Crystal-state 3D-structural characterization of novel, Aib-based, turn and helical peptides.

The crystal-state conformations of the hexapeptide amide Pht-(Aib)(6)-NH-C(CH(3))(2)-O-OtBu (7), the hexapeptide Ac-L-aIle-(Aib)(5)-OtBu (6), the pentapeptide Z-(Aib)(3)-L-Glu(OtBu)-Aib-O-(CH(2))(2)-(1)Nap (5), the tetrapeptides Z-(Aib)(2)-L-His(N(tau)-Trt)-Aib-OMe (4 I) and Z-(Aib)(2)-L-Nva-Aib-OtBu (4 II), the tripeptide Pyr-(Aib)(3)-OtBu (3 I), the dipeptide amides Pyr-(Aib)(2)-(4)NH-TEMPO (3 II) and Piv-(Aib)(2)-NH-C(CH(3))(2)-O-OtBu (3 III), and the dipeptides Pht-Aib-betaAc(6)c-OtBu (2 I), Pht-Aib-NH-C(CH(3))(2)-O-OtBu (2 II) and Boc-gGly-mAib-OH (2 III) have been determined by X-ray diffraction analyses. All peptides investigated are characterized by one or more turn/helix forming Aib residues. Except the three short dipeptides, all are folded into C==O...H--N intramolecularly H-bonded 3(10)-helices, or into various types of beta-turns. In the structure of 6, two independent molecules of opposite screw sense were observed in the asymmetric unit, generating diastereomeric 3(10)-helices.     

Structural and Biochemical Study of the Mono-ADP-Ribosyltransferase Domain of SdeA,a Ubiquitylating/Deubiquitylating Enzyme from Legionella pneumophila

Conventional ubiquitylation occurs through an ATP-dependent three-enzyme cascade (E1, E2, and E3) that mediates the covalent conjugation of the C-terminus of ubiquitin to a lysine on the substrate. SdeA, which belongs to the SidE effector family of Legionella pneumophila, can transfer ubiquitin to endoplasmic reticulum-associated Rab-family GTPases in a manner independent of E1 and E2 enzymes. The novel ubiquitin-modifying enzyme SdeA utilizes NAD⁺ as a cofactor to attach ubiquitin to a serine residue of the substrate. Here, to elucidate the coupled enzymatic reaction of NAD + hydrolysis and ADP-ribosylation of ubiquitin in SdeA, we characterized the mono-ADP-ribosyltransferase domain of SdeA and show that it consists of two sub-domains termed mART-N and mART-C. The crystal structure of the mART-C domain of SdeA was also determined in free form and in complex with NAD⁺ at high resolution. Furthermore, the spatial orientations of the N-terminal deubiquitylase, phosphodiesterase, mono-ADP-ribosyltransferase, and C-terminal coiled-coil domains within the 180-kDa full-length SdeA were determined. These results provide insight into the unusual ubiquitylation mechanism of SdeA and expand our knowledge on the structure–function of mono-ADP-ribosyltransferases.     

A Rare Lysozyme Crystal Form Solved Using Highly Redundant Multiple Electron Diffraction Datasets from Micron-Sized Crystals

1. Download high-res image (361KB)
2. Download full-size image

     

X射线衍射晶体法解析脱卤酶DehDIV-R结构的研究

R-2-卤代酸脱卤酶能立体选择性水解R-2-卤代酸。解析酶的单晶结构对提高酶的选择性和活性提供了直接的结构指导,是目前酶结构领域研究的前沿。以实验室前期得到的来自假单胞菌ZJU26的R-2-氯丙酸脱卤酶(Deh DIV-R)为研究对象,采用X射线衍射晶体法进行结构解析。采用pp SUMO载体融合表达Deh DIV-R蛋白,依次通过Ni-NTA亲和层析、透析酶切、二次NiNTA亲和层析以及凝胶过滤层析纯化得到单一条带,且均一性好的蛋白。接着对结晶条件进行初筛与优化,得到的最佳结晶条件为0.1mol/L HEPES p H 7,12%PEG 6 000,0.2mol/L Mg Cl2,8mmol/L CHAPS。晶体在上海同步辐射光源BL18U1线站上收集衍射数据,采用分子置换法成功解析获得了分辨率为2.35的Deh DIV-R的晶体结构。Ramachandran图表明98.02%的氨基酸位于最适区,证明了该结构的合理性。Deh DIV-R的纯化、结晶以及结构解析为进一步深入了解其结构和功能奠定了基础。     

Three‐dimensional correlative single‐cell imaging utilizing fluorescence and refractive index tomography

Cells alter the path of light, a fact that leads to well‐known aberrations in single cell or tissue imaging. Optical diffraction tomography (ODT) measures the biophysical property that causes these aberrations, the refractive index (RI). ODT is complementary to fluorescence imaging and does not require any markers. The present study introduces RI and fluorescence tomography with optofluidic rotation (RAFTOR) of suspended cells, facilitating the segmentation of the 3D‐correlated RI and fluorescence data for a quantitative interpretation of the nuclear RI. The technique is validated with cell phantoms and used to confirm a lower nuclear RI for HL60 cells. Furthermore, the nuclear inversion of adult mouse photoreceptor cells is observed in the RI distribution. The applications shown confirm predictions of previous studies and illustrate the potential of RAFTOR to improve our understanding of cells and tissues.      

The Structure of the Neisserial Lipooligosaccharide Phosphoethanolamine Transferase A (LptA) Required for Resistance to Polymyxin

Gram-negative bacteria possess an outer membrane envelope consisting of an outer leaflet of lipopolysaccharides, also called endotoxins, which protect the pathogen from antimicrobial peptides and have multifaceted roles in virulence. Lipopolysaccharide consists of a glycan moiety attached to lipid A, embedded in the outer membrane. Modification of the lipid A headgroups by phosphoethanolamine (PEA) or 4-amino-arabinose residues increases resistance to the cationic cyclic polypeptide antibiotic, polymyxin. Lipid A PEA transferases are members of the YhjW/YjdB/YijP superfamily and usually consist of a transmembrane domain anchoring the enzyme to the periplasmic face of the cytoplasmic membrane attached to a soluble catalytic domain. The crystal structure of the soluble domain of the protein of the lipid A PEA transferase from Neisseria meningitidis has been determined crystallographically and refined to 1.4 Å resolution. The structure reveals a core hydrolase fold similar to that of alkaline phosphatase. Loop regions in the structure differ, presumably to enable interaction with the membrane-localized substrates and to provide substrate specificity. A phosphorylated form of the putative nucleophile, Thr280, is observed. Metal ions present in the active site are coordinated to Thr280 and to residues conserved among the family of transferases. The structure reveals the protein components needed for the transferase chemistry; however, substrate-binding regions are not evident and are likely to reside in the transmembrane domain of the protein.     

Immobilization of Nickel by Bacterial Isolates from the Indian Ridge System and the Chemical Nature of the Accumulated Metal

Bacterial isolates from Carlsberg-Ridge waters were tested for their ability to immobilize Ni. For this, test cultures were suspended for 60 d in seawater with and without added Ni at concentrations of 10 to 10,000 μ M. At an initial concentration of 100 μ M, isolates CR35 and CR48 caused a 89.8 and 6.95 μ M decrease in dissolved Ni at 28 2°C and a 14.75 and 6.38 μ M at 3 1°C. Analyses showed the contribution of hydroxyl, carbonyl, sulfide and phosphoryl groups in Ni binding. The study proves that Ni immobilization can occur even in the absence of Mn and suggests their plausible role in the ridge ecosystem.     

The Absolute Configuration of Belactin A,a β-Lactone-Containing Serine Carboxypeptidase Inhibitor: Importance of the β-Lactone Structure for Serine Carboxypeptidase Inhibition

Abstract

The absolute configuration of belactin A, a β-lactone-containing serine carboxypeptidase inhibitor was studied by a crystal X-ray diffraction analysis and its absolute structure was determined to be (2R,3S)-2-{(3S)-3-[(2-amino-5-chlorophenyl)carboxamido]-1, 1-dimethy1-2-oxobutyl}-3-methyl-4-oxooxetane. The importance of the β-lactone structure for inhibitory activity was found by preparing several derivatives of belactin A.     

A radish seed antifungal peptide with a high amyloid fibril-forming propensity

The amyloid fibril-forming ability of two closely related antifungal and antimicrobial peptides derived from plant defensin proteins has been investigated. As assessed by sequence analysis, thioflavin T binding, transmission electron microscopy, atomic force microscopy and X-ray fiber diffraction, a 19 amino acid fragment from the C-terminal region of Raphanus sativus antifungal protein, known as RsAFP-19, is highly amyloidogenic. Further, its fibrillar morphology can be altered by externally controlled conditions. Freezing and thawing led to amyloid fibril formation which was accompanied by loss of RsAFP-19 antifungal activity. A second, closely related antifungal peptide displayed no fibril-forming capacity. It is concluded that while fibril formation is not associated with the antifungal properties of these peptides, the peptide RsAFP-19 is of potential use as a controllable, highly amyloidogenic small peptide for investigating the structure of amyloid fibrils and their mechanism of formation.     

Crystal structure of SsfS6, the putative C‐glycosyltransferase involved in SF2575 biosynthesis

The molecule known as SF2575 from Streptomyces sp. is a tetracycline polyketide natural product that displays antitumor activity against murine leukemia P388 in vivo. In the SF2575 biosynthetic pathway, SsfS6 has been implicated as the crucial C‐glycosyltransferase (C‐GT) that forms the C‐C glycosidic bond between the sugar and the SF2575 tetracycline‐like scaffold. Here, we report the crystal structure of SsfS6 in the free form and in complex with TDP, both at 2.4 Å resolution. The structures reveal SsfS6 to adopt a GT‐B fold wherein the TDP and docked putative aglycon are consistent with the overall C‐glycosylation reaction. As one of only a few existing structures for C‐glycosyltransferases, the structures described herein may serve as a guide to better understand and engineer C‐glycosylation. Proteins 2013; 81:1277–1282. © 2013 Wiley Periodicals, Inc.