Martin Zoltner

The Orientation of a Tandem POTRA Domain Pair, of the Beta-Barrel Assembly Protein BamA, Determined by PELDOR Spectroscopy

The outer membrane beta-barrel trans-membrane proteins in gram-negative bacteria are folded into the membrane with the aid of polypeptide transport-associated (POTRA) domains. These domains occur, and probably function, as a tandem array situated on the periplasmic side of the outer membrane. Two crystal structures and one NMR study have attempted to define the structure and articulation of the POTRA domains of the Escherichia coli, prototypic Omp85 protein BamA. We have used pulsed electron paramagnetic resonance (EPR) to determine the distance and distance distribution between (1-Oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate spin labels (MTSSL), placed across the domain interface of the first two POTRA domains of BamA. Our results show tightly defined interdomain distance distributions that indicate a well-defined domain orientation. Examination of the known structures revealed that none of them fitted the EPR data. A combination of EPR and NMR data was used to generate converged structures with defined domain-domain orientation.

Heterogeneity in ess transcriptional organization and variable contribution of the Ess/Type VII protein secretion system to virulence across closely related Staphylocccus aureus strains

The Type VII protein secretion system, found in Gram‐positive bacteria, secretes small proteins, containing a conserved W‐x‐G amino acid sequence motif, to the growth medium. Staphylococcus aureus has a conserved Type VII secretion system, termed Ess, which is dispensable for laboratory growth but required for virulence. In this study we show that there are unexpected differences in the organization of the ess gene cluster between closely related strains of S. aureus. We further show that in laboratory growth medium different strains of S. aureus secrete the EsxA and EsxC substrate proteins at different growth points, and that the Ess system in strain Newman is inactive under these conditions. Systematic deletion analysis in S. aureus RN6390 is consistent with the EsaA, EsaB, EssA, EssB, EssC and EsxA proteins comprising core components of the secretion machinery in this strain. Finally we demonstrate that the Ess secretion machinery of two S. aureus strains, RN6390 and COL, is important for nasal colonization and virulence in the murine lung pneumonia model. Surprisingly, however, the secretion system plays no role in the virulence of strain SA113 under the same conditions.

Characterization of Staphylococcus aureus EssB, an integral membrane component of the Type VII secretion system: atomic resolution crystal structure of the cytoplasmic segment.

The Type VII protein translocation/secretion system, unique to Gram-positive bacteria, is a key virulence determinant in Staphylococcus aureus. We aim to characterize the architecture of this secretion machinery and now describe the present study of S. aureus EssB, a 52 kDa bitopic membrane protein essential for secretion of the ESAT-6 (early secretory antigenic target of 6 kDa) family of proteins, the prototypic substrate of Type VII secretion. Full-length EssB was heterologously expressed in Escherichia coli, solubilized from the bacterial membrane, purified to homogeneity and shown to be dimeric. A C-terminal truncation, EssB∆C, and two soluble fragments termed EssB-N and EssB-C, predicted to occur on either side of the cytoplasmic membrane, have been successfully purified in a recombinant form, characterized and, together with the full-length protein, used in crystallization trials. EssB-N, the 25 kDa N-terminal cytoplasmic fragment, gave well-ordered crystals and we report the structure, determined by SAD (single-wavelength anomalous diffraction) targeting an SeMet (selenomethionine) derivative, refined to atomic (1.05 Å; 1 Å=0.1 nm) resolution. EssB-N is dimeric in solution, but crystallizes as a monomer and displays a fold comprised of two globular domains separated by a cleft. The structure is related to that of serine/threonine protein kinases and the present study identifies that the Type VII secretion system exploits and re-uses a stable modular entity and fold that has evolved to participate in protein–protein interactions in a similar fashion to the catalytically inert pseudokinases.

Membrane interactions and self-association of components of the Ess/Type VII secretion system of Staphylococcus aureus.

The Ess/Type VII protein secretion system, essential for virulence of pathogenic Staphylococcus aureus, is dependent upon the four core membrane proteins EssA, EssB, EssC and EsaA. Here, we use crosslinking and blue native PAGE analysis to show that the EssB, EssC and EsaA proteins individually form homomeric complexes. Surprisingly, these components appear unable to interact with each other, or with the EssA protein. We further show that two high molecular weight multimers of EssC detected in whole cells are not dependent upon the presence of EsxA, EsxB or any other Ess component for their assembly.

Modulation of the Surface Proteome through Multiple Ubiquitylation Pathways in African Trypanosomes.

Recently we identified multiple suramin-sensitivity genes with a genome wide screen in Trypanosoma brucei that includes the invariant surface glycoprotein ISG75, the adaptin-1 (AP-1) complex and two deubiquitylating enzymes (DUBs) orthologous to ScUbp15/HsHAUSP1 and pVHL-interacting DUB1 (type I), designated TbUsp7 and TbVdu1, respectively. Here we have examined the roles of these genes in trafficking of ISG75, which appears key to suramin uptake. We found that, while AP-1 does not influence ISG75 abundance, knockdown of TbUsp7 or TbVdu1 leads to reduced ISG75 abundance. Silencing TbVdu1 also reduced ISG65 abundance. TbVdu1 is a component of an evolutionarily conserved ubiquitylation switch and responsible for rapid receptor modulation, suggesting similar regulation of ISGs in T. brucei. Unexpectedly, TbUsp7 knockdown also blocked endocytosis. To integrate these observations we analysed the impact of TbUsp7 and TbVdu1 knockdown on the global proteome using SILAC. For TbVdu1, ISG65 and ISG75 are the only significantly modulated proteins, but for TbUsp7 a cohort of integral membrane proteins, including the acid phosphatase MBAP1, that is required for endocytosis, and additional ISG-related proteins are down-regulated. Furthermore, we find increased expression of the ESAG6/7 transferrin receptor and ESAG5, likely resulting from decreased endocytic activity. Therefore, multiple ubiquitylation pathways, with a complex interplay with trafficking pathways, control surface proteome expression in trypanosomes.

EssC: domain structures inform on the elusive translocation channel in the Type VII secretion system.

Structural dissection of EssC, a membrane-bound component of the bacterial Type VII secretion system, reveals two β-sandwich domains at the N-terminus and two ATPase domains at the C-terminus. A structure for the potential pore of the secretion system is proposed.

The Architecture of Essb, an Integral Membrane Component of the Type Vii Secretion System.

Summary The membrane-bound EssB is an integral and essential component of the bacterial type VII secretion system that can contribute to pathogenicity. The architecture of Geobacillus thermodenitrificans EssB has been investigated by combining crystallographic and EPR spectroscopic methods. The protein forms a dimer that straddles the cytoplasmic membrane. A helical fold is observed for the C-terminal segment, which is positioned on the exterior of the membrane. This segment contributes most to dimer formation. The N-terminal segment displays a structure related to the pseudokinase fold and may contribute to function by recognizing substrates or secretion system partners. The remaining part of EssB may serve as an anchor point for the secretion apparatus, which is embedded in the cytoplasmic membrane with the C-terminal domain protruding out to interact with partner proteins or components of peptidoglycan.

Exploiting the Achilles' heel of membrane trafficking in trypanosomes

Highlights • Development of new drugs against trypanosomes is a crucial but unmet need.• Membrane transport, endocytosis and related processes have been proposed as drug targets.• Recent insights uncovered the mode of action of two drugs that are already in the clinic.• Both of these drugs, suramin and pentamidine, bind surface proteins.• It is possible that endocytosis is a common component of sensitivity to suramin and pentamidine.

Structures of bacterial kynurenine formamidase reveal a crowded binuclear zinc catalytic site primed to generate a potent nucleophile.

Tryptophan is an important precursor for chemical entities that ultimately support the biosynthesis of key metabolites. The second stage of tryptophan catabolism is catalysed by kynurenine formamidase, an enzyme that is different between eukaryotes and prokaryotes. In the present study, we characterize the catalytic properties and present the crystal structures of three bacterial kynurenine formamidases. The structures reveal a new amidase protein fold, a highly organized and distinctive binuclear Zn2+ catalytic centre in a confined, hydrophobic and relatively rigid active site. The structure of a complex with 2-aminoacetophenone delineates aspects of molecular recognition extending to the observation that the substrate itself may be conformationally restricted to assist binding in the confined space of the active site and for subsequent processing. The cations occupy a crowded environment, and, unlike most Zn2+-dependent enzymes, there is little scope to increase co-ordination number during catalysis. We propose that the presence of a bridging water/hydroxide ligand in conjunction with the placement of an active site histidine supports a distinctive amidation mechanism.

Host-parasite co-metabolic activation of antitrypanosomal aminomethyl-benzoxaboroles

Recent development of benzoxaborole-based chemistry gave rise to a collection of compounds with great potential in targeting diverse infectious diseases, including human African Trypanosomiasis (HAT), a devastating neglected tropical disease. However, further medicinal development is largely restricted by a lack of insight into mechanism of action (MoA) in pathogenic kinetoplastids. We adopted a multidisciplinary approach, combining a high-throughput forward genetic screen with functional group focused chemical biological, structural biology and biochemical analyses, to tackle the complex MoAs of benzoxaboroles in Trypanosoma brucei. We describe an oxidative enzymatic pathway composed of host semicarbazide-sensitive amine oxidase and a trypanosomal aldehyde dehydrogenase TbALDH3. Two sequential reactions through this pathway serve as the key underlying mechanism for activating a series of 4-aminomethylphenoxy-benzoxaboroles as potent trypanocides; the methylamine parental compounds as pro-drugs are transformed first into intermediate aldehyde metabolites, and further into the carboxylate metabolites as effective forms. Moreover, comparative biochemical and crystallographic analyses elucidated the catalytic specificity of TbALDH3 towards the benzaldehyde benzoxaborole metabolites as xenogeneic substrates. Overall, this work proposes a novel drug activation mechanism dependent on both host and parasite metabolism of primary amine containing molecules, which contributes a new perspective to our understanding of the benzoxaborole MoA, and could be further exploited to improve the therapeutic index of antimicrobial compounds.