Jurgen Sygusch

Aldolase is essential for energy production and bridging adhesin-actin cytoskeletal interactions during parasite invasion of host cells.

Apicomplexan parasites rely on actin-based motility to drive host cell invasion. Prior in vitro studies implicated aldolase, a tetrameric glycolytic enzyme, in coupling actin filaments to the parasites surface adhesin microneme protein 2 (MIC2). Here, we test the essentiality of this interaction in host cell invasion. Based on in vitro studies and homology modeling, we generated a series of mutations in Toxoplasma gondii aldolase (TgALD1) that delineated MIC2 tail domain (MIC2t) binding function from its enzyme activity. We tested these mutants by complementing a conditional knockout of TgALD1. Mutations that affected glycolysis also reduced motility. Mutants only affecting binding to MIC2t had no motility phenotype, but were decreased in their efficiency of host cell invasion. Our studies demonstrate that aldolase is not only required for energy production but is also essential for efficient host cell invasion, based on its ability to bridge adhesin-cytoskeleton interactions in the parasite.

Crystal Structures of DNA-Whirly Complexes and Their Role in Arabidopsis Organelle Genome Repair

This work examines the repair of DNA double-strand breaks in the mitochondria and plastids of Arabidopsis. The crystal structures of a Whirly protein bound to single-stranded DNA suggest a role for these proteins in DNA damage tolerance in the organelles. DNA double-strand breaks are highly detrimental to all organisms and need to be quickly and accurately repaired. Although several proteins are known to maintain plastid and mitochondrial genome stability in plants, little is known about the mechanisms of DNA repair in these organelles and the roles of specific proteins. Here, using ciprofloxacin as a DNA damaging agent specific to the organelles, we show that plastids and mitochondria can repair DNA double-strand breaks through an error-prone pathway similar to the microhomology-mediated break-induced replication observed in humans, yeast, and bacteria. This pathway is negatively regulated by the single-stranded DNA (ssDNA) binding proteins from the Whirly family, thus indicating that these proteins could contribute to the accurate repair of plant organelle genomes. To understand the role of Whirly proteins in this process, we solved the crystal structures of several Whirly-DNA complexes. These reveal a nonsequence-specific ssDNA binding mechanism in which DNA is stabilized between domains of adjacent subunits and rendered unavailable for duplex formation and/or protein interactions. Our results suggest a model in which the binding of Whirly proteins to ssDNA would favor accurate repair of DNA double-strand breaks over an error-prone microhomology-mediated break-induced replication repair pathway.

A new family of plant transcription factors displays a novel ssDNA-binding surface

The crystal structure of p24, the single-stranded DNA (ssDNA) binding subunit of the plant defense transcription factor PBF-2, has been determined to 2.3 Å resolution. p24 is representative of a novel family of ubiquitous plant-specific proteins that we refer to as the Whirly family because of their quaternary structure. PBF-2 is composed of four p24 molecules that interact through a helix-loop-helix motif. This interaction produces a central pore, with β-strands radiating outwards, resulting in a whirligig appearance to the quaternary structure. The noncrystallographic C4 symmetry arrangement of p24 subunits is novel for ssDNA binding proteins and may explain the binding specificity of PBF-2. This structural arrangement also supports the role of PBF-2 in binding melted promoter regions to modulate gene expression.

Glycolytic and Non-glycolytic Functions of Mycobacterium tuberculosis Fructose-1,6-bisphosphate Aldolase, an Essential Enzyme Produced by Replicating and Non-replicating Bacilli

Background: New drugs active against persistent Mycobacterium tuberculosis are needed. Results: The fructose-1,6-bisphosphate aldolase (FBA-tb) is essential for growth of M. tuberculosis, is expressed by replicating and non-replicating bacilli, and displays plasminogen binding activity. Conclusion: FBA-tb is an essential TB enzyme that might also play a role in host/pathogen interactions. Significance: FBA-tb shows potential as a novel anti-TB therapeutic target. The search for antituberculosis drugs active against persistent bacilli has led to our interest in metallodependent class II fructose-1,6-bisphosphate aldolase (FBA-tb), a key enzyme of gluconeogenesis absent from mammalian cells. Knock-out experiments at the fba-tb locus indicated that this gene is required for the growth of Mycobacterium tuberculosis on gluconeogenetic substrates and in glucose-containing medium. Surface labeling and enzymatic activity measurements revealed that this enzyme was exported to the cell surface of M. tuberculosis and produced under various axenic growth conditions including oxygen depletion and hence by non-replicating bacilli. Importantly, FBA-tb was also produced in vivo in the lungs of infected guinea pigs and mice. FBA-tb bound human plasmin(ogen) and protected FBA-tb-bound plasmin from regulation by α2-antiplasmin, suggestive of an involvement of this enzyme in host/pathogen interactions. The crystal structures of FBA-tb in the native form and in complex with a hydroxamate substrate analog were determined to 2.35- and 1.9-Å resolution, respectively. Whereas inhibitor attachment had no effect on the plasminogen binding activity of FBA-tb, it competed with the natural substrate of the enzyme, fructose 1,6-bisphosphate, and substantiated a previously unknown reaction mechanism associated with metallodependent aldolases involving recruitment of the catalytic zinc ion by the substrate upon active site binding. Altogether, our results highlight the potential of FBA-tb as a novel therapeutic target against both replicating and non-replicating bacilli.

Covalent intermediate trapped in 2-keto-3-deoxy-6- phosphogluconate (KDPG) aldolase structure at 1.95-Å resolution

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase catalyzes the reversible cleavage of KDPG to pyruvate and glyceraldehyde-3-phosphate. The enzyme is a class I aldolase whose reaction mechanism involves formation of Schiff base intermediates between Lys-133 and a keto substrate. A covalent adduct was trapped by flash freezing KDPG aldolase crystals soaked with 10 mM pyruvate in acidic conditions at pH 4.6. Structure determination to 1.95-Å resolution showed that pyruvate had undergone nucleophilic attack with Lys-133, forming a protonated carbinolamine intermediate, a functional Schiff base precursor, which was stabilized by hydrogen bonding with active site residues. Carbinolamine interaction with Glu-45 indicates general base catalysis of several rate steps. Stereospecific addition is ensured by aromatic interaction of Phe-135 with the pyruvate methyl group. In the native structure, Lys-133 donates all of its hydrogen bonds, indicating the presence of an ɛ-ammonium salt group. Nucleophilic activation is postulated to occur by proton transfer in the monoprotonated zwitterionic pair (Glu-45/Lys-133). Formation of the zwitterionic pair requires prior side chain rearrangement by protonated Lys-133 to displace a water molecule, hydrogen bonded to the zwitterionic residues.

Identification of the Binding Site of Brucella VirB8 Interaction Inhibitors

Secretion systems translocate virulence factors of many bacterial pathogens, enabling their survival inside the host organism. Consequently, inhibition strongly attenuates pathogenicity and can be considered a target for novel antimicrobial drugs. The type IV secretion system (T4SS) of the intracellular pathogen Brucella is a prerequisite for its virulence, and in this work we targeted the interactions of the essential assembly factor protein, VirB8, using small-molecule inhibitors. High-throughput screening identified several potent and specific inhibitors, and the target-binding site of these inhibitors was identified by X-ray crystallography, in silico docking, and analysis of the derivates of the inhibitor B8I-2. VirB8 interaction inhibitors bind to a surface groove opposite to the dimerization interface, and by varying the binding-site residues, we were able to determine which residues are required for inhibitor activity. E115 and K182 were found to be especially important, and changes at R114, Y229, and L151 also reduced inhibitor efficiency.

Crystal Structures of the Organomercurial Lyase MerB in Its Free and Mercury-bound Forms INSIGHTS INTO THE MECHANISM OF METHYLMERCURY DEGRADATION

Bacteria resistant to methylmercury utilize two enzymes (MerA and MerB) to degrade methylmercury to the less toxic elemental mercury. The crucial step is the cleavage of the carbon-mercury bond of methylmercury by the organomercurial lyase (MerB). In this study, we determined high resolution crystal structures of MerB in both the free (1.76-Å resolution) and mercury-bound (1.64-Å resolution) states. The crystal structure of free MerB is very similar to the NMR structure, but important differences are observed when comparing the two structures. In the crystal structure, an amino-terminal α-helix that is not present in the NMR structure makes contact with the core region adjacent to the catalytic site. This interaction between the amino-terminal helix and the core serves to bury the active site of MerB. The crystal structures also provide detailed insights into the mechanism of carbon-mercury bond cleavage by MerB. The structures demonstrate that two conserved cysteines (Cys-96 and Cys-159) play a role in substrate binding, carbon-mercury bond cleavage, and controlled product (ionic mercury) release. In addition, the structures establish that an aspartic acid (Asp-99) in the active site plays a crucial role in the proton transfer step required for the cleavage of the carbon-mercury bond. These findings are an important step in understanding the mechanism of carbon-mercury bond cleavage by MerB.

A conserved lysine residue of plant Whirly proteins is necessary for higher order protein assembly and protection against DNA damage

All organisms have evolved specialized DNA repair mechanisms in order to protect their genome against detrimental lesions such as DNA double-strand breaks. In plant organelles, these damages are repaired either through recombination or through a microhomology-mediated break-induced replication pathway. Whirly proteins are modulators of this second pathway in both chloroplasts and mitochondria. In this precise pathway, tetrameric Whirly proteins are believed to bind single-stranded DNA and prevent spurious annealing of resected DNA molecules with other regions in the genome. In this study, we add a new layer of complexity to this model by showing through atomic force microscopy that tetramers of the potato Whirly protein WHY2 further assemble into hexamers of tetramers, or 24-mers, upon binding long DNA molecules. This process depends on tetramer–tetramer interactions mediated by K67, a highly conserved residue among plant Whirly proteins. Mutation of this residue abolishes the formation of 24-mers without affecting the protein structure or the binding to short DNA molecules. Importantly, we show that an Arabidopsis Whirly protein mutated for this lysine is unable to rescue the sensitivity of a Whirly-less mutant plant to a DNA double-strand break inducing agent.

Mechanism of Aldolase Control of Sorting Nexin 9 Function in Endocytosis

Sorting nexin 9 (SNX9) functions in a complex with the GTPase dynamin-2 at clathrin-coated pits, where it provokes fission of vesicles to complete endocytosis. Here the SNX9·dynamin-2 complex binds to clathrin and adapter protein complex 2 (AP-2) that line these pits, and this occurs through interactions of the low complexity domain (LC4) of SNX9 with AP-2. Intriguingly, localization of the SNX9·dynamin-2 complex to clathrin-coated pits is blocked by interactions with the abundant glycolytic enzyme aldolase, which also binds to the LC4 domain of SNX9. The crystal structure of the LC4 motif of human SNX9 in complex with aldolase explains the biochemistry and biology of this interaction, where SNX9 binds near the active site of aldolase via residues 165–171 that are also required for the interactions of SNX9 with AP-2. Accordingly, SNX9 binding to aldolase is structurally precluded by the binding of substrate to the active site. Interactions of SNX9 with aldolase are far more extensive and differ from those of the actin-nucleating factor WASP with aldolase, indicating considerable plasticity in mechanisms that direct the functions of the aldolase as a scaffold protein.

Stereospecific proton transfer by a mobile catalyst in mammalian fructose-1,6-bisphosphate aldolase

Class I fructose-1,6-bisphosphate aldolases catalyze the interconversion between the enamine and iminium covalent enzymatic intermediates by stereospecific exchange of the pro(S) proton of the dihydroxyacetone-phosphate C3 carbon, an obligatory reaction step during substrate cleavage. To investigate the mechanism of stereospecific proton exchange, high resolution crystal structures of native and a mutant Lys146 → Met aldolase were solved in complex with dihydroxyacetone phosphate. The structural analysis revealed trapping of the enamine intermediate at Lys229 in native aldolase. Mutation of conserved active site residue Lys146 to Met drastically decreased activity and enabled trapping of the putative iminium intermediate in the crystal structure showing active site attachment by C-terminal residues 360-363. Attachment positions the conserved C-terminal Tyr363 hydroxyl within 2.9Å of the C3 carbon in the iminium in an orientation consistent with incipient re face proton transfer. We propose a catalytic mechanism by which the mobile C-terminal Tyr363 is activated by the iminium phosphate via a structurally conserved water molecule to yield a transient phenate, whose developing negative charge is stabilized by a Lys146 positive charge, and which abstracts the C3 pro(S) proton forming the enamine. An identical C-terminal binding mode observed in the presence of phosphate in the native structure corroborates Tyr363 interaction with Lys146 and is consistent with transient C terminus binding in the enamine. The absence of charge stabilization and of a mobile C-terminal catalyst explains the extraordinary stability of enamine intermediates in transaldolases.