Dirk W Heinz

Helmholtz Centre for Infection Research, Brunswyck, Lower Saxony, Germany

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Publications (129)704.8 Total impact

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    ABSTRACT: 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-tRNA(Ala)-dependent alanyl-phosphatidylglycerol synthase (A-PGS) or by Lys-tRNA(Lys)-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.
    No preview · Article · Aug 2015 · Proceedings of the National Academy of Sciences
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    Full-text · Dataset · Jul 2015
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    ABSTRACT: The discovery of Streptomyces-produced streptomycin founded the age of tuberculosis therapy. Despite the subsequent development of a curative regimen for this disease, tuberculosis remains a worldwide problem, and the emergence of multidrug-resistant Mycobacterium tuberculosis has prioritized the need for new drugs. Here we show that new optimized derivatives from Streptomyces-derived griselimycin are highly active against M. tuberculosis, both in vitro and in vivo, by inhibiting the DNA polymerase sliding clamp DnaN. We discovered that resistance to griselimycins, occurring at very low frequency, is associated with amplification of a chromosomal segment containing dnaN, as well as the ori site. Our results demonstrate that griselimycins have high translational potential for tuberculosis treatment, validate DnaN as an antimicrobial target, and capture the process of antibiotic pressure-induced gene amplification. Copyright © 2015, American Association for the Advancement of Science.
    Full-text · Article · Jun 2015 · Science
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    ABSTRACT: The isobacteriochlorin heme d1 serves as an essential cofactor in the cytochrome cd1 nitrite reductase NirS which plays an important role for denitrification. During the biosynthesis of heme d1 the enzyme siroheme decarboxylase catalyzes the conversion of siroheme to 12,18-didecarboxysiroheme. This enzyme was discovered recently (Bali et al. (2011) Proc. Natl. Acad. Sci. USA 108, 18260-5) and is only scarcely characterized. Here, we present the crystal structure of the siroheme decarboxylase from Hydrogenobacter thermophilus representing the first three-dimensional structure for this type of enzyme. The overall structure strikingly resembles those of transcriptional regulators of the Lrp/AsnC-family. Moreover, the structure of the enzyme in complex with a substrate analog reveals first insights into its active site architecture. Through site-directed mutagenesis and subsequent biochemical characterization of the enzyme variants two conserved histidine residues within the active site are identified to be involved in substrate binding and catalysis. Based on our results we propose a potential catalytic mechanism for the enzymatic reaction catalyzed by the siroheme decarboxylase.
    No preview · Article · Jul 2014 · Journal of Molecular Biology
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    Dirk W Heinz
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    ABSTRACT: Secretins are major constituents of bacterial type III secretion systems (T3SS). In this issue of Structure, Kowal and colleagues report on the cryo-EM structure of the native YscC secretin from Yersinia, revealing its internal symmetry and mode of length adaptation.
    Preview · Article · Dec 2013 · Structure
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    ABSTRACT: A constitutively dimeric truncated variant of internalin B (InlB321-CD), acting as stimulator of the receptor tyrosine kinase MET, was tested for dermal wound-healing potential. Due to a lack of the endogenous MET agonist HGF/SF in chronic wounds, HGF/SF substitution by an InlB321-CD-loaded hydrogel might be beneficial in chronic wound therapy. In this study, InlB321-CD in solution and incorporated in a hydrogel was tested for mitogenic effects on immortalized human dermal keratinocytes (HaCaT) with an MTT assay. Cell migration was investigated with a scratch assay on primary keratinocytes (PHK) and on HaCaT. For the latter, scratching needed to be mitomycin C-controlled. InlB321-CD effects on a model of human skin were analyzed histologically with respect to viability. InlB321-CD led to dose-dependent proliferative effects on HaCaT cells whereas the equimolar dose of monomeric InlB321 did not. Upon hydrogel incorporation of InlB321-CD its mitogenic activity for HaCaT cells was maintained thus confirming the hydrogel as a promising drug delivery system. Motogenic effects were shown on both HaCaT and PHK cells. InlB321-CD neither possesses cytotoxic effects on the viability of a human skin model nor alters its organotypic cell morphology.
    No preview · Article · Oct 2013 · European journal of pharmaceutics and biopharmaceutics: official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V

  • No preview · Article · Aug 2013 · Acta Crystallographica Section A Foundations of Crystallography
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    ABSTRACT: Injectisomes are multi-protein transmembrane machines allowing pathogenic bacteria to inject effector proteins into eukaryotic host cells, a process called type III secretion. Here we present the first three-dimensional structure of Yersinia enterocolitica and Shigella flexneri injectisomes in situ and the first structural analysis of the Yersinia injectisome. Unexpectedly, basal bodies of injectisomes inside the bacterial cells showed length variations of 20%. The in situ structures of the Y. enterocolitica and S. flexneri injectisomes had similar dimensions and were significantly longer than the isolated structures of related injectisomes. The crystal structure of the inner membrane injectisome component YscD appeared elongated compared to a homologous protein, and molecular dynamics simulations documented its elongation elasticity. The ring-shaped secretin YscC at the outer membrane was stretched by 30–40% in situ, compared to its isolated liposome-embedded conformation. We suggest that elasticity is critical for some two-membrane spanning protein complexes to cope with variations in the intermembrane distance.
    Full-text · Dataset · Jul 2013
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    ABSTRACT: Injectisomes are multi-protein transmembrane machines allowing pathogenic bacteria to inject effector proteins into eukaryotic host cells, a process called type III secretion. Here we present the first three-dimensional structure of Yersinia enterocolitica and Shigella flexneri injectisomes in situ and the first structural analysis of the Yersinia injectisome. Unexpectedly, basal bodies of injectisomes inside the bacterial cells showed length variations of 20%. The in situ structures of the Y. enterocolitica and S. flexneri injectisomes had similar dimensions and were significantly longer than the isolated structures of related injectisomes. The crystal structure of the inner membrane injectisome component YscD appeared elongated compared to a homologous protein, and molecular dynamics simulations documented its elongation elasticity. The ring-shaped secretin YscC at the outer membrane was stretched by 30-40% in situ, compared to its isolated liposome-embedded conformation. We suggest that elasticity is critical for some two-membrane spanning protein complexes to cope with variations in the intermembrane distance. DOI:http://dx.doi.org/10.7554/eLife.00792.001.
    Full-text · Article · Jul 2013 · eLife Sciences
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    ABSTRACT: AlsR from Bacillus subtilis, a member of the LysR-type transcriptional regulator (LTTR) family, regulates the transcription of the alsSD operon encoding enzymes involved in acetoin biosynthesis. LTTRs represent the largest known family of transcriptional regulators in bacteria. In this study, AlsR82-302S100A, representing the effector domain, was produced in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method in the presence of 2.1 M DL-malic acid pH 7.0 at 293 K. The crystals belonged to space group C2, with unit-cell parameters a = 142.91, b = 74.96, c = 94.39 Å, β = 110.543°. X-ray data extending to a resolution of 2.6 Å were collected.
    No preview · Article · May 2013 · Acta Crystallographica Section F Structural Biology and Crystallization Communications
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    ABSTRACT: 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·AlF(3)-stabilized) transition state complex between the DPOR components L(2) and (NB)(2) from the marine cyanobacterium Prochlorococcus marinus. Our analysis permits a thorough investigation of the dynamic interplay between L(2) and (NB)(2). Upon complex formation, substantial ATP-dependent conformational rearrangements of L(2) 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.
    Full-text · Article · Jan 2013 · Proceedings of the National Academy of Sciences
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    ABSTRACT: In recent decades, several canonical serine protease inhibitor families have been classified and characterized. In contrast to most trypsin inhibitors, those from garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) do not share sequence similarity and have been proposed to form the new Mirabilis serine protease inhibitor family. These 30-40-amino-acid inhibitors possess a defined disulfide-bridge topology and belong to the cystine-knot miniproteins (knottins). To date, no atomic structure of this inhibitor family has been solved. Here, the first structure of S. oleracea trypsin inhibitor III (SOTI-III), in complex with bovine pancreatic trypsin, is reported. The inhibitor was synthesized by solid-phase peptide synthesis on a multi-milligram scale and was assayed to test its inhibitory activity and binding properties. The structure confirmed the proposed cystine-bridge topology. The structural features of SOTI-III suggest that it belongs to a new canonical serine protease inhibitor family with promising properties for use in protein-engineering and medical applications.
    No preview · Article · Jan 2013 · Acta Crystallographica Section D Biological Crystallography
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    Full-text · Article · Jan 2013
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    ABSTRACT: The complement system as major part of innate immunity is the first line of defense against invading microorganisms. Orchestrated by more than 60 proteins, its major task is to discriminate between host cells and pathogens and to initiate immune response. Additional recognition of necrotic or apoptotic cells demand a fine-tune regulation of this powerful system. C4b-binding protein (C4BP) is the major inhibitor of the classical complement and lectin pathway. The crystal structure of the human C4BP oligomerization domain in its 7α isoform and molecular simulations provide first structural insights of C4BP oligomerization. The heptameric core structure is stabilized by intermolecular disulfide bonds. In addition, thermal shift assays indicate that layers of electrostatic interactions mainly contribute to the extraordinary thermodynamic stability of the complex. These findings make C4BP a promising scaffold for multivalent ligand display with applications in immunology and biological chemistry.
    Full-text · Article · Dec 2012 · Journal of Molecular Biology
  • Nick Quade · Dirk W. Heinz · Rolf Müller
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    ABSTRACT: Polyketides are medically relevant molecules that are assembled from precursor molecules in a stepwise fashion. The variability of introduced sidechains was considered to be limited due to the availability of cell metabolites. Recently, a newly discovered family of proteins, the crotonyl-CoA carboxylases/reductases (CCR), have been shown to generate new precursor molecules. This explains the observed chemical diversity of polyketides and paves the way for engineering of novel drugs.
    No preview · Article · Nov 2012 · BioSpektrum
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    ABSTRACT: The physiological relevance of contacts in crystal lattices often remains elusive. This was also the case for the complex between the invasion protein internalin B (InlB) from Listeria monocytogenes and its host cell receptor, the human receptor tyrosine kinase (RTK) MET. InlB is a MET agonist and induces bacterial host cell invasion. Activation of RTKs generally involves ligand-induced dimerization of the receptor ectodomain. The two currently available crystal structures of the InlB:MET complex show the same arrangement of InlB and MET in a 1:1 complex, but different dimeric 2:2 assemblies. Only one of these 2:2 assemblies is predicted to be stable by a computational procedure. This assembly is mainly stabilized by a contact between the Cap domain of InlB from one and the Sema domain of MET from another 1:1 complex. Here, we probe the physiological relevance of this interaction. We generated variants of the leucine-rich repeat (LRR) protein InlB by inserting an additional repeat between the first and the second LRR. This should allow formation of the 1:1 complex but disrupt the potential 2:2 complex involving the Cap-Sema contact due to steric distortions. A crystal structure of one of the engineered proteins showed that it folded properly. Binding affinity to MET was comparable to that of wild-type InlB. The InlB variant induced MET phosphorylation and cell scatter like wild-type InlB. These results suggest that the Cap-Sema interaction is not physiologically relevant and support the previously proposed assembly, in which a 2:2 InlB:MET complex is built around a ligand dimer.
    Preview · Article · Oct 2012 · Protein Science
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    ABSTRACT: Hepatitis C virus (HCV) NS3-4A protease is essential for viral replication. All current small molecular weight drugs against NS3-4A are substrate peptidomimetics that have a similar binding and resistance profile. We developed inhibitory peptides (IPs) capping the active site and binding via a novel “tyrosine” finger at an alternative NS3-4A site that is of particular interest for further HCV drug development. The peptides are not cleaved due to a combination of geometrical constraints and impairment of the oxyanion hole function. Selection and optimization through combinatorial phagemid display, protein crystallography, and further modifications resulted in a 32-amino acid peptide with a Ki of 0.53 nm. Inhibition of viral replication in cell culture was demonstrated by fusion to a cell-penetrating peptide. Negligible susceptibility to known (A156V and R155K) resistance mutations of the NS3-4A protease was observed. This work shows for the first time that antiviral peptides can target an intracellular site and reveals a novel druggable site on the HCV protease.
    Full-text · Article · Sep 2012 · Journal of Biological Chemistry
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    ABSTRACT: Pathogens often rely on thermosensing to adjust virulence gene expression. In yersiniae, important virulence-associated traits are under the control of the master regulator RovA, which uses a built-in thermosensor to control its activity. Thermal upshifts encountered upon host entry induce conformational changes in the RovA dimer that attenuate DNA binding and render the protein more susceptible to proteolysis. Here, we report the crystal structure of RovA in the free and DNA-bound forms and provide evidence that thermo-induced loss of RovA activity is promoted mainly by a thermosensing loop in the dimerization domain and residues in the adjacent C-terminal helix. These determinants allow partial unfolding of the regulator upon an upshift to 37 °C. This structural distortion is transmitted to the flexible DNA-binding domain of RovA. RovA contacts mainly the DNA backbone in a low-affinity binding mode, which allows the immediate release of RovA from its operator sites. We also show that SlyA, a close homolog of RovA from Salmonella with a very similar structure, is not a thermosensor and remains active and stable at 37 °C. Strikingly, changes in only three amino acids, reflecting evolutionary replacements in SlyA, result in a complete loss of the thermosensing properties of RovA and prevent degradation. In conclusion, only minor alterations can transform a thermotolerant regulator into a thermosensor that allows adjustment of virulence and fitness determinants to their thermal environment.
    No preview · Article · Aug 2012 · Journal of Biological Chemistry
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    ABSTRACT: Flower power: Potent protease inhibitors containing triazolyl mimics of cis and trans backbone amides were engineered based on the structure of the sunflower trypsin inhibitor 1. The biologically relevant cis-Pro motif was successfully replaced with a non-prolyl unit. High-resolution crystal structures of 1,4- and 1,5-disubstituted 1,2,3-triazolyl peptidomimetics can serve in the design of tailor-made Bowman-Birk inhibitors.
    Full-text · Article · Apr 2012 · Angewandte Chemie International Edition
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    ABSTRACT: Flower‐Power: Basierend auf der Struktur des Sunflower‐Trypsininhibitors 1 wurden Proteaseinhibitoren hergestellt, die Triazolylmimetika von cis‐ und trans‐Rückgrat‐Amiden enthalten. Das biologisch relevante cis‐Pro‐Motiv wurde durch eine nicht‐Prolyl‐Einheit ersetzt und röntgenstrukturanalytisch charakterisiert. Hochaufgelöste Kristallstrukturen von 1,4‐ und 1,5‐disubstituierten 1,2,3‐Triazolyl‐Peptidomimetika können als Strukturmotive für die Entwicklung maßgeschneiderter Bowman‐Birk‐Inhibitoren dienen.
    Full-text · Article · Apr 2012 · Angewandte Chemie

Publication Stats

4k Citations
704.80 Total Impact Points

Institutions

  • 2007-2015
    • Helmholtz Centre for Infection Research
      • Department of Molecular Structural Biology (MOSB)
      Brunswyck, Lower Saxony, Germany
  • 2005
    • Technische Universität Braunschweig
      • Institute of Microbiology
      Braunschweig, Lower Saxony, Germany
  • 2001
    • Justus-Liebig-Universität Gießen
      • Institute of Physical Chemistry
      Gieben, Hesse, Germany
  • 1994-1998
    • University of Freiburg
      • Institute of Organic Chemistry and Biochemistry (Organic Chemistry)
      Freiburg, Baden-Württemberg, Germany
  • 1992-1996
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
    • Harvard Medical School
      Boston, Massachusetts, United States
  • 1992-1993
    • University of Oregon
      • • Institute of Molecular Biology
      • • Department of Physics
      Eugene, OR, United States