Publications by authors named "Milton T Stubbs"

67 Publications

Mammalian-like type II glutaminyl cyclases in Porphyromonas gingivalis and other oral pathogenic bacteria as targets for treatment of periodontitis.

J Biol Chem 2021 Jan 5. Epub 2021 Jan 5.

Institut für Biochemie und Biotechnologie, Martin Luther University Halle-Wittenberg, Germany.

The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer Disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens , and represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures  of the enzymes from (QC) and (QC) reveal a tertiary structure composed of an eight-stranded β-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a QC-selective inhibitor described here for the first time results in growth inhibition of two clinical isolates in a dose dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases.
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http://dx.doi.org/10.1074/jbc.RA120.016836DOI Listing
January 2021

2.7 Å cryo-EM structure of vitrified M. musculus H-chain apoferritin from a compact 200 keV cryo-microscope.

PLoS One 2020 6;15(5):e0232540. Epub 2020 May 6.

ZIK HALOmem, Charles-Tanford-Proteinzentrum, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany.

Here we present the structure of mouse H-chain apoferritin at 2.7 Å (FSC = 0.143) solved by single particle cryogenic electron microscopy (cryo-EM) using a 200 kV device, the Thermo Fisher Glacios®. This is a compact, two-lens illumination system with a constant power objective lens, without any energy filters or aberration correctors, often thought of as a "screening cryo-microscope". Coulomb potential maps reveal clear densities for main chain carbonyl oxygens, residue side chains (including alternative conformations) and bound solvent molecules. We used a quasi-crystallographic reciprocal space approach to fit model coordinates to the experimental cryo-EM map. We argue that the advantages offered by (a) the high electronic and mechanical stability of the microscope, (b) the high emission stability and low beam energy spread of the high brightness Field Emission Gun (X-FEG), (c) direct electron detection technology and (d) particle-based Contrast Transfer Function (CTF) refinement have contributed to achieving high resolution. Overall, we show that basic electron optical settings for automated cryo-electron microscopy imaging can be used to determine structures approaching atomic resolution.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0232540PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7202636PMC
July 2020

Outward open conformation of a Major Facilitator Superfamily multidrug/H antiporter provides insights into switching mechanism.

Nat Commun 2018 10 1;9(1):4005. Epub 2018 Oct 1.

ZIK HALOmem, Kurt-Mothes-Straße 3, D-06120, Halle/Saale, Germany.

Multidrug resistance (MDR) poses a major challenge to medicine. A principle cause of MDR is through active efflux by MDR transporters situated in the bacterial membrane. Here we present the crystal structure of the major facilitator superfamily (MFS) drug/H antiporter MdfA from Escherichia coli in an outward open conformation. Comparison with the inward facing (drug binding) state shows that, in addition to the expected change in relative orientations of the N- and C-terminal lobes of the antiporter, the conformation of TM5 is kinked and twisted. In vitro reconstitution experiments demonstrate the importance of selected residues for transport and molecular dynamics simulations are used to gain insights into antiporter switching. With the availability of structures of alternative conformational states, we anticipate that MdfA will serve as a model system for understanding drug efflux in MFS MDR antiporters.
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http://dx.doi.org/10.1038/s41467-018-06306-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6167325PMC
October 2018

The multidrug-resistance transporter MdfA from Escherichia coli: crystallization and X-ray diffraction analysis.

Acta Crystallogr F Struct Biol Commun 2017 Jul 20;73(Pt 7):423-430. Epub 2017 Jun 20.

ZIK HALOmem, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes Strasse 3, 06120 Halle (Saale), Germany.

The active efflux of antibiotics by multidrug-resistance (MDR) transporters is a major pathway of drug resistance and complicates the clinical treatment of bacterial infections. MdfA is a member of the major facilitator superfamily (MFS) from Escherichia coli and provides resistance to a wide variety of dissimilar toxic compounds, including neutral, cationic and zwitterionic substances. The 12-transmembrane-helix MdfA was expressed as a GFP-octahistidine fusion protein with a TEV protease cleavage site. Following tag removal, MdfA was purified using two chromatographic steps, complexed with a Fab fragment and further purified using size-exclusion chromatography. MdfA and MdfA-Fab complexes were subjected to both vapour-diffusion and lipidic cubic phase (LCP) crystallization techniques. Vapour-diffusion-grown crystals were of type II, with poor diffraction behaviour and weak crystal contacts. LCP lipid screening resulted in type I crystals that diffracted to 3.4 Å resolution and belonged to the hexagonal space group P622.
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http://dx.doi.org/10.1107/S2053230X17008500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5505248PMC
July 2017

Structural and functional analyses of pyroglutamate-amyloid-β-specific antibodies as a basis for Alzheimer immunotherapy.

J Biol Chem 2017 07 16;292(30):12713-12724. Epub 2017 Jun 16.

the Institute of Biotechnology, Martin Luther University, 06108 Halle-Wittenberg, Germany,

Alzheimer disease is associated with deposition of the amyloidogenic peptide Aβ in the brain. Passive immunization using Aβ-specific antibodies has been demonstrated to reduce amyloid deposition both and Because N-terminally truncated pyroglutamate (pE)-modified Aβ species (Aβ) exhibit enhanced aggregation potential and propensity to form toxic oligomers, they represent particularly attractive targets for antibody therapy. Here we present three separate monoclonal antibodies that specifically recognize Aβ with affinities of 1-10 nm and inhibit Aβ fibril formation application of one of these resulted in improved memory in Aβ oligomer-treated mice. Crystal structures of F-Aβ complexes revealed two distinct binding modes for the peptide. Juxtaposition of pyroglutamate pE3 and the F4 side chain (the "pEF head") confers a pronounced bulky hydrophobic nature to the Aβ N terminus that might explain the enhanced aggregation properties of the modified peptide. The deep burial of the pEF head by two of the antibodies explains their high target specificity and low cross-reactivity, making them promising candidates for the development of clinical antibodies.
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http://dx.doi.org/10.1074/jbc.M117.777839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5535044PMC
July 2017

Membrane composition influences the activity of in vitro refolded human vitamin K epoxide reductase.

Biochemistry 2015 Oct 9;54(42):6454-61. Epub 2015 Oct 9.

Institut für Biochemie und Biotechnologie, Martin-Luther Universität Halle-Wittenberg , Kurt-Mothes Strasse 3, D-06120 Halle/Saale, Germany.

Human vitamin K epoxide reductase (hVKOR) is an integral membrane protein responsible for the maintenance of reduced vitamin K pools, a prerequisite for the action of γ-glutamyl carboxylase and hence for hemostasis. Here we describe the recombinant expression of hVKOR as an insoluble fusion protein in Escherichia coli, followed by purification and chemical cleavage under denaturing conditions. In vitro renaturation and reconstitution of purified solubilized hVKOR in phospholipids could be established to yield active protein. Crucially, the renatured enzyme is inhibited by the powerful coumarin anticoagulant warfarin, and we demonstrate that enzyme activity depends on lipid composition. The completely synthetic system for protein production allows a rational investigation of the multiple variables in membrane protein folding and paves the way for the provision of pure, active membrane protein for structural studies.
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http://dx.doi.org/10.1021/acs.biochem.5b00716DOI Listing
October 2015

PspF-binding domain PspA1-144 and the PspA·F complex: New insights into the coiled-coil-dependent regulation of AAA+ proteins.

Mol Microbiol 2015 Nov 10;98(4):743-59. Epub 2015 Sep 10.

Institute of Microbiology, Leibniz Universität Hannover, Herrenhäuser Str. 2, Hannover, 30419, Germany.

Phage shock protein A (PspA) belongs to the highy conserved PspA/IM30 family and is a key component of the stress inducible Psp system in Escherichia coli. One of its central roles is the regulatory interaction with the transcriptional activator of this system, the σ(54) enhancer-binding protein PspF, a member of the AAA+ protein family. The PspA/F regulatory system has been intensively studied and serves as a paradigm for AAA+ enzyme regulation by trans-acting factors. However, the molecular mechanism of how exactly PspA controls the activity of PspF and hence σ(54) -dependent expression of the psp genes is still unclear. To approach this question, we identified the minimal PspF-interacting domain of PspA, solved its structure, determined its affinity to PspF and the dissociation kinetics, identified residues that are potentially important for PspF regulation and analyzed effects of their mutation on PspF in vivo and in vitro. Our data indicate that several characteristics of AAA+ regulation in the PspA·F complex resemble those of the AAA+ unfoldase ClpB, with both proteins being regulated by a structurally highly conserved coiled-coil domain. The convergent evolution of both regulatory domains points to a general mechanism to control AAA+ activity for divergent physiologic tasks via coiled-coil domains.
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http://dx.doi.org/10.1111/mmi.13154DOI Listing
November 2015

High-resolution structures of the D-alanyl carrier protein (Dcp) DltC from Bacillus subtilis reveal equivalent conformations of apo- and holo-forms.

FEBS Lett 2015 Aug 17;589(18):2283-9. Epub 2015 Jul 17.

Institut für Biochemie und Biotechnologie, Martin-Luther Universität Halle-Wittenberg, Kurt-Mothes Strasse 3, D-06120 Halle/Saale, Germany. Electronic address:

D-Alanylation of lipoteichoic acids plays an important role in modulating the properties of Gram-positive bacteria cell walls. The D-alanyl carrier protein DltC from Bacillus subtilis has been solved in apo- and two cofactor-modified holo-forms, whereby the entire phosphopantetheine moiety is defined in one. The atomic resolution of the apo-structure allows delineation of alternative conformations within the hydrophobic core of the 78 residue four helix bundle. In contrast to previous reports for a peptidyl carrier protein from a non-ribosomal peptide synthetase, no obvious structural differences between apo- and holo-DltC forms are observed. Solution NMR spectroscopy confirms these findings and demonstrates in addition that the two forms exhibit similar backbone dynamics on the ps-ns and ms timescales.
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http://dx.doi.org/10.1016/j.febslet.2015.07.008DOI Listing
August 2015

Thermodynamic signatures in macromolecular interactions involving conformational flexibility.

Biol Chem 2014 Jul;395(7-8):905-11

The energetics of macromolecular interactions are complex, particularly where protein flexibility is involved. Exploiting serendipitous differences in the plasticity of a series of closely related trypsin variants, we analyzed the enthalpic and entropic contributions accompanying interaction with L45K-eglin C. Binding of the four variants show significant differences in released heat, although the affinities vary little, in accordance with the principle of enthalpy-entropy compensation. Binding of the most disordered variant is almost entirely enthalpically driven, with practically no entropy change. As structures of the complexes reveal negligible differences in protein-inhibitor contacts, we conclude that solvent effects contribute significantly to binding affinities.
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http://dx.doi.org/10.1515/hsz-2014-0177DOI Listing
July 2014

Correlating structure and ligand affinity in drug discovery: a cautionary tale involving second shell residues.

Biol Chem 2014 Jul;395(7-8):891-903

A high-resolution crystallographic structure determination of a protein-ligand complex is generally accepted as the 'gold standard' for structure-based drug design, yet the relationship between structure and affinity is neither obvious nor straightforward. Here we analyze the interactions of a series of serine proteinase inhibitors with trypsin variants onto which the ligand-binding site of factor Xa has been grafted. Despite conservative mutations of only two residues not immediately in contact with ligands (second shell residues), significant differences in the affinity profiles of the variants are observed. Structural analyses demonstrate that these are due to multiple effects, including differences in the structure of the binding site, differences in target flexibility and differences in inhibitor binding modes. The data presented here highlight the myriad competing microscopic processes that contribute to protein-ligand interactions and emphasize the difficulties in predicting affinity from structure.
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http://dx.doi.org/10.1515/hsz-2014-0158DOI Listing
July 2014

Deadenylation-a piece of PANcake.

EMBO J 2014 Jul 23;33(14):1503-5. Epub 2014 Jun 23.

Institute of Biochemistry and Biotechnology, Martin Luther University Halle, Halle, Germany.

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http://dx.doi.org/10.15252/embj.201489136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4198045PMC
July 2014

Structure of the Toll-Spatzle complex, a molecular hub in Drosophila development and innate immunity.

Proc Natl Acad Sci U S A 2014 Apr 14;111(17):6281-6. Epub 2014 Apr 14.

Institut für Biochemie und Biotechnologie and Mitteldeutsches Zentrum für Struktur und Dynamik von Proteinen, Martin-Luther-Universität Halle-Wittenberg, 06120 Halle (Saale), Germany.

Drosophila Toll receptors are involved in embryonic development and the immune response of adult flies. In both processes, the only known Toll receptor ligand is the human nerve growth factor-like cystine knot protein Spätzle. Here we present the crystal structure of a 1:1 (nonsignaling) complex of the full-length Toll receptor ectodomain (ECD) with the Spätzle cystine knot domain dimer. The ECD is divided into two leucine-rich repeat (LRR) domains, each of which is capped by cysteine-rich domains. Spätzle binds to the concave surface of the membrane-distal LRR domain, in contrast to the flanking ligand interactions observed for mammalian Toll-like receptors, with asymmetric contributions from each Spätzle protomer. The structure allows rationalization of existing genetic and biochemical data and provides a framework for targeting the immune systems of insects of economic importance, as well as a variety of invertebrate disease vectors.
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http://dx.doi.org/10.1073/pnas.1320678111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4036000PMC
April 2014

Disulfide linkage and structure of highly stable yeast-derived virus-like particles of murine polyomavirus.

J Biol Chem 2014 Apr 24;289(15):10411-8. Epub 2014 Feb 24.

From the Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes Strasse 03, 06120 Halle, Germany.

VP1 is the major coat protein of murine polyomavirus and forms virus-like particles (VLPs) in vitro. VLPs consist of 72 pentameric VP1 subunits held together by a terminal clamp structure that is further stabilized by disulfide bonds and chelation of calcium ions. Yeast-derived VLPs (yVLPs) assemble intracellularly in vivo during recombinant protein production. These in vivo assembled yVLPs differ in several properties from VLPs assembled in vitro from bacterially produced pentamers. We found several intermolecular disulfide linkages in yVLPs involving 5 of the 6 cysteines of VP1 (Cys(115)-Cys(20), Cys(12)-Cys(20), Cys(16)-Cys(16), Cys(12)/ Cys(16)-Cys(115), and Cys(274)-Cys(274)), indicating a highly coordinated disulfide network within the in vivo assembled particles involving the N-terminal region of VP1. Cryoelectron microscopy revealed structured termini not resolved in the published crystal structure of the bacterially expressed VLP that appear to clamp the pentameric subunits together. These structural features are probably the reason for the observed higher stability of in vivo assembled yVLPs compared with in vitro assembled bacterially expressed VLPs as monitored by increased thermal stability, higher resistance to trypsin cleavage, and a higher activation enthalpy of the disassembly reaction. This high stability is decreased following disassembly of yVLPs and subsequent in vitro reassembly, suggesting a role for cellular components in optimal assembly.
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http://dx.doi.org/10.1074/jbc.M113.484162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4036163PMC
April 2014

N-terminal protein modification by substrate-activated reverse proteolysis.

Angew Chem Int Ed Engl 2014 Mar 12;53(11):3024-8. Epub 2014 Feb 12.

Institute of Biochemistry/Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle/Saale (Germany).

Although site-specific incorporation of artificial functionalities into proteins is an important tool in both basic and applied research, it can be a major challenge to protein chemists. Enzymatic protein modification is an attractive goal due to the inherent regio- and stereoselectivity of enzymes, yet their specificity remains a problem. As a result of the intrinsic reversibility of enzymatic reactions, proteinases can in principle catalyze ligation reactions. While this makes them attractive tools for site-specific protein bioconjugation, competing hydrolysis reactions limits their general use. Here we describe the design and application of a highly specific trypsin variant for the selective modification of N-terminal residues of diverse proteins with various reagents. The modification proceeds quantitatively under native (aqueous) conditions. We show that the variant has a disordered zymogen-like activation domain, effectively suppressing the hydrolysis reaction, which is converted to an active conformation in the presence of appropriate substrates.
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http://dx.doi.org/10.1002/anie.201307736DOI Listing
March 2014

In vitro maturation of Drosophila melanogaster Spätzle protein with refolded Easter reveals a novel cleavage site within the prodomain.

Biol Chem 2013 Aug;394(8):1069-75

Institut für Biochemie und Biotechnologie, Martin-Luther- Universität Halle-Wittenberg, Kurt-Mothes-Strasse 3, D-06120 Halle Saale, Germany

Dorsoventral patterning during Drosophila melanogaster embryogenesis is mediated by a well-defined gradient of the mature NGF-like ligand Spätzle. Easter, the ultimate protease of a ventrally-restricted serine protease cascade, plays a key role in the regulation of the morphogenic gradient, catalyzing the activation cleavage of proSpätzle. As a result of alternative splicing, proSpätzle exists in multiple isoforms, almost all of which differ only in their prodomain. Although this domain is unstructured in isolation, it has a stabilizing influence on the mature cystine knot domain and is involved in the binding to the Toll receptor. Here, we report the expression and refolding of Easter, and show that the renatured enzyme performs the activation cleavage of two Spätzle isoforms. We determine the affinity of the prodomain for the cystine knot domain, and show that Easter performs a previously unknown secondary cleavage in each prodomain.
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http://dx.doi.org/10.1515/hsz-2013-0131DOI Listing
August 2013

High level expression of the Drosophila Toll receptor ectodomain and crystallization of its complex with the morphogen Spätzle.

Biol Chem 2013 Aug;394(8):1091-6

Martin-Luther- Universität Halle-Wittenberg, Institut f u r Biotechnologie, Kurt-Mothes-Strasse 3, D-06120 Halle Saale, Germany.

Drosophila Toll receptors are involved in embryonic development and in the immune response of adult flies. In both processes, the Toll receptor ligand is the NGF-like cystine knot protein Spätzle. Here we present the expression of Toll receptor ectodomain in Schneider cells at high yields and demonstrate a high affinity interaction with the refolded and trypsin-processed Spätzle cystine knot domain dimer. Poorly and anisotropically diffracting crystals of the complex could be improved by deglycosylation and dehydration, paving the way for structural analyses of the Toll-Spätzle interaction.
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http://dx.doi.org/10.1515/hsz-2013-0136DOI Listing
August 2013

Structure of glutaminyl cyclase from Drosophila melanogaster in space group I4.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2013 Apr 28;69(Pt 4):358-61. Epub 2013 Mar 28.

Department of Physical Biochemistry, Institute of Biochemistry and Biotechnology, MLU, Kurt-Mothes-Strasse 3, 06120 Halle, Germany.

The structure of ligand-free glutaminyl cyclase (QC) from Drosophila melanogaster (DmQC) has been determined in a novel crystal form. The protein crystallized in space group I4, with unit-cell parameters a = b = 122.3, c = 72.7 Å. The crystal diffracted to a resolution of 2 Å at the home source. The structure was solved by molecular replacement and was refined to an R factor of 0.169. DmQC exhibits a typical α/β-hydrolase fold. The electron density of three monosaccharides could be localized. The accessibility of the active site will facilitate structural studies of novel inhibitor-binding modes.
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http://dx.doi.org/10.1107/S1744309113005575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3614157PMC
April 2013

Peptide binding by catalytic domains of the protein disulfide isomerase-related protein ERp46.

J Mol Biol 2013 Apr 30;425(8):1340-62. Epub 2013 Jan 30.

Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle (Saale), Germany.

The protein disulfide isomerase (PDI) family member ERp46/endoPDI/thioredoxin domain-containing protein 5 is preferentially expressed in a limited number of tissues, where it may function as a survival factor for nitrosative stress in vivo. It is involved in insulin production as well as in adiponectin signaling and interacts specifically with the redox-regulatory endoplasmic reticulum proteins endoplasmic oxidoreductin 1α (Ero1α) and peroxiredoxin-4. Here, we show that ERp46, although lacking a PDI-like redox-inactive b'-thioredoxin domain with its hydrophobic substrate binding site, is able to bind to a large pool of peptides containing aromatic and basic residues via all three of its catalytic domains (a(0), a and a'), though the a(0) domain may contain the primary binding site. ERp46, which shows relatively higher activity as a disulfide-reductase than as an oxidase/isomerase in vitro compared to PDI and ERp57, possesses chaperone activity in vivo, a property also shared by the C-terminal a' domain. A crystal structure of the a' domain is also presented, offering a view of possible substrate binding sites within catalytic domains of PDI proteins.
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http://dx.doi.org/10.1016/j.jmb.2013.01.029DOI Listing
April 2013

Crystal structures of glutaminyl cyclases (QCs) from Drosophila melanogaster reveal active site conservation between insect and mammalian QCs.

Biochemistry 2012 Sep 5;51(37):7383-92. Epub 2012 Sep 5.

Probiodrug AG, Weinbergweg 22, D-06120 Halle (Saale), Germany.

Glutaminyl cyclases (QCs), which catalyze the formation of pyroglutamic acid (pGlu) at the N-terminus of a variety of peptides and proteins, have attracted particular attention for their potential role in Alzheimer's disease. In a transgenic Drosophila melanogaster (Dm) fruit fly model, oral application of the potent competitive QC inhibitor PBD150 was shown to reduce the burden of pGlu-modified Aβ. In contrast to mammals such as humans and rodents, there are at least three DmQC species, one of which (isoDromeQC) is localized to mitochondria, whereas DromeQC and an isoDromeQC splice variant possess signal peptides for secretion. Here we present the recombinant expression, characterization, and crystal structure determination of mature DromeQC and isoDromeQC, revealing an overall fold similar to that of mammalian QCs. In the case of isoDromeQC, the putative extended substrate binding site might be affected by the proximity of the N-terminal residues. PBD150 inhibition of DromeQC is roughly 1 order of magnitude weaker than that of the human and murine QCs. The inhibitor binds to isoDromeQC in a fashion similar to that observed for human QCs, whereas it adopts alternative binding modes in a DromeQC variant lacking the conserved cysteines near the active center and shows a disordered dimethoxyphenyl moiety in wild-type DromeQC, providing an explanation for the lower affinity. Our biophysical and structural data suggest that isoDromeQC and human QC are similar with regard to functional aspects. The two Dm enzymes represent a suitable model for further in-depth analysis of the catalytic mechanism of animal QCs, and isoDromeQC might serve as a model system for the structure-based design of potential AD therapeutics.
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http://dx.doi.org/10.1021/bi300687gDOI Listing
September 2012

Molecular basis of β-amyloid oligomer recognition with a conformational antibody fragment.

Proc Natl Acad Sci U S A 2012 Jul 18;109(31):12503-8. Epub 2012 Jul 18.

Institute for Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, 06120 Halle, Saale, Germany.

Oligomers are intermediates of the β-amyloid (Aβ) peptide fibrillogenic pathway and are putative pathogenic culprits in Alzheimer's disease (AD). Here we report the biotechnological generation and biochemical characterization of an oligomer-specific antibody fragment, KW1. KW1 not only discriminates between oligomers and other Aβ conformations, such as fibrils or disaggregated peptide; it also differentiates between different types of Aβ oligomers, such as those formed by Aβ (1-40) and Aβ (1-42) peptide. This high selectivity of binding contrasts sharply with many other conformational antibodies that interact with a large number of structurally analogous but sequentially different antigens. X-ray crystallography, NMR spectroscopy, and peptide array measurements imply that KW1 recognizes oligomers through a hydrophobic and significantly aromatic surface motif that includes Aβ residues 18-20. KW1-positive oligomers occur in human AD brain samples and induce synaptic dysfunctions in living brain tissues. Bivalent KW1 potently neutralizes this effect and interferes with Aβ assembly. By altering a specific step of the fibrillogenic cascade, it prevents the formation of mature Aβ fibrils and induces the accumulation of nonfibrillar aggregates. Our data illuminate significant mechanistic differences in oligomeric and fibril recognition and suggest the considerable potential of KW1 in future studies to detect or inhibit specific types of Aβ conformers.
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http://dx.doi.org/10.1073/pnas.1206433109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3412029PMC
July 2012

Electron microscopy of biotinylated protein complexes bound to streptavidin monolayer crystals.

J Struct Biol 2012 Oct 11;180(1):249-53. Epub 2012 May 11.

Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA.

Chemical biotinylation of protein complexes followed by binding to two-dimensional (monolayer) crystals of streptavidin is shown to be an effective way to prepare cryo-EM specimens from samples at low protein concentration. Three different multiprotein complexes are used to demonstrate the generality of this method. In addition, native thermosomes, purified from Sulfolobus solfataricus P2, are used to demonstrate that a uniform distribution of Euler angles is produced, even though this particle is known to adopt a preferred orientation when other methods of cryo-EM specimen preparation are used.
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http://dx.doi.org/10.1016/j.jsb.2012.04.025DOI Listing
October 2012

The O-carbamoyltransferase TobZ catalyzes an ancient enzymatic reaction.

Angew Chem Int Ed Engl 2012 Apr 1;51(17):4046-52. Epub 2012 Mar 1.

Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle (Saale), Germany.

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http://dx.doi.org/10.1002/anie.201108896DOI Listing
April 2012

Flexing and stretching in nonribosomal Peptide synthetases.

Chem Biol 2012 Feb;19(2):167-9

Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 3, D-06120 Halle (Saale), Germany.

Re-engineering of nonribosomal peptide synthetase molecular assembly lines has been hampered by a lack of detailed knowledge concerning inter-domain substrate transfer. Recent structural studies of catalytically relevant domain-domain interactions provide valuable insights into this problem (Liu et al., 2011; Sundlov et al., 2012 [in this issue of Chemistry & Biology]).
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http://dx.doi.org/10.1016/j.chembiol.2012.02.002DOI Listing
February 2012

Structures of glycosylated mammalian glutaminyl cyclases reveal conformational variability near the active center.

Biochemistry 2011 Jul 27;50(28):6280-8. Epub 2011 Jun 27.

Probiodrug AG, Weinbergweg 22, D-06120 Halle, Saale, Germany.

Formation of N-terminal pyroglutamate (pGlu or pE) from glutaminyl or glutamyl precursors is catalyzed by glutaminyl cyclases (QC). As the formation of pGlu-amyloid has been linked with Alzheimer's disease, inhibitors of QCs are currently the subject of intense development. Here, we report three crystal structures of N-glycosylated mammalian QC from humans (hQC) and mice (mQC). Whereas the overall structures of the enzymes are similar to those reported previously, two surface loops in the neighborhood of the active center exhibit conformational variability. Furthermore, two conserved cysteine residues form a disulfide bond at the base of the active center that was not present in previous reports of hQC structure. Site-directed mutagenesis suggests a structure-stabilizing role of the disulfide bond. At the entrance to the active center, the conserved tryptophan residue, W(207), which displayed multiple orientations in previous structure, shows a single conformation in both glycosylated human and murine QCs. Although mutagenesis of W(207) into leucine or glutamine altered substrate conversion significantly, the binding constants of inhibitors such as the highly potent PQ50 (PBD150) were minimally affected. The crystal structure of PQ50 bound to the active center of murine QC reveals principal binding determinants provided by the catalytic zinc ion and a hydrophobic funnel. This study presents a first comparison of two mammalian QCs containing typical, conserved post-translational modifications.
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http://dx.doi.org/10.1021/bi200249hDOI Listing
July 2011

Towards a restriction proteinase: construction of a self-activating enzyme.

Chembiochem 2011 Jul 8;12(10):1523-7. Epub 2011 Mar 8.

Institut für Biochemie und Biotechnologie, Martin-Luther Universität Halle-Wittenberg, Halle, Germany.

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http://dx.doi.org/10.1002/cbic.201000787DOI Listing
July 2011

Amyloid fibril recognition with the conformational B10 antibody fragment depends on electrostatic interactions.

J Mol Biol 2011 Jan 6;405(2):341-8. Epub 2010 Nov 6.

Max-Planck Research Unit for Enzymology of Protein Folding, 06120 Halle, Germany.

Amyloid fibrils are naturally occurring polypeptide scaffolds with considerable importance for human health and disease. These supermolecular assemblies are β-sheet rich and characterized by a high structural order. Clinical diagnosis and emerging therapeutic strategies of amyloid-dependent diseases, such as Alzheimer's, rely on the specific recognition of amyloid structures by other molecules. Recently, we generated the B10 antibody fragment, which selectively binds to Alzheimer's Aβ(1-40) amyloid fibrils but does not explicitly recognize other protein conformers, such as oligomers and disaggregated Aβ peptide. B10 presents poly-amyloid specific binding and interacts with fibrillar structures consisting of different polypeptide chains. To determine the molecular basis behind its specificity, we have analyzed the molecular properties of B10 with a battery of biochemical and biophysical techniques, ranging from X-ray crystallography to chemical modification studies. We find that fibril recognition depends on positively charged residues within the B10 antigen binding site. Mutation of these basic residues into alanine potently impairs fibril binding, and reduced B10-fibril interactions are also observed when the fibril carboxyl groups are covalently masked by a chemical modification approach. These data imply that the B10 conformational specificity for amyloid fibrils depends upon specific electrostatic interactions with an acidic moiety, which is common to different amyloid fibrils.
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http://dx.doi.org/10.1016/j.jmb.2010.10.059DOI Listing
January 2011

Kinetic and structural characterization of bacterial glutaminyl cyclases from Zymomonas mobilis and Myxococcus xanthus.

Biol Chem 2010 Dec;391(12):1419-28

Probiodrug AG, Halle/Saale, Germany.

Although enzymes responsible for the cyclization of amino-terminal glutamine residues are present in both plant and mammal species, none have yet been characterized in bacteria. Based on low sequence homologies to plant glutaminyl cyclases (QCs), we cloned the coding sequences of putative microbial QCs from Zymomonas mobilis (ZmQC) and Myxococcus xanthus (MxQC). The two recombinant enzymes exhibited distinct QC activity, with specificity constants k(cat)/K(m) of 1.47±0.33 mm⁻¹ s⁻¹ (ZmQC) and 142±32.7 mm⁻¹ s⁻¹ (MxQC) towards the fluorescent substrate glutamine-7-amino-4-methyl-coumarine. The measured pH-rate profile of the second order rate constant displayed an interesting deviation towards the acidic limb of the pH chart in the case of ZmQC, whereas MxQC showed maximum activity in the mild alkaline pH range. Analysis of the enzyme variants ZmQCGlu⁴⁶Gln and MxQCGln⁴⁶Glu show that the exchanged residues play a significant role in the pH behaviour of the respective enzymes. In addition, we determined the three dimensional crystal structures of both enzymes. The tertiary structure is defined by a five-bladed β-propeller anchored by a core cation. The structures corroborate the putative location of the active site and confirm the proposed relation between bacterial and plant glutaminyl cyclases.
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http://dx.doi.org/10.1515/BC.2010.130DOI Listing
December 2010

Impact of the C-terminal disulfide bond on the folding and stability of onconase.

Chembiochem 2010 May;11(7):978-86

Institute of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle, Germany.

The two homologous proteins ribonuclease A and onconase fold through conserved initial contacts but differ significantly in their thermodynamic stability. A disulfide bond is located in the folding initiation site of onconase (the C-terminal part of the protein molecule) that is missing in ribonuclease A, whereas the other three disulfide bonds of onconase are conserved in ribonuclease A. Consequently, the deletion of this C-terminal disulfide bond (C87-C104) allows the impact of the contacts in this region on the folding of onconase to be studied. We found the C87A/C104A-onconase variant to be less active and less stable than the wild-type protein, whereas the tertiary structure, which was determined by both X-ray crystallography and NMR spectroscopy, was only marginally affected. The folding kinetics of the variant, however, were found to be changed considerably in comparison to wild-type onconase. Proton exchange experiments in combination with two-dimensional NMR spectroscopy revealed differences in the native-state dynamics of the two proteins in the folding initiation site, which are held responsible for the changed folding mechanism. Likewise, the molecular dynamics simulation of the unfolding reaction indicated disparities for both proteins. Our results show that the high stability of onconase is based on the efficient stabilization of the folding initiation site by the C-terminal disulfide bond. The formation of the on-pathway intermediate, which is detectable during the folding of the wild-type protein and promotes the fast and efficient refolding reaction, requires the presence of this covalent bond.
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http://dx.doi.org/10.1002/cbic.200900773DOI Listing
May 2010