Joern Krausze

Structure of ADP-aluminium fluoride-stabilized protochlorophyllide oxidoreductase complex

Photosynthesis uses chlorophylls for the conversion of light into chemical energy, the driving force of life on Earth. During chlorophyll biosynthesis in photosynthetic bacteria, cyanobacteria, green algae and gymnosperms, dark-operative protochlorophyllide oxidoreductase (DPOR), a nitrogenase-like metalloenzyme, catalyzes the chemically challenging two-electron reduction of the fully conjugated ring system of protochlorophyllide a. The reduction of the C-17=C-18 double bond results in the characteristic ring architecture of all chlorophylls, thereby altering the absorption properties of the molecule and providing the basis for light-capturing and energy-transduction processes of photosynthesis. We report the X-ray crystallographic structure of the substrate-bound, ADP-aluminium fluoride–stabilized (ADP·AlF3-stabilized) transition state complex between the DPOR components L2 and (NB)2 from the marine cyanobacterium Prochlorococcus marinus. Our analysis permits a thorough investigation of the dynamic interplay between L2 and (NB)2. Upon complex formation, substantial ATP-dependent conformational rearrangements of L2 trigger the protein–protein interactions with (NB)2 as well as the electron transduction via redox-active [4Fe–4S] clusters. We also present the identification of artificial “small-molecule substrates” of DPOR in correlation with those of nitrogenase. The catalytic differences and similarities between DPOR and nitrogenase have broad implications for the energy transduction mechanism of related multiprotein complexes that are involved in the reduction of chemically stable double and/or triple bonds.

The 3D Structure of Kaposi Sarcoma Herpesvirus Lana C-Terminal Domain Bound to DNA.

Significance KSHV is the etiological agent of Kaposi sarcoma, primary effusion lymphoma, and the plasma cell variant of multicentric Castleman disease. During latency, this dsDNA tumor virus expresses only a small subset of its more than 90 ORFs. Among these is ORF73/latency-associated nuclear antigen (LANA), which acts as the origin binding protein and chromatin anchor of the extrachromosomal viral genome. This work provides detailed structural insights into the DNA-binding characteristics of LANA. We also report a previously unrecognized, third LANA binding site within the minimal replicator of Kaposi sarcoma herpesvirus. In addition to its mechanistic implications for latent viral persistence, the X-ray crystal structure of LANA bound to LANA binding site 1 DNA may assist in the tailored development of therapeutic LANA inhibitors. Kaposi sarcoma herpesvirus (KSHV) persists as a latent nuclear episome in dividing host cells. This episome is tethered to host chromatin to ensure proper segregation during mitosis. For duplication of the latent genome, the cellular replication machinery is recruited. Both of these functions rely on the constitutively expressed latency-associated nuclear antigen (LANA) of the virus. Here, we report the crystal structure of the KSHV LANA DNA-binding domain (DBD) in complex with its high-affinity viral target DNA, LANA binding site 1 (LBS1), at 2.9 Å resolution. In contrast to homologous proteins such as Epstein-Barr virus nuclear antigen 1 (EBNA-1) of the related γ-herpesvirus Epstein-Barr virus, specific DNA recognition by LANA is highly asymmetric. In addition to solving the crystal structure, we found that apart from the two known LANA binding sites, LBS1 and LBS2, LANA also binds to a novel site, denoted LBS3. All three sites are located in a region of the KSHV terminal repeat subunit previously recognized as a minimal replicator. Moreover, we show that the LANA DBD can coat DNA of arbitrary sequence by virtue of a characteristic lysine patch, which is absent in EBNA-1 of the Epstein-Barr virus. Likely, these higher-order assemblies involve the self-association of LANA into supermolecular spirals. One such spiral assembly was solved as a crystal structure of 3.7 Å resolution in the absence of DNA. On the basis of our data, we propose a model for the controlled nucleation of higher-order LANA oligomers that might contribute to the characteristic subnuclear KSHV microdomains (“LANA speckles”), a hallmark of KSHV latency.

Crystal Structure of the Heme d1 Biosynthesis Enzyme NirE in Complex with Its Substrate Reveals New Insights into the Catalytic Mechanism of S-Adenosyl-l-methionine-dependent Uroporphyrinogen III Methyltransferases

During the biosynthesis of heme d1, the essential cofactor of cytochrome cd1 nitrite reductase, the NirE protein catalyzes the methylation of uroporphyrinogen III to precorrin-2 using S-adenosyl-l-methionine (SAM) as the methyl group donor. The crystal structure of Pseudomonas aeruginosa NirE in complex with its substrate uroporphyrinogen III and the reaction by-product S-adenosyl-l-homocysteine (SAH) was solved to 2.0 Å resolution. This represents the first enzyme-substrate complex structure for a SAM-dependent uroporphyrinogen III methyltransferase. The large substrate binds on top of the SAH in a “puckered” conformation in which the two pyrrole rings facing each other point into the same direction either upward or downward. Three arginine residues, a histidine, and a methionine are involved in the coordination of uroporphyrinogen III. Through site-directed mutagenesis of the nirE gene and biochemical characterization of the corresponding NirE variants the amino acid residues Arg-111, Glu-114, and Arg-149 were identified to be involved in NirE catalysis. Based on our structural and biochemical findings, we propose a potential catalytic mechanism for NirE in which the methyl transfer reaction is initiated by an arginine catalyzed proton abstraction from the C-20 position of the substrate.

Glycoprotein production for structure analysis with stable, glycosylation mutant CHO cell lines established by fluorescence-activated cell sorting

Stable mammalian cell lines are excellent tools for the expression of secreted and membrane glycoproteins. However, structural analysis of these molecules is generally hampered by the complexity of N‐linked carbohydrate side chains. Cell lines with mutations are available that result in shorter and more homogenous carbohydrate chains. Here, we use preparative fluorescence‐activated cell sorting (FACS) and site‐specific gene excision to establish high‐yield glycoprotein expression for structural studies with stable clones derived from the well‐established Lec3.2.8.1 glycosylation mutant of the Chinese hamster ovary (CHO) cell line. We exemplify the strategy by describing novel clones expressing single‐chain hepatocyte growth factor/scatter factor (HGF/SF, a secreted glycoprotein) and a domain of lysosome‐associated membrane protein 3 (LAMP3d). In both cases, stable GFP‐expressing cell lines were established by transfection with a genetic construct including a GFP marker and two rounds of cell sorting after 1 and 2 weeks. The GFP marker was subsequently removed by heterologous expression of Flp recombinase. Production of HGF/SF and LAMP3d was stable over several months. 1.2 mg HGF/SF and 0.9 mg LAMP3d were purified per litre of culture, respectively. Homogenous glycoprotein preparations were amenable to enzymatic deglycosylation under native conditions. Purified and deglycosylated LAMP3d protein was readily crystallized. The combination of FACS and gene excision described here constitutes a robust and fast procedure for maximizing the yield of glycoproteins for structural analysis from glycosylation mutant cell lines.

Structures of two bacterial resistance factors mediating tRNA-dependent aminoacylation of phosphatidylglycerol with lysine or alanine

Significance Lipid homeostasis is a fundamental process for understanding antimicrobial susceptibility. Modification of the polar head group of phosphatidylglycerol into the respective aminoacyl-ester of phosphatidylglycerol is a widely used strategy to mediate bacterial resistance. Here we present the structures of the catalytic domains of aminoacyl-phosphatidylglycerol synthases from Pseudomonas aeruginosa and Bacillus licheniformis. These prototypical enzymes specifically catalyze the tRNA-dependent synthesis of alanyl-phosphatidylglycerol and lysyl-phosphatidylglycerol, respectively. A central tunnel architecture facilitates binding of the polar aminoacyl-tRNA molecule opposite the hydrophobic lipid substrate as a fundamental principle for the catalysis at the water–lipid interface. Specific inhibition of aminoacyl-phosphatidylglycerol synthases might be a promising strategy to render Gram-positive and Gram-negative pathogenic bacteria more susceptible to antimicrobial treatment. The cytoplasmic membrane is probably the most important physical barrier between microbes and the surrounding habitat. Aminoacylation of the polar head group of the phospholipid phosphatidylglycerol (PG) catalyzed by Ala-tRNAAla–dependent alanyl-phosphatidylglycerol synthase (A-PGS) or by Lys-tRNALys–dependent lysyl-phosphatidylglycerol synthase (L-PGS) enables bacteria to cope with cationic peptides that are harmful to the integrity of the cell membrane. Accordingly, these synthases also have been designated as multiple peptide resistance factors (MprF). They consist of a separable C-terminal catalytic domain and an N-terminal transmembrane flippase domain. Here we present the X-ray crystallographic structure of the catalytic domain of A-PGS from the opportunistic human pathogen Pseudomonas aeruginosa. In parallel, the structure of the related lysyl-phosphatidylglycerol–specific L-PGS domain from Bacillus licheniformis in complex with the substrate analog L-lysine amide is presented. Both proteins reveal a continuous tunnel that allows the hydrophobic lipid substrate PG and the polar aminoacyl-tRNA substrate to access the catalytic site from opposite directions. Substrate recognition of A-PGS versus L-PGS was investigated using misacylated tRNA variants. The structural work presented here in combination with biochemical experiments using artificial tRNA or artificial lipid substrates reveals the tRNA acceptor stem, the aminoacyl moiety, and the polar head group of PG as the main determinants for substrate recognition. A mutagenesis approach yielded the complementary amino acid determinants of tRNA interaction. These results have broad implications for the design of L-PGS and A-PGS inhibitors that could render microbial pathogens more susceptible to antimicrobial compounds.

Oligomerization Inhibits Legionella pneumophila PlaB Phospholipase A Activity

Background: PlaB is the most prominent phospholipase A of Legionella pneumophila. Results: PlaB possesses high activity at nanomolar but low activity at micromolar concentrations where it forms tetramers. Conclusion: PlaB tetramerization inhibits and oligomer dissociation activates phospholipase activity. Significance: Our data highlight the first example of concentration-dependent phospholipase inactivation by tetramerization, which may be a mechanism for self-protection. The intracellularly replicating lung pathogen Legionella pneumophila consists of an extraordinary variety of phospholipases, including at least 15 different phospholipases A (PLA). Among them, PlaB, the first characterized member of a novel lipase family, is a hemolytic virulence factor that exhibits the most prominent PLA activity in L. pneumophila. We analyzed here protein oligomerization, the importance of oligomerization for activity, addressed further essential regions for activity within the PlaB C terminus, and the significance of PlaB-derived lipolytic activity for L. pneumophila intracellular replication. We determined by means of analytical ultracentrifugation and small angle x-ray scattering analysis that PlaB forms homodimers and homotetramers. The C-terminal 5, 10, or 15 amino acids, although the individual regions contributed to PLA activity, were not essential for protein tetramerization. Infection of mouse macrophages with L. pneumophila wild type, plaB knock-out mutant, and plaB complementing or various mutated plaB-harboring strains showed that catalytic activity of PlaB promotes intracellular replication. We observed that PlaB was most active in the lower nanomolar concentration range but not at or only at a low level at concentration above 0.1 μm where it exists in a dimer/tetramer equilibrium. We therefore conclude that PlaB is a virulence factor that, on the one hand, assembles in inactive tetramers at micromolar concentrations. On the other hand, oligomer dissociation at nanomolar concentrations activates PLA activity. Our data highlight the first example of concentration-dependent phospholipase inactivation by tetramerization, which may protect the bacterium from internal PLA activity, but enzyme dissociation may allow its activation after export.

Biochemical Characterization of Molybdenum Cofactor-free Nitrate Reductase from Neurospora crassa

Background: Eukaryotic nitrate reductase maturation is poorly understood. Results: Binding of molybdenum cofactor to apo-nitrate reductase is independent from other prosthetic groups. Conclusion: Active site formation of eukaryotic nitrate reductase is an autonomous process intrinsically tied to nitrate reductase dimerization. Significance: The understanding of molybdenum cofactor-dependent enzyme maturation is of significance as molybdenum enzymes are involved in essential cellular processes. Nitrate reductase (NR) is a complex molybdenum cofactor (Moco)-dependent homodimeric metalloenzyme that is vitally important for autotrophic organism as it catalyzes the first and rate-limiting step of nitrate assimilation. Beside Moco, eukaryotic NR also binds FAD and heme as additional redox active cofactors, and these are involved in electron transfer from NAD(P)H to the enzyme molybdenum center where reduction of nitrate to nitrite takes place. We report the first biochemical characterization of a Moco-free eukaryotic NR from the fungus Neurospora crassa, documenting that Moco is necessary and sufficient to induce dimer formation. The molybdenum center of NR reconstituted in vitro from apo-NR and Moco showed an EPR spectrum identical to holo-NR. Analysis of mutants unable to bind heme or FAD revealed that insertion of Moco into NR occurs independent from the insertion of any other NR redox cofactor. Furthermore, we showed that at least in vitro the active site formation of NR is an autonomous process.

Dimerization of the plant molybdenum insertase Cnx1E is required for synthesis of the molybdenum cofactor

The molybdenum cofactor (Moco) is a redox active prosthetic group, essentially required for numerous enzyme-catalyzed two electron transfer reactions. Moco is synthesized by an evolutionarily old and highly conserved multistep pathway. In the last step of Moco biosynthesis, the molybdenum center is inserted into the final Moco precursor adenylated molybdopterin (MPT-AMP). This unique and yet poorly characterized maturation reaction finally yields physiologically active Moco. In the model plant Arabidopsis, the two domain enzyme, Cnx1, is required for Moco formation. Recently, a genetic screen identified novel Arabidopsis cnx1 mutant plant lines each harboring a single amino acid exchange in the N-terminal Cnx1E domain. Biochemical characterization of the respective recombinant Cnx1E variants revealed two different amino acid exchanges (S197F and G175D) that impair Cnx1E dimerization, thus linking Cnx1E oligomerization to Cnx1 functionality. Analysis of the Cnx1E structure identified Cnx1E active site-bound molybdate and magnesium ions, which allowed to fine-map the Cnx1E MPT-AMP-binding site.

Biosynthesis of Violacein: Structure and Function of L-Tryptophan Oxidase VioA from Chromobacterium violaceum

Violacein is a natural purple pigment of Chromobacterium violaceum with potential medical applications as antimicrobial, antiviral, and anticancer drugs. The initial step of violacein biosynthesis is the oxidative conversion of l-tryptophan into the corresponding α-imine catalyzed by the flavoenzyme l-tryptophan oxidase (VioA). A substrate-related (3-(1H-indol-3-yl)-2-methylpropanoic acid) and a product-related (2-(1H-indol-3-ylmethyl)prop-2-enoic acid) competitive VioA inhibitor was synthesized for subsequent kinetic and x-ray crystallographic investigations. Structures of the binary VioA·FADH2 and of the ternary VioA·FADH2·2-(1H-indol-3-ylmethyl)prop-2-enoic acid complex were resolved. VioA forms a “loosely associated” homodimer as indicated by small-angle x-ray scattering experiments. VioA belongs to the glutathione reductase family 2 of FAD-dependent oxidoreductases according to the structurally conserved cofactor binding domain. The substrate-binding domain of VioA is mainly responsible for the specific recognition of l-tryptophan. Other canonical amino acids were efficiently discriminated with a minor conversion of l-phenylalanine. Furthermore, 7-aza-tryptophan, 1-methyl-tryptophan, 5-methyl-tryptophan, and 5-fluoro-tryptophan were efficient substrates of VioA. The ternary product-related VioA structure indicated involvement of protein domain movement during enzyme catalysis. Extensive structure-based mutagenesis in combination with enzyme kinetics (using l-tryptophan and substrate analogs) identified Arg64, Lys269, and Tyr309 as key catalytic residues of VioA. An increased enzyme activity of protein variant H163A in the presence of l-phenylalanine indicated a functional role of His163 in substrate binding. The combined structural and mutational analyses lead to the detailed understanding of VioA substrate recognition. Related strategies for the in vivo synthesis of novel violacein derivatives are discussed.

Crystallization, room-temperature X-ray diffraction and preliminary analysis of Kaposi's sarcoma herpesvirus LANA bound to DNA

The latency-associated nuclear antigen (LANA) is the latent origin-binding protein and chromatin anchor of the Kaposis sarcoma herpesvirus (KSHV/HHV-8) genome. Its C-terminal domain (CTD) binds sequence-specifically to the viral origin of replication, whereas the N-terminal domain links it to nucleosomes of cellular chromatin for long-term persistence in dividing host cells. Here, the crystallization and X-ray data acquisition of a mutant LANA CTD in complex with its wild-type target DNA LBS1 is described. This report describes the rational protein engineering for successful co-crystallization with DNA and X-ray diffraction data collection at room temperature on the high-brilliance third-generation synchrotron PETRA III at DESY, Germany.