Structural and functional insights into the activation of the dual incision activity of UvrC, a key player in bacterial NER.

Bacterial nucleotide excision repair (NER), mediated by the UvrA, UvrB and UvrC proteins is a multistep, ATP-dependent process, that is responsible for the removal of a very wide range of chemically and structurally diverse DNA lesions. DNA damage removal is performed by UvrC, an enzyme possessing a dual endonuclease activity, capable of incising the DNA on either side of the damaged site to release a short single-stranded DNA fragment containing the lesion. Using biochemical and biophysical approaches, we have probed the oligomeric state, UvrB- and DNA-binding abilities and incision activities of wild-type and mutant constructs of UvrC from the radiation resistant bacterium, Deinococcus radiodurans. Moreover, by combining the power of new structure prediction algorithms and experimental crystallographic data, we have assembled the first model of a complete UvrC, revealing several unexpected structural motifs and in particular, a central inactive RNase H domain acting as a platform for the surrounding domains. In this configuration, UvrC is maintained in a 'closed' inactive state that needs to undergo a major rearrangement to adopt an 'open' active state capable of performing the dual incision reaction. Taken together, this study provides important insight into the mechanism of recruitment and activation of UvrC during NER.

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.

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.

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.

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.

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.

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.

Human L-ficolin recognizes phosphocholine moieties of pneumococcal teichoic acid.

Human L-ficolin is a soluble protein of the innate immune system able to sense pathogens through its fibrinogen (FBG) recognition domains and to trigger activation of the lectin complement pathway through associated serine proteases. L-Ficolin has been previously shown to recognize pneumococcal clinical isolates, but its ligands and especially its molecular specificity remain to be identified. Using solid-phase binding assays, serum and recombinant L-ficolins were shown to interact with serotype 2 pneumococcal strain D39 and its unencapsulated R6 derivative. Incubation of both strains with serum triggered complement activation, as measured by C4b and C3b deposition, which was decreased by using ficolin-depleted serum. Recombinant L-ficolin and its FBG-like recognition domain bound to isolated pneumococcal cell wall extracts, whereas binding to cell walls depleted of teichoic acid (TA) was decreased. Both proteins were also shown to interact with two synthetic TA compounds, each comprising part structures of the complete lipoteichoic acid molecule with two PCho residues. Competition studies and direct interaction measurements by surface plasmon resonance identified PCho as a novel L-ficolin ligand. Structural analysis of complexes of the FBG domain of L-ficolin and PCho revealed that the phosphate moiety interacts with amino acids previously shown to define an acetyl binding site. Consequently, binding of L-ficolin to immobilized acetylated BSA was inhibited by PCho and synthetic TA. Binding of serum L-ficolin to immobilized synthetic TA and PCho-conjugated BSA triggered activation of the lectin complement pathway, thus further supporting the hypothesis of L-ficolin involvement in host antipneumococcal defense.

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.

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.

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.

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.

Oxygen-Sensitive Metalloprotein Structure Determination by Cryo-Electron Microscopy.

Metalloproteins are involved in key cell processes such as photosynthesis, respiration, and oxygen transport. However, the presence of transition metals (notably iron as a component of [Fe-S] clusters) often makes these proteins sensitive to oxygen-induced degradation. Consequently, their study usually requires strict anaerobic conditions. Although X-ray crystallography has been the method of choice for solving macromolecular structures for many years, recently electron microscopy has also become an increasingly powerful structure-solving technique. We have used our previous experience with cryo-crystallography to develop a method to prepare cryo-EM grids in an anaerobic chamber and have applied it to solve the structures of apoferritin and the 3 [Fe4S4]-containing pyruvate ferredoxin oxidoreductase (PFOR) at 2.40 Å and 2.90 Å resolution, respectively. The maps are of similar quality to the ones obtained under air, thereby validating our method as an improvement in the structural investigation of oxygen-sensitive metalloproteins by cryo-EM.

Carbohydrate recognition properties of human ficolins: glycan array screening reveals the sialic acid binding specificity of M-ficolin.

Ficolins are oligomeric innate immune recognition proteins consisting of a collagen-like region and a fibrinogen-like recognition domain that bind to pathogen- and apoptotic cell-associated molecular patterns. To investigate their carbohydrate binding specificities, serum-derived L-ficolin and recombinant H- and M-ficolins were fluorescently labeled, and their carbohydrate binding ability was analyzed by glycan array screening. L-ficolin preferentially recognized disulfated N-acetyllactosamine and tri- and tetrasaccharides containing terminal galactose or N-acetylglucosamine. Binding was sensitive to the position and orientation of the bond between N-acetyllactosamine and the adjacent carbohydrate. No significant binding of H-ficolin to any of the 377 glycans probed could be detected, providing further evidence for its poor lectin activity. M-ficolin bound preferentially to 9-O-acetylated 2-6-linked sialic acid derivatives and to various glycans containing sialic acid engaged in a 2-3 linkage. To further investigate the structural basis of sialic acid recognition by M-ficolin, point mutants were produced in which three residues of the fibrinogen domain were replaced by their counterparts in L-ficolin. Mutations G221F and A256V inhibited binding to the 9-O-acetylated sialic acid derivatives, whereas Y271F abolished interaction with all sialic acid-containing glycans. The crystal structure of the Y271F mutant fibrinogen domain was solved, showing that the mutation does not alter the structure of the ligand binding pocket. These analyses reveal novel ficolin ligands such as sulfated N-acetyllactosamine (L-ficolin) and gangliosides (M-ficolin) and provide precise insights into the sialic acid binding specificity of M-ficolin, emphasizing the essential role of Tyr(271) in this respect.

Experimental approaches to kinetics of gas diffusion in hydrogenase.

Hydrogenases, which catalyze H(2) to H(+) conversion as part of the bioenergetic metabolism of many microorganisms, are among the metalloenzymes for which a gas-substrate tunnel has been described by using crystallography and molecular dynamics. However, the correlation between protein structure and gas-diffusion kinetics is unexplored. Here, we introduce two quantitative methods for probing the rates of diffusion within hydrogenases. One uses protein film voltammetry to resolve the kinetics of binding and release of the competitive inhibitor CO; the other is based on interpreting the yield in the isotope exchange assay. We study structurally characterized mutants of a NiFe hydrogenase, and we show that two mutations, which significantly narrow the tunnel near the entrance of the catalytic center, decrease the rates of diffusion of CO and H(2) toward and from the active site by up to 2 orders of magnitude. This proves the existence of a functional channel, which matches the hydrophobic cavity found in the crystal. However, the changes in diffusion rates do not fully correlate with the obstruction induced by the mutation and deduced from the x-ray structures. Our results demonstrate the necessity of measuring diffusion rates and emphasize the role of side-chain dynamics in determining these.

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.

Transient Formation of a Second Active Site Cavity during Quinolinic Acid Synthesis by NadA.

Quinolinate synthase, also called NadA, is a [4Fe-4S]-containing enzyme that uses what is probably the oldest pathway to generate quinolinic acid (QA), the universal precursor of the biologically essential cofactor nicotinamide adenine dinucleotide (NAD). Its synthesis comprises the condensation of dihydroxyacetone phosphate (DHAP) and iminoaspartate (IA), which involves dephosphorylation, isomerization, cyclization, and two dehydration steps. The convergence of the three homologous domains of NadA defines a narrow active site that contains a catalytically essential [4Fe-4S] cluster. A tunnel, which can be opened or closed depending on the nature (or absence) of the bound ligand, connects this cofactor to the protein surface. One outstanding riddle has been the observation that the so far characterized active site is too small to bind IA and DHAP simultaneously. Here, we have used site-directed mutagenesis, X-ray crystallography, functional analyses, and molecular dynamics simulations to propose a condensation mechanism that involves the transient formation of a second active site cavity to which one of the substrates can migrate before this reaction takes place.

High-resolution structure of a modular hyperthermostable endo-ß-1,4-mannanase from Thermotoga petrophila: The ancillary immunoglobulin-like module is a thermostabilizing domain.

The endo-ß-1,4-mannanase from the hyperthermostable bacterium Thermotoga petrophila (TpMan) is an enzyme that catalyzes the hydrolysis of mannan and heteromannan polysaccharides. Of the three domains that comprise TpMan, the N-terminal GH5 catalytic domain and the C-terminal carbohydrate-binding domain are connected through a central ancillary domain of unknown structure and function. In this study, we report the partial crystal structure of the TpMan at 1.45 Å resolution, so far, the first modular hyperthermostable endo-ß-1,4-mannanase structure determined. The structure exhibits two domains, a (ß/α)8-barrel GH5 catalytic domain connected via a linker to the central domain with an immunoglobulin-like ß-sandwich fold formed of seven ß-strands. Functional analysis showed that whereas the immunoglobulin-like domain does not have the carbohydrate-binding function, it stacks on the GH5 catalytic domain acting as a thermostabilizing domain and allowing operation at hyperthermophilic conditions. The carbohydrate-binding domain is absent in the crystal structure most likely due to its high flexibility around the immunoglobulin-like domain which may act also as a pivot. These results represent new structural and functional information useful on biotechnological applications for biofuel and food industries.