The macromolecular crystallography beamlines at BESSY II of the Helmholtz-Zentrum Berlin: Current status and perspectives

Muêller, U.; Förster, Ronald; Hellmig, Michael; Huschmann, F.; Kastner, Alexandra; Malecki, P.; Pühringer, Sandra; Röwer, Martin; Sparta, Karine; Steffien, Michael; Ühlein, Monika; Wilk, P.; Weiss, M.S.

doi:10.1140/epjp/i2015-15141-2

Cited by 266 publications

(240 citation statements)

References 29 publications

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“…Crystals grew over the course of 3 mo and were cryoprotected with 25% (vol/vol) glycerol in crystallization solution before flash-cooling in liquid nitrogen. For X-ray experiments, crystals were placed into a nitrogen gas stream at 100 K, and data were collected at a wavelength of 13.5 KeV (0.91841 Å) at beamline BL14.1 (19) operated by the Helmholtz-Zentrum Berlin at the BESSY II electron storage ring.…”

Section: Resultsmentioning

confidence: 99%

Capturing LTA ₄ hydrolase in action: Insights to the chemistry and dynamics of chemotactic LTB ₄ synthesis

Stsiapanava

Samuelsson

Haeggström

2017

Proc. Natl. Acad. Sci. U.S.A.

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Human leukotriene (LT) A 4 hydrolase/aminopeptidase (LTA 4 H) is a bifunctional enzyme that converts the highly unstable epoxide intermediate LTA 4 into LTB 4 , a potent leukocyte activating agent, while the aminopeptidase activity cleaves and inactivates the chemotactic tripeptide Pro-Gly-Pro. Here, we describe high-resolution crystal structures of LTA 4 H complexed with LTA 4 , providing the structural underpinnings of the enzyme's unique epoxide hydrolase (EH) activity, involving Zn 2+ , Y383, E271, D375, and two catalytic waters. The structures reveal that a single catalytic water is involved in both catalytic activities of LTA 4 H, alternating between epoxide ring opening and peptide bond hydrolysis, assisted by E271 and E296, respectively. Moreover, we have found two conformations of LTA 4 H, uncovering significant domain movements. The resulting structural alterations indicate that LTA 4 entrance into the active site is a dynamic process that includes rearrangement of three moving domains to provide fast and efficient alignment and processing of the substrate. Thus, the movement of one dynamic domain widens the active site entrance, while another domain acts like a lid, opening and closing access to the hydrophobic tunnel, which accommodates the aliphatic tale of LTA 4 during EH reaction. The enzyme-LTA 4 complex structures and dynamic domain movements provide critical insights for development of drugs targeting LTA 4 H. is a 69-kDa cytosolic bifunctional zinc metalloenzyme that converts the transient epoxide LTA 4 (5S-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid) into the dihydroxy acid LTB 4 (5S,12R-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid) via a unique epoxide hydrolase (EH) reaction (1). LTB 4 , a potent chemotactic and immune-activating agent, is implicated in acute and chronic inflammatory diseases (2, 3), cardiovascular disorders (4, 5), and carcinogenesis (6). In addition to its proinflammatory EH activity, LTA 4 H may also counteract inflammation by its aminopeptidase activity, which inactivates by cleavage another neutrophil attractant, the tripeptide Pro-Gly-Pro (PGP) (7). In addition, the fatty acid epoxide LTA 4 is also the key precursor for biosynthesis of lipoxins, a family of anti-inflammatory, proresolving lipid mediators (8). Hence, LTA 4 H is a checkpoint in lipid mediator biosynthesis that regulates both the initiation and resolution phases of inflammation.LTA 4 H consists of N-terminal, catalytic, and C-terminal domains whose interface delimits a deep cleft harboring an L-shaped pocket, with a wide polar arm that binds PGP and a narrow hydrophobic arm that is believed to accommodate LTA 4 (9). The elbow of the pocket includes H295, H299, and E318, which coordinate a Zn 2+ ion essential for both LTA 4 H activities.LTA 4 H is the only EH reported to form a nonvicinal distant 5,12 diol (10), requiring an atypical catalytic mechanism, the details of which have remained elusive in the absence of a crystal structure with bound substrate (11). Acquirement of an enzymesubstr...

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Section: Resultsmentioning

confidence: 99%

Capturing LTA ₄ hydrolase in action: Insights to the chemistry and dynamics of chemotactic LTB ₄ synthesis

Stsiapanava

Samuelsson

Haeggström

2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Diffraction experiments were performed on the beamline X06DA, operated by the Paul Scherrer Institute at the Swiss Light Source (Villigen, Switzerland), on beamline P11, operated by DESY at PETRA III synchrotron (Hamburg, Germany) (58), and on beamline 14.1, operated by the Helmholtz-Zentrum Berlin at the BESSY II electron storage ring (Berlin-Adlershof, Germany) (59). Data processing was carried out with XDS (60), and the upper resolution limits were assessed through careful observation of I/I and CC 1/2 (61).…”

Section: Data Collection Structure Determination and Refinementmentioning

confidence: 99%

Biosynthesis of Violacein, Structure and Function of l-Tryptophan Oxidase VioA from Chromobacterium violaceum

Füller

Röpke

Krausze

et al. 2016

Journal of Biological Chemistry

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Violacein is a natural purple pigment of Chromobacterium violaceum with potential medical applications as antimicrobial, antiviral, and anticancer drugs. The initial step of violacein biosynthesis is the oxidative conversion of L-tryptophan into the corresponding ␣-imine catalyzed by the flavoenzyme L-tryptophan oxidase (VioA). A substrate-related (3-(1H-indol-3-yl)-2-methylpropanoic acid) and a product-related (2-(1H-indol-3-ylmethyl)prop-2-enoic acid) competitive VioA inhibitor was synthesized for subsequent kinetic and x-ray crystallographic investigations. Structures of the binary VioA⅐FADH 2 and of the ternary VioA⅐FADH 2 ⅐2-(1H-indol-3-ylmethyl)prop-2-enoic acid complex were resolved. VioA forms a "loosely associated" homodimer as indicated by small-angle x-ray scattering experiments. VioA belongs to the glutathione reductase family 2 of The amino acid tryptophan (Trp) is a versatile metabolite that is shunted into several microbial secondary pathways. The oxidative dimerization of two Trp scaffolds is an essential step for the synthesis of the natural bisindole compounds violacein, rebeccamycin, and staurosporine. The water-insoluble purple pigment violacein is mainly produced by the bacteria Chromobacterium violaceum and Janthinobacterium lividum in tropical habitats where it might be responsible for the shielding against UV radiation (1-3). Various biological activities of violacein, including antibacterial, antiviral, as well as cytotoxic effects against several tumor cell lines, have been demonstrated (4). The related compounds rebeccamycin and staurosporine show strong antitumorigenic activity because of DNA topoisomerase or protein kinase inhibition (5, 6).Synthesis of violacein, rebeccamycin, or staurosporine is based on a closely related initial pathway in which orthologous enzymes catalyze the oxidation of a Trp moiety into the related indole-3-pyruvic acid (IPA) 2 imine by the enzymes VioA, RebO, or StaO ( Fig. 1) (7-9). Subsequently, oxidative coupling of two imines by VioB, RebD, or StaD results in the formation of a short-lived compound that was proposed to be an IPA imine dimer (7, 10). For the synthesis of rebeccamycin and staurosporine, this reactive intermediate is spontaneously converted into chromopyrrolic acid (11-13). By contrast, violacein biosynthesis requires a key intramolecular rearrangement. The postulated IPA imine dimer is the substrate of VioE, which is catalyzing a [1,2]-shift of the indole ring to produce protodeoxyviolaceinic acid (7,14). Fig. 1 gives a schematic overview about the related pathways as follows: common enzymatic reactions and the involved cofactors are highlighted (gray shading), subsequent steps for the synthesis of violacein (VioE, VioD, VioC, and one autocatalytic step) (3, 7), rebeccamycin (RebP, RebC, RebG, and RebM) (15, 16), and staurosporine (StaP, StaC, StaG, StaN, StaMA, and StaMB) (16) are indicated.Synthesis of violacein starts with the FAD-dependent oxidation of L-Trp to IPA imine catalyzed by VioA. The VioA protein from C. violaceum shares a su...

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“…Crystals were cryoprotected by transfer into mother liquor containing 22.5% (v/v) ethylene glycol and flash-cooled in liquid nitrogen. Diffraction data were collected at 100 K on beamline 14.1 of the BESSY II storage ring (Berlin, Germany) (Mueller et al 2015) and on EMBL beamline P14 of the Petra III synchrotron (Hamburg, Germany) using a monochromated X-ray beam (λ = 0.9184 Å) and processed with XDS (Table 1; Kabsch 2010). The structure was solved by molecular replacement with Molrep (Vagin and Teplyakov 2010) using a truncated yeast Brr2-Jab1 structure as the search model (Protein Data Bank [PDB] ID 4BGD) (Nguyen et al 2013) and completed by manual model building in Coot (Emsley and Cowtan 2004), guided in part by the crystal structure of a C. thermophilum Brr2 PWI domain (PDB ID 4RVQ) (Absmeier et al 2015).…”

Section: Crystallographic Proceduresmentioning

confidence: 99%

The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation

et al. 2015

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The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 disnRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation.[Keywords: pre-mRNA splicing; RNA helicase structure and function; remodeling of RNA-protein complexes; spliceosome catalytic activation; X-ray crystallography] Supplemental material is available for this article.Received September 14, 2015; revised version accepted November 13, 2015.Splicing entails the removal of noncoding sequences (introns) from primary transcripts and the concomitant ligation of neighboring coding regions (exons). It is mediated by a highly dynamic, multimegadalton RNA protein (RNP) molecular machine, the spliceosome, which consists of five small nuclear RNPs (snRNPs; U1, U2, U4, U5, and U6 in the case of the major spliceosome) and numerous non-snRNPs (Wahl et al. 2009). For each round of splicing, a spliceosome is assembled de novo on a substrate by the stepwise recruitment of snRNPs and nonsnRNPs. After assembly of a precatalytic complex, the spliceosome is catalytically activated and carries out the two consecutive steps of a splicing reaction before it is disassembled and its subunits are recycled. Each assembly, activation, catalysis, and disassembly step involves profound rearrangements of the spliceosomal RNP interaction networks, mediated predominantly by eight conserved superfamily 2 (SF2) NTPases/RNA helicases (Staley and Guthrie 1998). The most extensive rearrangements occur during spliceosome activation. In the precatalytic spliceosome, U4 and U6 snRNPs form a disnRNP by base-pairing of their snRNAs and are associated with U5 snRNP via protein-protein interactions. During spliceosome activation, the U5 snRNP-specific Brr2 helicase unwinds the U4/U6 ...

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The macromolecular crystallography beamlines at BESSY II of the Helmholtz-Zentrum Berlin: Current status and perspectives

Cited by 266 publications

References 29 publications

Capturing LTA ₄ hydrolase in action: Insights to the chemistry and dynamics of chemotactic LTB ₄ synthesis

Capturing LTA ₄ hydrolase in action: Insights to the chemistry and dynamics of chemotactic LTB ₄ synthesis

Biosynthesis of Violacein, Structure and Function of l-Tryptophan Oxidase VioA from Chromobacterium violaceum

The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation

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The macromolecular crystallography beamlines at BESSY II of the Helmholtz-Zentrum Berlin: Current status and perspectives

Cited by 266 publications

References 29 publications

Capturing LTA 4 hydrolase in action: Insights to the chemistry and dynamics of chemotactic LTB 4 synthesis

Capturing LTA 4 hydrolase in action: Insights to the chemistry and dynamics of chemotactic LTB 4 synthesis

Biosynthesis of Violacein, Structure and Function of l-Tryptophan Oxidase VioA from Chromobacterium violaceum

The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation

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Capturing LTA ₄ hydrolase in action: Insights to the chemistry and dynamics of chemotactic LTB ₄ synthesis

Capturing LTA ₄ hydrolase in action: Insights to the chemistry and dynamics of chemotactic LTB ₄ synthesis