Wolf-Dieter Schubert

Structure of Internalin, a Major Invasion Protein of Listeria monocytogenes, in Complex with Its Human Receptor E-Cadherin

Listeria monocytogenes, a food-borne bacterial pathogen, enters mammalian cells by inducing its own phagocytosis. The listerial protein internalin (InlA) mediates bacterial adhesion and invasion of epithelial cells in the human intestine through specific interaction with its host cell receptor E-cadherin. We present the crystal structures of the functional domain of InlA alone and in a complex with the extracellular, N-terminal domain of human E-cadherin (hEC1). The leucine rich repeat (LRR) domain of InlA surrounds and specifically recognizes hEC1. Individual interactions were probed by mutagenesis and analytical ultracentrifugation. These include Pro16 of hEC1, a major determinant for human susceptibility to L. monocytogenes infection that is essential for intermolecular recognition. Our studies reveal the structural basis for host tro-pism of this bacterium and the molecular deception L. monocytogenes employs to exploit the E-cadherin system.

Crystal structure of coproporphyrinogen III oxidase reveals cofactor geometry of Radical SAM enzymes.

‘Radical SAM’ enzymes generate catalytic radicals by combining a 4Fe–4S cluster and S‐adenosylmethionine (SAM) in close proximity. We present the first crystal structure of a Radical SAM enzyme, that of HemN, the Escherichia coli oxygen‐independent coproporphyrinogen III oxidase, at 2.07 Å resolution. HemN catalyzes the essential conversion of coproporphyrinogen III to protoporphyrinogen IX during heme biosynthesis. HemN binds a 4Fe–4S cluster through three cysteine residues conserved in all Radical SAM enzymes. A juxtaposed SAM coordinates the fourth Fe ion through its amide nitrogen and carboxylate oxygen. The SAM sulfonium sulfur is near both the Fe (3.5 Å) and a neighboring sulfur of the cluster (3.6 Å), allowing single electron transfer from the 4Fe–4S cluster to the SAM sulfonium. SAM is cleaved yielding a highly oxidizing 5′‐deoxyadenosyl radical. HemN, strikingly, binds a second SAM immediately adjacent to the first. It may thus successively catalyze two propionate decarboxylations. The structure of HemN reveals the cofactor geometry required for Radical SAM catalysis and sets the stage for the development of inhibitors with antibacterial function due to the uniquely bacterial occurrence of the enzyme.

Extending the Host Range of Listeria monocytogenes by Rational Protein Design

In causing disease, pathogens outmaneuver host defenses through a dedicated arsenal of virulence determinants that specifically bind or modify individual host molecules. This dedication limits the intruder to a defined range of hosts. Newly emerging diseases mostly involve existing pathogens whose arsenal has been altered to allow them to infect previously inaccessible hosts. We have emulated this chance occurrence by extending the host range accessible to the human pathogen Listeria monocytogenes by the intestinal route to include the mouse. Analyzing the recognition complex of the listerial invasion protein InlA and its human receptor E-cadherin, we postulated and verified amino acid substitutions in InlA to increase its affinity for E-cadherin. Two single substitutions increase binding affinity by four orders of magnitude and extend binding specificity to include formerly incompatible murine E-cadherin. By rationally adapting a single protein, we thus create a versatile murine model of human listeriosis.

Crystal structure of 5‐aminolevulinate synthase, the first enzyme of heme biosynthesis, and its link to XLSA in humans

5‐Aminolevulinate synthase (ALAS) is the first and rate‐limiting enzyme of heme biosynthesis in humans, animals, other non‐plant eukaryotes, and α‐proteobacteria. It catalyzes the synthesis of 5‐aminolevulinic acid, the first common precursor of all tetrapyrroles, from glycine and succinyl‐coenzyme A (sCoA) in a pyridoxal 5′‐phosphate (PLP)‐dependent manner. X‐linked sideroblastic anemias (XLSAs), a group of severe disorders in humans characterized by inadequate formation of heme in erythroblast mitochondria, are caused by mutations in the gene for erythroid eALAS, one of two human genes for ALAS. We present the first crystal structure of homodimeric ALAS from Rhodobacter capsulatus (ALASRc) binding its cofactor PLP. We, furthermore, present structures of ALASRc in complex with the substrates glycine or sCoA. The sequence identity of ALAS from R. capsulatus and human eALAS is 49%. XLSA‐causing mutations may thus be mapped, revealing the molecular basis of XLSA in humans. Mutations are found to obstruct substrate binding, disrupt the dimer interface, or hamper the correct folding. The structure of ALAS completes the structural analysis of enzymes in heme biosynthesis.

V-Shaped Structure of Glutamyl-tRNA Reductase, the First Enzyme of tRNA-Dependent Tetrapyrrole Biosynthesis.

Processes vital to life such as respiration and photosynthesis critically depend on the availability of tetrapyrroles including hemes and chlorophylls. tRNA‐dependent catalysis generally is associated with protein biosynthesis. An exception is the reduction of glutamyl‐tRNA to glutamate‐1‐semialdehyde by the enzyme glutamyl‐tRNA reductase. This reaction is the indispensable initiating step of tetrapyrrole biosynthesis in plants and most prokaryotes. The crystal structure of glutamyl‐tRNA reductase from the archaeon Methanopyrus kandleri in complex with the substrate‐like inhibitor glutamycin at 1.9 Å resolution reveals an extended yet planar V‐shaped dimer. The well defined interactions of the inhibitor with the active site support a thioester‐mediated reduction process. Modeling the glutamyl‐tRNA onto each monomer reveals an extensive protein–tRNA interface. We furthermore propose a model whereby the large void of glutamyl‐tRNA reductase is occupied by glutamate‐1‐semialdehyde‐1,2‐mutase, the subsequent enzyme of this pathway, allowing for the efficient synthesis of 5‐aminolevulinic acid, the common precursor of all tetrapyrroles.

The crystal structure of SdsA1, an alkylsulfatase from Pseudomonas aeruginosa, defines a third class of sulfatases

Pseudomonas aeruginosa is both a ubiquitous environmental bacterium and an opportunistic human pathogen. A remarkable metabolic versatility allows it to occupy a multitude of ecological niches, including wastewater treatment plants and such hostile environments as the human respiratory tract. P. aeruginosa is able to degrade and metabolize biocidic SDS, the detergent of most commercial personal hygiene products. We identify SdsA1 of P. aeruginosa as a secreted SDS hydrolase that allows the bacterium to use primary sulfates such as SDS as a sole carbon or sulfur source. Homologues of SdsA1 are found in many pathogenic and some nonpathogenic bacteria. The crystal structure of SdsA1 reveals three distinct domains. The N-terminal catalytic domain with a binuclear Zn2+ cluster is a distinct member of the metallo-β-lactamase fold family, the central dimerization domain ensures resistance to high concentrations of SDS, whereas the C-terminal domain provides a hydrophobic groove, presumably to recruit long aliphatic substrates. Crystal structures of apo-SdsA1 and complexes with substrate analog and products indicate an enzymatic mechanism involving a water molecule indirectly activated by the Zn2+ cluster. The enzyme SdsA1 thus represents a previously undescribed class of sulfatases that allows P. aeruginosa to survive and thrive under otherwise bacteriocidal conditions.

Structure of Photosystem I at 4.5 A resolution: a short review including evolutionary aspects

In photosynthesis of higher plants and cyanobacteria two photosystems are responsible for light induced charge separation. Photosystem II catalyses the electron transfer from water at the lumenal side to quinone at the stromal side, during this process oxygen is evolved. Photosystem I catalyses the electron transport from the soluble electron carrier plastocyanin or cytochrome c 6 at the lumenal side to ferredoxin at the stromal side of the membrane. Photosystem II shows homology to the reaction centre of purple bacteria in sequence as well as in content of electron carriers, yet purple bacteria are not able to use water as electron donor. Photosystem I has nearly no sequence homology to the reaction centre of purple bacteria, there are furthermore differences in the content of electron carriers and in the fact that Photosystem I carries its own antenna system of 90 chlorophyll a molecules. Green sulfur bacteria and heliobacteria are related to Photosystem I (for review on evolutionary relationship see Ref. [1 ]). The major part of Photosystem I is constituted by the two large subunits PsaA and PsaB ( = 83 kDa, each), carrying most of the electron transport chain: P700 (a Chl a dimer), A0 (a monomer of Chl a ), AI (phylloquinone/Vit K l ) and the first of the three [4Fe-4S] clusters F x. In addition, the 90 Chl a molecules of the antenna are bound by these subunits. Three subunits are

The mutation G145S in PrfA, a key virulence regulator of Listeria monocytogenes, increases DNA‐binding affinity by stabilizing the HTH motif

Listeria monocytogenes, a Gram‐positive, facultative intracellular human pathogen, causes systemic infections with high mortality rate. The majority of the known pathogenicity factors of L. monocytogenes is regulated by a single transcription factor, PrfA. Hyperhaemolytic laboratory strains of L. monocytogenes express the constitutively active mutant PrfAG145S inducing virulence gene overexpression independent of environmental conditions. PrfA belongs to the Crp/Fnr family of transcription factors generally activated by a small effector, such as cAMP or O2. We present the crystal structures of wild‐type PrfA, the first Gram‐positive member of the Crp/Fnr family, and of the constitutively active mutant PrfAG145S. Cap (Crp) has previously been described exclusively in the cAMP‐induced (DNA‐free and ‐bound) conformation. By contrast, the PrfA structures present views both of the non‐induced state and of the mutationally activated form. The low DNA‐binding affinity of wild‐type PrfA is supported both structurally (partly disordered helix–turn–helix motif, overall geometry of the HTH α‐helices deviates from Cap) and by surface plasmon resonance analyses (KD = 0.9 µM). In PrfAG145S the HTH motifs dramatically rearrange to adopt a conformation comparable to cAMP‐induced Cap and hence favourable for DNA binding, supported by a DNA‐binding affinity of 50 nM. Finally, the hypothesis that wild‐type PrfA, like other Crp/Fnr family members, may require an as yet unidentified cofactor for activation is supported by the presence of a distinct tunnel in PrfA, located at the interface of the β‐barrel and the DNA‐binding domain.

The Phosphotyrosine Peptide Binding Specificity of Nck1 and Nck2 Src Homology 2 Domains

Nck proteins are essential Src homology (SH) 2 and SH3 domain-bearing adapters that modulate actin cytoskeleton dynamics by linking proline-rich effector molecules to tyrosine kinases or phosphorylated signaling intermediates. Two mammalian pathogens, enteropathogenic Escherichia coli and vaccinia virus, exploit Nck as part of their infection strategy. Conflicting data indicate potential differences in the recognition specificities of the SH2 domains of the isoproteins Nck1 (Nckα) and Nck2 (Nckβ and Grb4). We have characterized the binding specificities of both SH2 domains and find them to be essentially indistinguishable. Crystal structures of both domains in complex with phosphopeptides derived from the enteropathogenic E. coli protein Tir concur in identifying highly conserved, specific recognition of the phosphopeptide. Differential peptide recognition can therefore not account for the preference of either Nck in particular signaling pathways. Binding studies using sequentially mutated, high affinity phosphopeptides establish the sequence variability tolerated in peptide recognition. Based on this binding motif, we identify potential new binding partners of Nck1 and Nck2 and confirm this experimentally for the Arf-GAP GIT1.

Crystal Structure of the Nitrogenase-like Dark Operative Protochlorophyllide Oxidoreductase Catalytic Complex (ChlN/ChlB)2

During (bacterio)chlorophyll biosynthesis of many photosynthetically active organisms, dark operative protochlorophyllide oxidoreductase (DPOR) catalyzes the two-electron reduction of ring D of protochlorophyllide to form chlorophyllide. DPOR is composed of the subunits ChlL, ChlN, and ChlB. Homodimeric ChlL2 bearing an intersubunit [4Fe-4S] cluster is an ATP-dependent reductase transferring single electrons to the heterotetrameric (ChlN/ChlB)2 complex. The latter contains two intersubunit [4Fe-4S] clusters and two protochlorophyllide binding sites, respectively. Here we present the crystal structure of the catalytic (ChlN/ChlB)2 complex of DPOR from the cyanobacterium Thermosynechococcus elongatus at a resolution of 2.4 Å. Subunits ChlN and ChlB exhibit a related architecture of three subdomains each built around a central, parallel β-sheet surrounded by α-helices. The (ChlN/ChlB)2 crystal structure reveals a [4Fe-4S] cluster coordinated by an aspartate oxygen alongside three cysteine ligands. Two equivalent substrate binding sites enriched in aromatic residues for protochlorophyllide substrate binding are located at the interface of each ChlN/ChlB half-tetramer. The complete octameric (ChlN/ChlB)2(ChlL2)2 complex of DPOR was modeled based on the crystal structure and earlier functional studies. The electron transfer pathway via the various redox centers of DPOR to the substrate is proposed.