Stepwise conversion of the Cys6[4Fe-3S] to a Cys4[4Fe-4S] cluster and its impact on the oxygen tolerance of [NiFe]-hydrogenase.

The membrane-bound [NiFe]-hydrogenase of Cupriavidus necator is a rare example of a truly O2-tolerant hydrogenase. It catalyzes the oxidation of H2 into 2e- and 2H+ in the presence of high O2 concentrations. This characteristic trait is intimately linked to the unique Cys6[4Fe-3S] cluster located in the proximal position to the catalytic center and coordinated by six cysteine residues. Two of these cysteines play an essential role in redox-dependent cluster plasticity, which bestows the cofactor with the capacity to mediate two redox transitions at physiological potentials. Here, we investigated the individual roles of the two additional cysteines by replacing them individually as well as simultaneously with glycine. The crystal structures of the corresponding MBH variants revealed the presence of Cys5[4Fe-4S] or Cys4[4Fe-4S] clusters of different architecture. The protein X-ray crystallography results were correlated with accompanying biochemical, spectroscopic and electrochemical data. The exchanges resulted in a diminished O2 tolerance of all MBH variants, which was attributed to the fact that the modified proximal clusters mediated only one redox transition. The previously proposed O2 protection mechanism that detoxifies O2 to H2O using four protons and four electrons supplied by the cofactor infrastructure, is extended by our results, which suggest efficient shutdown of enzyme function by formation of a hydroxy ligand in the active site that protects the enzyme from O2 binding under electron-deficient conditions.

Identification and characterisation of anti-IL-13 inhibitory single domain antibodies provides new insights into receptor selectivity and attractive opportunities for drug discovery.

Interleukin-13 (IL-13) is a cytokine involved in T-cell immune responses and is a well validated therapeutic target for the treatment of asthma, along with other allergic and inflammatory diseases. IL-13 signals through a ternary signalling complex formed with the receptors IL-13Rα1 and IL-4Rα. This complex is assembled by IL-13 initially binding IL-13Rα1, followed by association of the binary IL-13:IL-13Rα1 complex with IL-4Rα. The receptors are shared with IL-4, but IL-4 initially binds IL-4Rα. Here we report the identification and characterisation of a diverse panel of single-domain antibodies (VHHs) that bind to IL-13 (KD 40 nM-5.5 µM) and inhibit downstream IL-13 signalling (IC50 0.2-53.8 µM). NMR mapping showed that the VHHs recognise a number of epitopes on IL-13, including previously unknown allosteric sites. Further NMR investigation of VHH204 bound to IL-13 revealed a novel allosteric mechanism of inhibition, with the antibody stabilising IL-13 in a conformation incompatible with receptor binding. This also led to the identification of a conformational equilibrium for free IL-13, providing insights into differing receptor signalling complex assembly seen for IL-13 compared to IL-4, with formation of the IL-13:IL-13Rα1 complex required to stabilise IL-13 in a conformation with high affinity for IL-4Rα. These findings highlight new opportunities for therapeutic targeting of IL-13 and we report a successful 19F fragment screen of the IL-13:VHH204 complex, including binding sites identified for several hits. To our knowledge, these 19F containing fragments represent the first small-molecules shown to bind to IL-13 and could provide starting points for a small-molecule drug discovery programme.

Crystal structure and functional characterization of selenocysteine-containing glutathione peroxidase 4 suggests an alternative mechanism of peroxide reduction.

Glutathione peroxidases (GPX) are anti-oxidative enzymes that reduce organic and inorganic hydroperoxides to the corresponding alcohols at the expense of reduced glutathione. The human genome involves eight GPX genes and five of them encode for selenocysteine-containing enzymes. Among the human GPX-isoforms, GPX4 is unique since it is capable of reducing complex hydroperoxy ester lipids such as hydroperoxy phospholipids and hydroperoxy cholesterolesters. Using a number of genetically modified mouse strains the biological role of GPX4 has comprehensively characterized but the molecular enzymology is less well explored. This lack of knowledge is partly related to the fact that mammalian selenoproteins are not high-level expressed in conventional overexpression systems. To explore the structural and functional properties of human GPX4 we expressed this selenoprotein in a cysteine-auxotrophic E. coli strain using a semi-chemical expression strategy. The recombinant enzyme was purified in mg amounts from the bacterial lysate to electrophoretic homogeneity and characterized with respect to its protein-chemical and enzymatic properties. Its crystal structure was solved at 1.3 Šresolution and the X-ray data indicated a monomeric protein, which contains the catalytic selenium at the redox level of the seleninic acid. These data suggest an alternative reaction mechanism involving three different redox states (selenol, selenenic acid, seleninic acid) of the catalytically active selenocysteine.

Tracking the route of molecular oxygen in O2-tolerant membrane-bound [NiFe] hydrogenase.

[NiFe] hydrogenases catalyze the reversible splitting of H2 into protons and electrons at a deeply buried active site. The catalytic center can be accessed by gas molecules through a hydrophobic tunnel network. While most [NiFe] hydrogenases are inactivated by O2, a small subgroup, including the membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha, is able to overcome aerobic inactivation by catalytic reduction of O2 to water. This O2 tolerance relies on a special [4Fe3S] cluster that is capable of releasing two electrons upon O2 attack. Here, the O2 accessibility of the MBH gas tunnel network has been probed experimentally using a "soak-and-freeze" derivatization method, accompanied by protein X-ray crystallography and computational studies. This combined approach revealed several sites of O2 molecules within a hydrophobic tunnel network leading, via two tunnel entrances, to the catalytic center of MBH. The corresponding site occupancies were related to the O2 concentrations used for MBH crystal derivatization. The examination of the O2-derivatized data furthermore uncovered two unexpected structural alterations at the [4Fe3S] cluster, which might be related to the O2 tolerance of the enzyme.

Substrate specificity and reaction mechanism of human prolidase.

Prolidase is a ubiquitously distributed dipeptidase and the only dipeptidase in humans capable of cleaving the peptide bond preceding the amino acids proline (Pro) or hydroxyproline (Hyp). It is mainly implicated in the degradation of dietary and endogenous proteins. It is also involved in the terminal steps of collagen catabolism by hydrolyzing Pro and Hyp-containing dipeptides. Finally, it is believed to play a role in the regulation of peptidic hormones. Diminished or absent prolidase activity is related to a rare autosomal disease, referred to as prolidase deficiency (PD). This disease manifests itself by a variety of clinical symptoms. To date, there is no definitive cure to PD. This may in part be due to an incomplete understanding of the wild-type (wt) enzyme with respect to substrate-binding mode and consequently the mechanism of the catalyzed reaction. In this work, we describe the high-resolution crystal structures of the wt human prolidase in the ligand-free form as well as in substrate-bound states and in complex with the cleavage product Pro. This series of structures provides much relevant information for the definition of substrate-binding and the reaction mechanism. A recent study on Escherichia coli prolidase revealed how substrates of different length are discriminated. Here, based on our own structural results, we evaluate and extend this analysis. Moreover, we describe and analyze substrate and product binding in the active site and we propose that the crucial catalytic moiety is actually a hydroxide ion. This information significantly advances our understanding of prolidase-based pathologies. DATABASE: The refined structure coordinates as well as the corresponding structure factor amplitudes have been deposited in the PDB under the accession numbers 5M4G, 5M4J, 5M4L, and 5M4Q.

The crystal structure of Pseudomonas aeruginosa lipoxygenase Ala420Gly mutant explains the improved oxygen affinity and the altered reaction specificity.

Secreted LOX from Pseudomonas aeruginosa (PA-LOX) has previously been identified as arachidonic acid 15S-lipoxygenating enzyme. Here we report that the substitution of Ala420Gly in PA-LOX leads to an enzyme variant with pronounced dual specificity favoring arachidonic acid 11R-oxygenation. When compared with other LOX-isoforms the molecular oxygen affinity of wild-type PA-LOX is 1-2 orders of magnitude lower (Km O2 of 0.4mM) but Ala420Gly exchange improved the molecular oxygen affinity (Km O2 of 0.2mM). Experiments with stereo-specifically deuterated linoleic acid indicated that the formation of both 13S- and 9R-HpODE involves abstraction of the proS-hydrogen from C11 of the fatty acid backbone. To explore the structural basis for the observed functional changes (altered specificity, improved molecular oxygen affinity) we solved the crystal structure of the Ala420Gly mutant of PA-LOX at 1.8Å resolution and compared it with the wild-type enzyme. Modeling of fatty acid alignment at the catalytic center suggested that in the wild-type enzyme dioxygen is directed to C15 of arachidonic acid by a protein tunnel, which interconnects the catalytic center with the protein surface. Ala420Gly exchange redirects intra-enzyme O2 diffusion by bifurcating this tunnel so that C11 of arachidonic acid also becomes accessible for O2 insertion.

Structural and functional basis of phospholipid oxygenase activity of bacterial lipoxygenase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa expresses a secreted LOX-isoform (PA-LOX, LoxA) capable of oxidizing polyenoic fatty acids to hydroperoxy derivatives. Here we report high-level expression of this enzyme in E. coli and its structural and functional characterization. Recombinant PA-LOX oxygenates polyenoic fatty acids including eicosapentaenoic acid and docosahexaenoic acid to the corresponding (n-6)S-hydroperoxy derivatives. This reaction involves abstraction of the proS-hydrogen from the n-8 bisallylic methylene. PA-LOX lacks major leukotriene synthase activity but converts 5S-HETE and 5S,6R/S-DiHETE to anti-inflammatory and pro-resolving lipoxins. It also exhibits phospholipid oxygenase activity as indicated by the formation of a specific pattern of oxygenation products from different phospholipid subspecies. Multiple mutagenesis studies revealed that PA-LOX does not follow classical concepts explaining the reaction specificity of mammalian LOXs. The crystal structure of PA-LOX was solved with resolutions of up to 1.48Å and its polypeptide chain is folded as single domain. The substrate-binding pocket consists of two fatty acid binding subcavities and lobby. Subcavity-1 contains the catalytic non-heme iron. A phosphatidylethanolamine molecule occupies the substrate-binding pocket and its sn1 fatty acid is located close to the catalytic non-heme iron. His377, His382, His555, Asn559 and the C-terminal Ile685 function as direct iron ligands and a water molecule (hydroxyl) completes the octahedral ligand sphere. Although the biological relevance of PA-LOX is still unknown its functional characteristics (lipoxin synthase activity) implicate this enzyme in a bacterial evasion strategy aimed at downregulating the hosts' immune system.

Krypton Derivatization of an O2 -Tolerant Membrane-Bound [NiFe] Hydrogenase Reveals a Hydrophobic Tunnel Network for Gas Transport.

[NiFe] hydrogenases are metalloenzymes catalyzing the reversible heterolytic cleavage of hydrogen into protons and electrons. Gas tunnels make the deeply buried active site accessible to substrates and inhibitors. Understanding the architecture and function of the tunnels is pivotal to modulating the feature of O2 tolerance in a subgroup of these [NiFe] hydrogenases, as they are interesting for developments in renewable energy technologies. Here we describe the crystal structure of the O2 -tolerant membrane-bound [NiFe] hydrogenase of Ralstonia eutropha (ReMBH), using krypton-pressurized crystals. The positions of the krypton atoms allow a comprehensive description of the tunnel network within the enzyme. A detailed overview of tunnel sizes, lengths, and routes is presented from tunnel calculations. A comparison of the ReMBH tunnel characteristics with crystal structures of other O2 -tolerant and O2 -sensitive [NiFe] hydrogenases revealed considerable differences in tunnel size and quantity between the two groups, which might be related to the striking feature of O2 tolerance.

Resonance Raman Spectroscopic Analysis of the [NiFe] Active Site and the Proximal [4Fe-3S] Cluster of an O2-Tolerant Membrane-Bound Hydrogenase in the Crystalline State.

We have applied resonance Raman (RR) spectroscopy on single protein crystals of the O2-tolerant membrane-bound [NiFe] hydrogenase (MBH from Ralstonia eutropha) which catalyzes the splitting of H2 into protons and electrons. RR spectra taken from 65 MBH samples in different redox states were analyzed in terms of the respective component spectra of the active site and the unprecedented proximal [4Fe-3S] cluster using a combination of statistical methods and global fitting procedures. These component spectra of the individual cofactors were compared with calculated spectra obtained by quantum mechanics/molecular mechanics (QM/MM) methods. Thus, the recently discovered hydroxyl-coordination of one iron in the [4Fe-3S] cluster was confirmed. Infrared (IR) microscopy of oxidized MBH crystals revealed the [NiFe] active site to be in the Nir-B [Ni(III)] and Nir-S [Ni(II)] states, whereas RR measurements of these crystals uncovered the Nia-S [Ni(II)] state as the main spectral component, suggesting its in situ formation via photodissociation of the assumed bridging hydroxyl or water ligand. On the basis of QM/MM calculations, individual band frequencies could be correlated with structural parameters for the Nia-S state as well as for the Ni-L state, which is formed upon photodissociation of the bridging hydride of H2-reduced active site states.

The class III cyclobutane pyrimidine dimer photolyase structure reveals a new antenna chromophore binding site and alternative photoreduction pathways.

Photolyases are proteins with an FAD chromophore that repair UV-induced pyrimidine dimers on the DNA in a light-dependent manner. The cyclobutane pyrimidine dimer class III photolyases are structurally unknown but closely related to plant cryptochromes, which serve as blue-light photoreceptors. Here we present the crystal structure of a class III photolyase termed photolyase-related protein A (PhrA) of Agrobacterium tumefaciens at 1.67-Å resolution. PhrA contains 5,10-methenyltetrahydrofolate (MTHF) as an antenna chromophore with a unique binding site and mode. Two Trp residues play pivotal roles for stabilizing MTHF by a double π-stacking sandwich. Plant cryptochrome I forms a pocket at the same site that could accommodate MTHF or a similar molecule. The PhrA structure and mutant studies showed that electrons flow during FAD photoreduction proceeds via two Trp triads. The structural studies on PhrA give a clearer picture on the evolutionary transition from photolyase to photoreceptor.

Reversible [4Fe-3S] cluster morphing in an O(2)-tolerant [NiFe] hydrogenase.

Hydrogenases catalyze the reversible oxidation of H(2) into protons and electrons and are usually readily inactivated by O(2). However, a subgroup of the [NiFe] hydrogenases, including the membrane-bound [NiFe] hydrogenase from Ralstonia eutropha, has evolved remarkable tolerance toward O(2) that enables their host organisms to utilize H(2) as an energy source at high O(2). This feature is crucially based on a unique six cysteine-coordinated [4Fe-3S] cluster located close to the catalytic center, whose properties were investigated in this study using a multidisciplinary approach. The [4Fe-3S] cluster undergoes redox-dependent reversible transformations, namely iron swapping between a sulfide and a peptide amide N. Moreover, our investigations unraveled the redox-dependent and reversible occurence of an oxygen ligand located at a different iron. This ligand is hydrogen bonded to a conserved histidine that is essential for H(2) oxidation at high O(2). We propose that these transformations, reminiscent of those of the P-cluster of nitrogenase, enable the consecutive transfer of two electrons within a physiological potential range.