A Versatile Virus-Mimetic Engineering Approach for Concurrent Protein Nanocage Surface-Functionalization and Cargo Encapsulation.

Naturally occurring protein nanocages like ferritin are self-assembled from multiple subunits. Because of their unique cage-like structure and biocompatibility, there is a growing interest in their biomedical use. A multipurpose and straightforward engineering approach does not exist for using nanocages to make drug-delivery systems by encapsulating hydrophilic or hydrophobic drugs and developing vaccines by surface functionalization with a protein like an antigen. Here, a versatile engineering approach is described by mimicking the HIV-1 Gap polyprotein precursor. Various PREcursors of nanoCages (PREC) are designed and created by linking two ferritin subunits via a flexible linker peptide containing a protease cleavage site. These precursors can have additional proteins at their N-terminus, and their protease cleavage generates ferritin-like nanocages named protease-induced nanocages (PINCs). It is demonstrated that PINC formation allows concurrent surface decoration with a protein and hydrophilic or hydrophobic drug encapsulation up to fourfold more than the amount achieved using other methods. The PINCs/Drug complex is stable and efficiently kills cancer cells. This work provides insight into the precursors' design rules and the mechanism of PINCs formation. The engineering approach and mechanistic insight described here will facilitate nanocages' applications in drug delivery or as a platform for making multifunctional therapeutics like mosaic vaccines.

Radical S-Adenosyl-l-Methionine Enzyme PylB: A C-Centered Radical to Convert l-Lysine into (3R)-3-Methyl-d-Ornithine.

PylB is a radical S-adenosyl-l-methionine (SAM) enzyme predicted to convert l-lysine into (3R)-3-methyl-d-ornithine, a precursor in the biosynthesis of the 22nd proteogenic amino acid pyrrolysine. This protein highly resembles that of the radical SAM tyrosine and tryptophan lyases, which activate their substrate by abstracting a H atom from the amino-nitrogen position. Here, combining in vitro assays, analytical methods, electron paramagnetic resonance spectroscopy, and theoretical methods, we demonstrated that instead, PylB activates its substrate by abstracting a H atom from the Cγ position of l-lysine to afford the radical-based ß-scission. Strikingly, we also showed that PylB catalyzes the reverse reaction, converting (3R)-3-methyl-d-ornithine into l-lysine and using catalytic amounts of the 5'-deoxyadenosyl radical. Finally, we identified significant in vitro production of 5'-thioadenosine, an unexpected shunt product that we propose to result from the quenching of the 5'-deoxyadenosyl radical species by the nearby [Fe4S4] cluster.

Maturation of the [FeFe]-Hydrogenase: Direct Transfer of the (κ3 -cysteinate)FeII (CN)(CO)2 Complex B from HydG to HydE.

[FeFe]-hydrogenases efficiently catalyze the reversible oxidation of molecular hydrogen. Their prowess stems from the intricate H-cluster, combining a [Fe4 S4 ] center with a binuclear iron center ([2Fe]H ). In the latter, each iron atom is coordinated by a CO and CN ligand, connected by a CO and an azadithiolate ligand. The synthesis of this active site involves a unique multiprotein assembly, featuring radical SAM proteins HydG and HydE. HydG initiates the transformation of L-tyrosine into cyanide and carbon monoxide to generate complex B, which is subsequently transferred to HydE to continue the biosynthesis of the [2Fe]H -subcluster. Due to its instability, complex B isolation for structural or spectroscopic characterization has been elusive thus far. Nevertheless, the use of a biomimetic analogue of complex B allowed circumvention of the need for the HydG protein during in vitro functional investigations, implying a similar structure for complex B. Herein, we used the HydE protein as a nanocage to encapsulate and stabilize the complex B product generated by HydG. Using X-ray crystallography, we successfully determined its structure at 1.3 Šresolution. Furthermore, we demonstrated that complex B is directly transferred from HydG to HydE, thus not being released into the solution post-synthesis, highlighting a transient interaction between the two proteins.

L-tyrosine-bound ThiH structure reveals C-C bond break differences within radical SAM aromatic amino acid lyases.

2-iminoacetate synthase ThiH is a radical S-adenosyl-L-methionine (SAM) L-tyrosine lyase and catalyzes the L-tyrosine Cα-Cß bond break to produce dehydroglycine and p-cresol while the radical SAM L-tryptophan lyase NosL cleaves the L-tryptophan Cα-C bond to produce 3-methylindole-2-carboxylic acid. It has been difficult to understand the features that condition one C-C bond break over the other one because the two enzymes display significant primary structure similarities and presumably similar substrate-binding modes. Here, we report the crystal structure of L-tyrosine bound ThiH from Thermosinus carboxydivorans revealing an unusual protonation state of L-tyrosine upon binding. Structural comparison of ThiH with NosL and computational studies of the respective reactions they catalyze show that substrate activation is eased by tunneling effect and that subtle structural changes between the two enzymes affect, in particular, the hydrogen-atom abstraction by the 5´-deoxyadenosyl radical species, driving the difference in reaction specificity.

Methods to Screen for Radical SAM Enzyme Crystallization Conditions.

Radical S-adenosyl-L-methionine proteins most probably belong to the widest superfamily of metalloenzymes. Thanks to their ability to catalyze difficult reactions, combined with their involvement in the biosynthesis of numbers of natural products, they sound promising for various biotechnological applications. Their structural study is often limited because they are usually challenging to crystallize. This chapter presents protocols and equipment developed to quickly screen for crystallization conditions under anaerobic conditions, as exemplified by our recent study of the nitrogenase maturase NifB.

Crystal Structure of the [FeFe]-Hydrogenase Maturase HydE Bound to Complex-B.

[FeFe]-hydrogenases use a unique organometallic complex, termed the H cluster, to reversibly convert H2 into protons and low-potential electrons. It can be best described as a [Fe4S4] cluster coupled to a unique [2Fe]H center where the reaction actually takes place. The latter corresponds to two iron atoms, each of which is bound by one CN- ligand and one CO ligand. The two iron atoms are connected by a unique azadithiolate molecule (-S-CH2-NH-CH2-S-) and an additional bridging CO. This [2Fe]H center is built stepwise thanks to the well-orchestrated action of maturating enzymes that belong to the Hyd machinery. Among them, HydG converts l-tyrosine into CO and CN- to produce a unique l-cysteine-Fe(CO)2CN species termed complex-B. Very recently, HydE was shown to perform radical-based chemistry using synthetic complex-B as a substrate. Here we report the high-resolution crystal structure that establishes the identity of the complex-B-bound HydE. By triggering the reaction prior to crystallization, we trapped a new five-coordinate Fe species, supporting the proposal that HydE performs complex modifications of complex-B to produce a monomeric "SFe(CO)2CN" precursor to the [2Fe]H center. Substrate access, product release, and intermediate transfer are also discussed.

Structural Insights into the Mechanism of the Radical SAM Carbide Synthase NifB, a Key Nitrogenase Cofactor Maturating Enzyme.

Nitrogenase is a key player in the global nitrogen cycle, as it catalyzes the reduction of dinitrogen into ammonia. The active site of the nitrogenase MoFe protein corresponds to a [MoFe7S9C-(R)-homocitrate] species designated FeMo-cofactor, whose biosynthesis and insertion requires the action of over a dozen maturation proteins provided by the NIF (for NItrogen Fixation) assembly machinery. Among them, the radical SAM protein NifB plays an essential role, concomitantly inserting a carbide ion and coupling two [Fe4S4] clusters to form a [Fe8S9C] precursor called NifB-co. Here we report on the X-ray structure of NifB from Methanotrix thermoacetophila at 1.95 Å resolution in a state pending the binding of one [Fe4S4] cluster substrate. The overall NifB architecture indicates that this enzyme has a single SAM binding site, which at this stage is occupied by cysteine residue 62. The structure reveals a unique ligand binding mode for the K1-cluster involving cysteine residues 29 and 128 in addition to histidine 42 and glutamate 65. The latter, together with cysteine 62, belongs to a loop inserted in the active site, likely protecting the already present [Fe4S4] clusters. These two residues regulate the sequence of events, controlling SAM dual reactivity and preventing unwanted radical-based chemistry before the K2 [Fe4S4] cluster substrate is loaded into the protein. The location of the K1-cluster, too far away from the SAM binding site, supports a mechanism in which the K2-cluster is the site of methylation.

Radical S-Adenosyl-l-methionine Tryptophan Lyase (NosL): How the Protein Controls the Carboxyl Radical •CO2- Migration.

The radical S-adenosyl-l-methionine tryptophan lyase uses radical-based chemistry to convert l-tryptophan into 3-methyl-2-indolic acid, a fragment in the biosynthesis of the thiopeptide antibiotic nosiheptide. This complex reaction involves several successive steps corresponding to (i) the activation by a specific hydrogen-atom abstraction, (ii) an unprecedented •CO2- radical migration, (iii) a cyanide fragment release, and (iv) the termination of the radical-based reaction. In vitro study of this reaction is made more difficult because the enzyme produces a significant amount of a shunt product instead of the natural product. Here, using a combination of X-ray crystallography, electron paramagnetic resonance spectroscopy, and quantum and hybrid quantum mechanical/molecular mechanical calculations, we have deciphered the fine mechanism of the key •CO2- radical migration, highlighting how the preorganized active site of the protein tightly controls this reaction.

1,2-Diol Dehydration by the Radical SAM Enzyme AprD4: A Matter of Proton Circulation and Substrate Flexibility.

Regiospecific dehydration of vicinal diols by enzymes is a difficult reaction that usually requires activation by dedicated organic cofactors. The enzymatic use of radical-based chemistry is an effective but challenging alternative as radical intermediates are difficult to control. Here we report the X-ray structure of the radical S-adenosyl-l-methionine (SAM) dehydratase AprD4 involved in the biosynthesis of the aminoglycoside (AG) antibiotic apramycin. Using in vitro characterizations and theoretical calculations based on our crystal structure, we have been able to propose a detailed mechanism of AprD4 catalysis, which involves a complex partially substrate-induced proton relay network in the enzyme active site and highlights the key role of the protein matrix in driving high-energy intermediates.

Carbon-sulfur bond-forming reaction catalysed by the radical SAM enzyme HydE.

Carbon-sulfur bond formation at aliphatic positions is a challenging reaction that is performed efficiently by radical S-adenosyl-L-methionine (SAM) enzymes. Here we report that 1,3-thiazolidines can act as ligands and substrates for the radical SAM enzyme HydE, which is involved in the assembly of the active site of [FeFe]-hydrogenase. Using X-ray crystallography, in vitro assays and NMR spectroscopy we identified a radical-based reaction mechanism that is best described as the formation of a C-centred radical that concomitantly attacks the sulfur atom of a thioether. To the best of our knowledge, this is the first example of a radical SAM enzyme that reacts directly on a sulfur atom instead of abstracting a hydrogen atom. Using theoretical calculations based on our high-resolution structures we followed the evolution of the electronic structure from SAM through to the formation of S-adenosyl-L-cysteine. Our results suggest that, at least in this case, the widely proposed and highly reactive 5'-deoxyadenosyl radical species that triggers the reaction in radical SAM enzymes is not an isolable intermediate.

Fine-tuning of a radical-based reaction by radical S-adenosyl-L-methionine tryptophan lyase.

The radical S-adenosyl-L-methionine tryptophan lyase NosL converts L-tryptophan into 3-methylindolic acid, which is a precursor in the synthesis of the thiopeptide antibiotic nosiheptide. Using electron paramagnetic resonance spectroscopy and multiple L-tryptophan isotopologues, we trapped and characterized radical intermediates that indicate a carboxyl fragment migration mechanism for NosL. This is in contrast to a proposed fragmentation-recombination mechanism that implied Cα-Cß bond cleavage of L-tryptophan. Although NosL resembles related tyrosine lyases, subtle substrate motions in its active site are responsible for a fine-tuned radical chemistry, which selects the Cα-C bond for disruption. This mechanism highlights evolutionary adaptation to structural constraints in proteins as a route to alternative enzyme function.

CO and CN- syntheses by [FeFe]-hydrogenase maturase HydG are catalytically differentiated events.

The synthesis and assembly of the active site [FeFe] unit of [FeFe]-hydrogenases require at least three maturases. The radical S-adenosyl-l-methionine HydG, the best characterized of these proteins, is responsible for the synthesis of the hydrogenase CO and CN(-) ligands from tyrosine-derived dehydroglycine (DHG). We speculated that CN(-) and the CO precursor (-):CO2H may be generated through an elimination reaction. We tested this hypothesis with both wild type and HydG variants defective in second iron-sulfur cluster coordination by measuring the in vitro production of CO, CN(-), and (-):CO2H-derived formate. We indeed observed formate production under these conditions. We conclude that HydG is a multifunctional enzyme that produces DHG, CN(-), and CO at three well-differentiated catalytic sites. We also speculate that homocysteine, cysteine, or a related ligand could be involved in Fe(CO)x(CN)y transfer to the HydF carrier/scaffold.

[NiFe]-hydrogenases revisited: nickel-carboxamido bond formation in a variant with accrued O2-tolerance and a tentative re-interpretation of Ni-SI states.

[NiFe]-hydrogenases are well-studied enzymes capable of oxidizing molecular hydrogen and reducing protons. EPR and FTIR spectroscopic studies have shown that these enzymes can be isolated in several redox states that include paramagnetic oxidized inactive Ni-A and Ni-B species and a reduced Ni-C form. The latter and the diamagnetic respectively more oxidized Ni-SI and more reduced Ni-R forms are generally thought to be involved in the catalytic cycle of [NiFe]-hydrogenases. With the exception of Ni-SI, these different stable states have been well characterized. Here, based on the crystal structure of a partially reduced Desulfovibrio fructosovorans (Df) enzyme and data from the literature we propose that at least one of the Ni-SI sub-states contains an unexpected combination of hydride and sulfenic acid moieties. We have also determined the structure of the less oxygen-sensitive Df [NiFe]-hydrogenase V74C mutant and found that more than half of the active site nickel occupies a novel position, called Ni'. In this new position, the metal ion is coordinated by two cysteine thiolates, a bridging species modeled as SH(-) and a main chain carboxamido N atom. The Ni' coordination is similar to the one found in Ni superoxide dismutase, an enzyme that operates at significantly more positive potentials than [NiFe]-hydrogenases. We propose that the oxygen-tolerance of the V74C variant results from a high potential stabilization of a Ni'(iii) species induced by the change in the metal ion coordination sphere. We also propose that transient Ni'(iii) species can rapidly attract successive electrons from the Fe4S4 proximal cluster accelerating the reduction of oxygen to water and hydroxide. The naturally occurring oxygen-tolerant [NiFe]-hydrogenases have an unusual proximal cluster that has been shown to be exceptionally plastic and capable of undergoing two successive one-electron oxidations. This double oxidation is modulated by the migration of one of the iron atoms in the cluster to the main chain where, as Fe(iii), it forms a bond with a carboxamido N ligand. Like in the Df V74C variant the electrons from the proximal cluster help reducing O2 to H2O and OH(-). In conclusion, in both cases a metal-carboxamido bond may explain, at least partially, the observed oxygen tolerance.

Crystallographic studies of [NiFe]-hydrogenase mutants: towards consensus structures for the elusive unready oxidized states.

Catalytically inactive oxidized O2-sensitive [NiFe]-hydrogenases are characterized by a mixture of the paramagnetic Ni-A and Ni-B states. Upon O2 exposure, enzymes in a partially reduced state preferentially form the unready Ni-A state. Because partial O2 reduction should generate a peroxide intermediate, this species was previously assigned to the elongated Ni-Fe bridging electron density observed for preparations of [NiFe]-hydrogenases known to contain the Ni-A state. However, this proposition has been challenged based on the stability of this state to UV light exposure and the possibility of generating it anaerobically under either chemical or electrochemical oxidizing conditions. Consequently, we have considered alternative structures for the Ni-A species including oxidation of thiolate ligands to either sulfenate or sulfenic acid. Here, we report both new and revised [NiFe]-hydrogenases structures and conclude, taking into account corresponding characterizations by Fourier transform infrared spectroscopy (FTIR), that the Ni-A species contains oxidized cysteine and bridging hydroxide ligands instead of the peroxide ligand we proposed earlier. Our analysis was rendered difficult by the typical formation of mixtures of unready oxidized states that, furthermore, can be reduced by X-ray induced photoelectrons. The present study could be carried out thanks to the use of Desulfovibrio fructosovorans [NiFe]-hydrogenase mutants with special properties. In addition to the Ni-A state, crystallographic results are also reported for two diamagnetic unready states, allowing the proposal of a revised oxidized inactive Ni-SU model and a new structure characterized by a persulfide ion that is assigned to an Ni-'Sox' species.

Crystal structure of HydG from Carboxydothermus hydrogenoformans: a trifunctional [FeFe]-hydrogenase maturase.

The structure of the radical S-adenosyl-L-methionine (SAM) [FeFe]-hydrogenase maturase HydG involved in CN(-) /CO synthesis is characterized by two internal tunnels connecting its tyrosine-binding pocket with the external medium and the C-terminal Fe4 S4 cluster-containing region. A comparison with a tryptophan-bound NosL structure suggests that substrate binding causes the closing of the first tunnel and, along with mutagenesis studies, that tyrosine binds to HydG with its amino group well positioned for H-abstraction by SAM. In this orientation the dehydroglycine (DHG) fragment caused by tyrosine Cα-Cß bond scission can readily migrate through the second tunnel towards the C-terminal domain where both CN(-) and CO are synthesized. Our HydG structure appears to be in a relaxed state with its C-terminal cluster CysX2 CysX22 Cys motif exposed to solvent. A rotation of this domain coupled to Fe4 S4 cluster assembly would bury its putatively reactive unique Fe ion thereby allowing it to interact with DHG.

Sustained activation of interferon regulatory factor 3 during infection by paramyxoviruses requires MDA5.

Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are the main cytosolic sensors of single-stranded RNA viruses, including paramyxoviruses, and are required to initiate a quick and robust innate antiviral response. Despite different ligand-binding properties, the consensus view is that RIG-I and MDA5 trigger common signal(s) to activate interferon regulatory factor 3 (IRF-3) and NF-κB, and downstream antiviral and proinflammatory cytokine expression. Here, we performed a thorough analysis of the temporal involvement of RIG-I and MDA5 in the regulation of IRF-3 during respiratory syncytial virus (RSV) infection. Based on specific RNA interference-mediated knockdown of RIG-I and MDA5 in A549 cells, we confirmed that RIG-I is critical for the initiation of IRF-3 phosphorylation, dimerization and downstream gene expression. On the other hand, our experiments yielded the first evidence that knockdown of MDA5 leads to early ubiquitination and proteasomal degradation of active IRF-3. Conversely, ectopic expression of MDA5 prolonged RIG-I-induced IRF-3 activation. Altogether, we provide novel mechanistic insight into the temporal involvement of RIG-I and MDA5 in the innate antiviral response. While RIG-I is essential for initial IRF-3 activation, engagement of induced MDA5 is essential to prevent early degradation of IRF-3, thereby sustaining IRF-3-dependent antiviral gene expression. MDA5 plays a similar role during Sendai virus infection suggesting that this model is not restricted to RSV amongst paramyxoviruses.

IFNß/TNFα synergism induces a non-canonical STAT2/IRF9-dependent pathway triggering a novel DUOX2 NADPH oxidase-mediated airway antiviral response.

Airway epithelial cells are key initial innate immune responders in the fight against respiratory viruses, primarily via the secretion of antiviral and proinflammatory cytokines that act in an autocrine/paracrine fashion to trigger the establishment of an antiviral state. It is currently thought that the early antiviral state in airway epithelial cells primarily relies on IFNß secretion and the subsequent activation of the interferon-stimulated gene factor 3 (ISGF3) transcription factor complex, composed of STAT1, STAT2 and IRF9, which regulates the expression of a panoply of interferon-stimulated genes encoding proteins with antiviral activities. However, the specific pathways engaged by the synergistic action of different cytokines during viral infections, and the resulting physiological outcomes are still ill-defined. Here, we unveil a novel delayed antiviral response in the airways, which is initiated by the synergistic autocrine/paracrine action of IFNß and TNFα, and signals through a non-canonical STAT2- and IRF9-dependent, but STAT1-independent cascade. This pathway ultimately leads to the late induction of the DUOX2 NADPH oxidase expression. Importantly, our study uncovers that the development of the antiviral state relies on DUOX2-dependent H2O2 production. Key antiviral pathways are often targeted by evasion strategies evolved by various pathogenic viruses. In this regard, the importance of the novel DUOX2-dependent antiviral pathway is further underlined by the observation that the human respiratory syncytial virus is able to subvert DUOX2 induction.

X-ray snapshots of possible intermediates in the time course of synthesis and degradation of protein-bound Fe4S4 clusters.

Fe4S4 clusters are very common versatile prosthetic groups in proteins. Their redox property of being sensitive to O2-induced oxidative damage is, for instance, used by the cell to sense oxygen levels and switch between aerobic and anaerobic metabolisms, as exemplified by the fumarate, nitrate reduction regulator (FNR). Using the hydrogenase maturase HydE from Thermotoga maritima as a template, we obtained several unusual forms of FeS clusters, some of which are associated with important structural changes. These structures represent intermediate states relevant to both FeS cluster assembly and degradation. We observe one Fe2S2 cluster bound by two cysteine persulfide residues. This observation lends structural support to a very recent Raman study, which reported that Fe4S4-to-Fe2S2 cluster conversion upon oxygen exposure in FNR resulted in concomitant production of cysteine persulfide as cluster ligands. Similar persulfide ligands have been observed in vitro for several other Fe4S4 cluster-containing proteins. We have also monitored FeS cluster conversion directly in our protein crystals. Our structures indicate that the Fe4S4-to-Fe2S2 change requires large structural modifications, which are most likely responsible for the dimer-monomer transition in FNR.

Histidine 416 of the periplasmic binding protein NikA is essential for nickel uptake in Escherichia coli.

Escherichia coli require nickel for the synthesis of [NiFe] hydrogenases under anaerobic growth conditions. Nickel import depends on the specific ABC-transporter NikABCDE encoded by the nik operon, which deletion causes the complete abolition of hydrogenase activity. We have previously postulated that the periplasmic binding protein NikA binds a natural metallophore containing three carboxylate functions that coordinate a Ni(II) ion, the fourth ligand being His416, the only direct metal-protein contact, completing a square-planar coordination for the metal. The crystal structure of the H416I mutant showed no electron density corresponding to a metal-chelator complex. In vivo experiments indicate that the mutation causes a significant decrease in nickel uptake and hydrogenase activity. These results confirm the essential role of His416 in nickel transport by NikA.

Crystallographic snapshots of the reaction of aromatic C-H with O(2) catalysed by a protein-bound iron complex.

Chemical reactions inside single crystals are quite rare because crystallinity is difficult to retain owing to atomic rearrangements. Protein crystals in general have a high solvent content. This allows for some molecular flexibility, which makes it possible to trap reaction intermediates of enzymatic reactions without disrupting the crystal lattice. A similar approach has not yet been fully implemented in the field of inorganic chemistry. Here, we have combined model chemistry and protein X-ray crystallography to study the intramolecular aromatic dihydroxylation by an arene-containing protein-bound iron complex. The bound complex was able to activate dioxygen in the presence of a reductant, leading to the formation of catechol as the sole product. The structure determination of four of the catalytic cycle intermediates and the end product showed that the hydroxylation reaction implicates an iron peroxo, generated by reductive O(2) activation, an intermediate already observed in iron monooxygenases. This strategy also provided unexpected mechanistic details such as the rearrangement of the iron coordination sphere on metal reduction.