here, we introduce a multi-objective enzyme engineering strategy for the design of next-generation thrombolytic proteases, explicitly targeting multiple properties required for therapeutic performance. Our approach combines computer-aided design, evolutionary reconstruction, and literature-guided mutation selection to improve catalytic activity, fibrin selectivity, inhibition resistance, and functional lifetime within a single workflow. This framework is coupled with systematic biochemical characterization, in vitro evaluation of clot penetration and fibrinolytic activity, and in vivo validation of efficacy and safety. By addressing multiple performance parameters simultaneously, this strategy enables efficient navigation of trade-offs that typically limit enzyme optimization. Using this approach, we identify Brnoteplase as a lead variant with enhanced fibrin selectivity, improved resistance to inhibition, and superior clot penetration, resulting in increased effective catalytic lifetime and enabling bolus administration. In vivo studies demonstrate enhanced thrombolysis and recanalization with reduced hemorrhagic complications. These findings provide a broadly applicable framework for designing proteolytic biocatalysts suitable for complex biological environments.

biochemistry↗

bioRxiv2025

Molecular trick to reverse SN2 mechanism in hydrolytic enzyme

Hydrolytic haloalkane dehalogenase enzymes catalyze an SN2 nucleophilic substitution to erase halogen substituents in organohalogen compounds. The acid-base-nucleophile triad secures irreversible SN2 displacement of the halogen for the hydroxyl derived from the water. Catalysis relies on the protonatable imidazole ring of the histidine base, and its substitution with an asparagine traps the enzyme in a covalently bound intermediate state, a principle exploited in the widely used HaloTag technology. In contrast, the histidine-to-phenylalanine substitution triggers reversibility of the SN2 mechanism, but the molecular trick by which it reprograms the catalytic pathway remains unknown. Here, we show that the phenylalanine at the site of the histidine base spatially disturbs the adjacent residues, leading to the remodeling of surrounding active-site loops. Consequently, rerouting of the access tunnels imparts distinctive kinetic behavior, featuring a reversible SN2 chemical step that facilitates transhalogenation reactions. This information is crucial for engineering next-generation biocatalysts for sustainable chemistry. HighlightsO_LICatalytic triad (glutamate-histidine-aspartate) secures irreversible SN2 mechanism C_LIO_LIHistidine-to-phenylalanine substitution in the triad leads to a reversible SN2 reaction C_LIO_LIEnzyme active site rerouting is a hallmark of the catalytic reprogramming C_LIO_LIBasis for designing biocatalysts for sustainable transhalogenation chemistry C_LI The bigger pictureHaloalkane dehalogenases catalyze the hydrolytic cleavage of the carbon-halogen bond in halogenated hydrocarbons, a feature used in a wide variety of industrial and biotechnological processes. In HaloTag technology, the catalytic histidine is replaced by an asparagine to enable a stable covalent bonding between a probe and a protein for biological imaging, affinity purification, etc. Remarkably, this originally irreversible SN2 process becomes fully reversible in a histidine-to-phenylalanine mutant. Moreover, halogen ion product is released at this stage, offering a greener alternative for transhalogenation reactions compared to the conventional synthetic approach and a way to recycle environmentally harmful halogenated compounds. However, the optimization of enzymes is needed for applications on an industrial scale. Therefore, we investigated the kinetics of the SN2 step and structural features of these two enzyme mutants, providing a basis for subsequent optimization efforts.

bioengineering↗

bioRxiv2025

Kinetic mechanism of Renilla luciferase guides induced-fit engineering for improved bioluminescence

Luciferases are widely used bioluminescent reporters, yet the molecular determinants of their catalytic efficiency and light-emission stability remain incompletely understood. Here, we reconstruct the complete catalytic pathway of Renilla luciferase by combining steady-state, transient, and temperature-dependent kinetics with crystallography and molecular simulations. We show that the enzyme is substantially undersaturated with oxygen (Km,O2 = 719 M), causing its true turnover number (kcat = 21.9 s-1) to be systematically underestimated. Concurrently, elevated oxygen drives irreversible enzyme inactivation after ~1,500 turnovers, revealing a fundamental trade-off that limits oxygen-affinity engineering. Instead, the genuine bottleneck of the catalytic cycle is the induced-fit conformational opening of the product-bound enzyme. Selective engineering of this transition step through rational loop grafting yielded a variant AncFT-L14 with enhanced catalytic efficiency and glow-type bioluminescence with substantially slower signal decay in cell lysates. Collectively, our results identify conformational dynamics as a primary tunable determinant of luciferase function. More broadly, this work establishes a mechanistically grounded framework for the development of next-generation bioluminescent tools and for engineering enzymes controlled by dynamically gated ligand exchange.

biochemistry↗

bioRxiv2025

Structural basis of pro-inflammatory signaling via the Interleukin-36 receptor (IL-36R) mediated by IL-36γ and IL-37

Interleukin-36 receptor (IL-36R) is activated by IL-36, IL-36{beta}, and IL-36{gamma} to elicit pro-inflammatory signaling and is targeted in acute skin inflammation by the approved antibody spesolimab. IL-37 was recently proposed as a new IL-36R agonist. Such diverse agonist repertoire together with the antagonistic IL-36Ra and IL-38 create a fascinating structure-function landscape for IL-36R, albeit one that is poorly understood. Here, we elucidate how IL-36R grapples IL-36{gamma} with low affinity to enable facile recruitment of the shared receptor IL-1RAcP with high-affinity. In contrast, IL-36R interacts with IL-37 via the exact opposite binding signature. Comparative interrogation of IL-36R activation by IL-36{gamma} and IL-37 confirmed their common pro-inflammatory signature and distinguished IL-36{gamma} as markedly more pro-inflammatory. Structural comparisons of cytokine-activated versus spesolimab-antagonized IL-36R revealed spesolimabs mode of action as an allosteric antagonist. Collectively, our study provides the structural and mechanistic blueprint of IL-36R activation by distinct cytokines and will facilitate its therapeutic targeting. One Sentence SummaryStructural blueprint for IL-36R activation by cognate cytokines and its antagonism by spesolimab in generalized pustular psoriasis.

immunology↗

bioRxiv2024

Improved protein binder design using -pairing targeted RFdiffusion

Despite recent advances in the computational design of protein binders, designing proteins that bind with high affinity to polar protein targets remains an outstanding problem. Here we show that RFdiffusion can be conditioned to efficiently generate protein scaffolds that form geometrically matched extended beta-sheets with target protein edge beta-strands in which polar groups on the target are nearly perfectly complemented with hydrogen bonding groups on the design. We use this approach to design binders against a set of therapeutically relevant polar targets (KIT, PDGFR[a], ALK-2, ALK-3, FCRL5, and NRP1) and find that beta-strand-targeted design yields higher affinities and success rates than unconditioned RFdiffusion. All by all binding experiments show that the designs have affinities ranging from 137 pM to mid nM for their targets and essentially no off target binding despite the sharing of beta-strand interactions, likely reflecting the precise customization of interacting beta-strand geometry and additional designed binder-target interactions. A co-crystal structure of one such design in complex with the KIT receptor is nearly identical to the computational design model confirming the accuracy of the design approach. The ability to robustly generate binders displaying high affinity and specificity to polar interaction surfaces with exposed beta-strands considerably increases the range and capabilities of computational binder design.

biochemistry↗

bioRxiv2022

Illuminating the mechanism and allosteric behavior of NanoLuc luciferase

NanoLuc, a superior {beta}-barrel fold luciferase, was engineered 10 years ago but the nature of its catalysis remains puzzling. Here experimental and computational techniques were combined, revealing that imidazopyrazinone luciferins bind to an intra-barrel catalytic site but also to an allosteric site shaped on the enzyme surface. Structurally, binding to the allosteric site prevents simultaneous binding to the catalytic site, and vice versa, through concerted conformational changes. We demonstrate that restructuration of the allosteric site can boost the luminescent reaction in the remote active site. Mechanistically, an intra-barrel arginine coordinates the imidazopyrazinone component of luciferin, which reacts with O2 via a radical charge-transfer mechanism, and then it also protonates the resulting excited amide product to form a light-emitting neutral species. Concomitantly, an aspartate, supported by two tyrosines, fine-tunes the blue color emitter to secure a high emission intensity. This information is critical to engineering the next-generation of ultrasensitive bioluminescent reporters.

biochemistry↗

bioRxiv2022

A catalytic mechanism for Renilla-type bioluminescence

The widely used coelenterazine-powered Renilla luciferase was discovered over 40 years ago but the oxidative mechanism by which it generates blue photons remains unclear. Here we decipher Renilla-type bioluminescence through crystallographic, spectroscopic, and computational experiments. Structures of ancestral and extant luciferases complexed with the substrate-like analogue azacoelenterazine or a reaction product were obtained, providing unprecedented snapshots of coelenterazine-to-coelenteramide oxidation. Bound coelenterazine adopts a Y-shaped conformation, enabling the deprotonated imidazopyrazinone component to attack O2 via a radical charge-transfer mechanism. A high emission intensity is secured by an aspartate from a conserved proton-relay system, which protonates the excited coelenteramide product. Another aspartate on the rim of the catalytic pocket fine-tunes the electronic state of coelenteramide and promotes the formation of the blue light-emitting phenolate anion. The results obtained also reveal structural features distinguishing flash-type from glow-type bioluminescence, providing insights that will guide the engineering of next-generation luciferase-luciferin pairs for ultrasensitive optical bioassays.

biochemistry↗