Crystal structure of YfeU protein from Haemophilus influenzae: a predicted etherase involved in peptidoglycan recycling

The bacterial periplasmic protein LpoA is an outer membrane lipoprotein and an activator for the cross-linking activity of PBP1A, a bifunctional peptidoglycan synthase. Previous structures of the amino-terminal (N) domain of LpoA showed it to consist entirely of helices and loops, with at least four tetratricopeptide-like repeats. Although the previously determined orthorhombic crystal structure of the N domain of Haemophilus influenzae LpoA showed a typical curved structure with a concave groove, an NMR structure of the same domain from Escherichia coli was relatively flat. Here, a crystal structure of the N domain of E. coli LpoA was determined to a resolution of 2.1 Å and was found to be more similar to the H. influenzae crystal structure than to the E. coli NMR structure. To provide a quantitative description for these comparisons, the various structures were superimposed pairwise by fitting the first half of each structure to its pairwise partner and then calculating the rotation axis that would optimally superimpose the second half. Differences in both the magnitude of the rotation and the direction of the rotation axis were observed between different pairs of structures. A 1.35 Å resolution structure of a monoclinic crystal form of the N domain of H. influenzae LpoA was also determined. In this structure, the subdomains rotate 10° relative to those in the original orthorhombic H. influenzae crystal structure to further narrow the groove between the subdomains. To accommodate this, a bound chloride ion (in place of sulfate) allowed the closer approach of a helix that forms one side of the groove.

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Crystal Structure of VapBC-1 from NontypeableHaemophilus influenzaeand the Effect of PIN Domain Mutations on Survival during Infection

Journal of Bacteriology ◽

10.1128/jb.00026-19 ◽

2019 ◽

Vol 201 (12) ◽

Cited By ~ 2

Author(s):

Ashley L. Molinaro ◽

Maithri M. Kashipathy ◽

Scott Lovell ◽

Kevin P. Battaile ◽

Nathan P. Coussens ◽

...

Keyword(s):

Crystal Structure ◽

Haemophilus Influenzae ◽

Protein Interactions ◽

Ex Vivo ◽

Single Copy ◽

Human Tissues ◽

Type Ii ◽

Wild Type ◽

Content Type ◽

Native Promoter

ABSTRACTToxin-antitoxin (TA) gene pairs have been identified in nearly all bacterial genomes sequenced to date and are thought to facilitate persistence and antibiotic tolerance. TA loci are classified into various types based upon the characteristics of their antitoxins, with those in type II expressing proteic antitoxins. Many toxins from type II modules are ribonucleases that maintain a PilT N-terminal (PIN) domain containing conserved amino acids considered essential for activity. ThevapBC(virulence-associatedprotein) TA system is the largest subfamily in this class and has been linked to pathogenesis of nontypeableHaemophilus influenzae(NTHi). In this study, the crystal structure of the VapBC-1 complex from NTHi was determined to 2.20 Å resolution. Based on this structure, aspartate-to-asparagine and glutamate-to-glutamine mutations of four conserved residues in the PIN domain of the VapC-1 toxin were constructed and the effects of the mutations on protein-protein interactions, growth ofEscherichia coli, and pathogenesisex vivowere tested. Finally, a novel model system was designed and utilized that consists of an NTHi ΔvapBC-1strain complemented inciswith the TA module containing a mutated or wild-type toxin at an ectopic site on the chromosome. This enabled the analysis of the effect of PIN domain toxin mutants in tandem with their wild-type antitoxin under the control of thevapBC-1native promoter and in single copy. This is the first report of a system facilitating the study of TA mutant operons in the background of NTHi during infections of primary human tissuesex vivo.IMPORTANCEHerein the crystal structure of the VapBC-1 complex from nontypeableHaemophilus influenzae(NTHi) is described. Our results show that some of the mutations in the PIN domain of the VapC-1 toxin were associated with decreased toxicity inE. coli, but the mutants retained the ability to homodimerize and to heterodimerize with the wild-type cognate antitoxin, VapB-1. A new system was designed and constructed to quantify the effects of these mutations on NTHi survival during infections of primary human tissuesex vivo. Any mutation to a conserved amino acid in the PIN domain significantly decreased the number of survivors compared to that of the inciswild-type toxin under the same conditions.

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Crystal structure of Haemophilus influenzae 3-isopropylmalate dehydrogenase (LeuB) in complex with the inhibitor O-isobutenyl oxalylhydroxamate

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2020.02.022 ◽

2020 ◽

Vol 524 (4) ◽

pp. 996-1002

Author(s):

Riccardo Miggiano ◽

Sara Martignon ◽

Alberto Minassi ◽

Franca Rossi ◽

Menico Rizzi

Keyword(s):

Crystal Structure ◽

Haemophilus Influenzae ◽

Isopropylmalate Dehydrogenase

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Crystal Structure of a Complex of Surfactant Protein D (SP-D) and Haemophilus influenzae Lipopolysaccharide Reveals Shielding of Core Structures in SP-D-Resistant Strains

Infection and Immunity ◽

10.1128/iai.01239-15 ◽

2016 ◽

Vol 84 (5) ◽

pp. 1585-1592 ◽

Cited By ~ 9

Author(s):

Howard W. Clark ◽

Rose-Marie Mackay ◽

Mary E. Deadman ◽

Derek W. Hood ◽

Jens Madsen ◽

...

Keyword(s):

Crystal Structure ◽

Haemophilus Influenzae ◽

Inner Core ◽

Binding Pocket ◽

Surfactant Protein ◽

Immune Defense ◽

Biologically Active ◽

Surfactant Protein D ◽

Dominant Form ◽

Content Type

The carbohydrate recognition domains (CRDs) of lung collectin surfactant protein D (SP-D) recognize sugar patterns on the surface of lung pathogens and promote phagocytosis. UsingHaemophilus influenzaeEagan strains expressing well-characterized lipopolysaccharide (LPS) surface structures of various levels of complexity, we show that bacterial recognition and binding by SP-D is inversely related to LPS chain extent and complexity. The crystal structure of a biologically active recombinant trimeric SP-D CRD complexed with a delipidated Eagan 4A LPS suggests that efficient LPS recognition by SP-D requires multiple binding interactions utilizing the three major ligand-binding determinants in the SP-D binding pocket, with Ca-dependent binding of inner-core heptose accompanied by interaction of anhydro-Kdo (4,7-anhydro-3-deoxy-d-manno-oct-2-ulosonic acid) with Arg343 and Asp325. Combined with enzyme-linked immunosorbent assays (ELISAs) and fluorescence-activated cell sorter (FACS) binding analyses, our results show that extended LPS structures previously thought to be targets for collectins are important in shielding the more vulnerable sites in the LPS core, revealing a mechanism by which pathogens with complex LPS extensions efficiently evade a first-line mucosal innate immune defense. The structure also reveals for the first time the dominant form of anhydro-Kdo.

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Crystal Structure Determination of Glycerol-Containing Lipids from Electron Diffraction Data. Use of Analog Compounds Based upon Configurational Isomers of Cyclopentane-1, 2, 3-TRIOL

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100077761 ◽

1977 ◽

Vol 35 ◽

pp. 24-25

Author(s):

Douglas L. Dorset ◽

Anthony J. Hancock

Keyword(s):

Crystal Structure ◽

Electron Diffraction ◽

Diffraction Data ◽

Structure Determination ◽

Crystal Surface ◽

Coherent Scattering ◽

Crystal Structure Determination ◽

Electron Diffraction Data ◽

Polar Group ◽

Axis Orientation

Lipids containing long polymethylene chains were among the first compounds subjected to electron diffraction structure analysis. It was only recently realized, however, that various distortions of thin lipid microcrystal plates, e.g. bends, polar group and methyl end plane disorders, etc. (1-3), restrict coherent scattering to the methylene subcell alone, particularly if undistorted molecular layers have well-defined end planes. Thus, ab initio crystal structure determination on a given single uncharacterized natural lipid using electron diffraction data can only hope to identify the subcell packing and the chain axis orientation with respect to the crystal surface. In lipids based on glycerol, for example, conformations of long chains and polar groups about the C-C bonds of this moiety still would remain unknown.One possible means of surmounting this difficulty is to investigate structural analogs of the material of interest in conjunction with the natural compound itself. Suitable analogs to the glycerol lipids are compounds based on the three configurational isomers of cyclopentane-1,2,3-triol shown in Fig. 1, in which three rotameric forms of the natural glycerol derivatives are fixed by the ring structure (4-7).

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Spherulitic crystal growth in the prodissoconch larval of Crassostrea virginica

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100076263 ◽

1983 ◽

Vol 41 ◽

pp. 518-519

Author(s):

George G. Cocks ◽

Louis Leibovitz ◽

DoSuk D. Lee

Keyword(s):

Crystal Structure ◽

Crassostrea Virginica ◽

Crystal Orientation ◽

Developmental Changes ◽

Gastrula Stage ◽

Orthorhombic Crystal ◽

Orthorhombic Crystal Structure ◽

Stages Of Growth ◽

Early Stages ◽

Initial Deposition

Our understanding of the structure and the formation of inorganic minerals in the bivalve shells has been considerably advanced by the use of electron microscope. However, very little is known about the ultrastructure of valves in the larval stage of the oysters. The present study examines the developmental changes which occur between the time of conception to the early stages of Dissoconch in the Crassostrea virginica(Gmelin), focusing on the initial deposition of inorganic crystals by the oysters.The spawning was induced by elevating the temperature of the seawater where the adult oysters were conditioned. The eggs and sperm were collected separately, then immediately mixed for the fertilizations to occur. Fertilized animals were kept in the incubator where various stages of development were stopped and observed. The detailed analysis of the early stages of growth showed that CaCO3 crystals(aragonite), with orthorhombic crystal structure, are deposited as early as gastrula stage(Figuresla-b). The next stage in development, the prodissoconch, revealed that the crystal orientation is in the form of spherulites.

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Transmission Electron Microscopy studies of the vacancy ordering in YSI2-X thin films

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100087124 ◽

1991 ◽

Vol 49 ◽

pp. 562-563

Author(s):

F.-R. Chen ◽

T. L. Lee ◽

L. J. Chen

Keyword(s):

Crystal Structure ◽

Electron Microscopy ◽

Thin Films ◽

Diffraction Pattern ◽

Annealing Temperature ◽

Lattice Mismatch ◽

Si Substrate ◽

Metal Thin Films ◽

Transmission Electron ◽

Vacancy Ordering

YSi2-x thin films were grown by depositing the yttrium metal thin films on (111)Si substrate followed by a rapid thermal annealing (RTA) at 450 to 1100°C. The x value of the YSi2-x films ranges from 0 to 0.3. The (0001) plane of the YSi2-x films have an ideal zero lattice mismatch relative to (111)Si surface lattice. The YSi2 has the hexagonal AlB2 crystal structure. The orientation relationship with Si was determined from the diffraction pattern shown in figure 1(a) to be and . The diffraction pattern in figure 1(a) was taken from a specimen annealed at 500°C for 15 second. As the annealing temperature was increased to 600°C, superlattice diffraction spots appear at position as seen in figure 1(b) which may be due to vacancy ordering in the YSi2-x films. The ordered vacancies in YSi2-x form a mesh in Si plane suggested by a LEED experiment.

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Crystal Structure Analysis of Cu-Zr Alloys by Convergent Beam Diffraction

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100098277 ◽

1981 ◽

Vol 39 ◽

pp. 354-355

Author(s):

A. F. Marshall ◽

J. W. Steeds ◽

D. Bouchet ◽

S. L. Shinde ◽

R. G. Walmsley

Keyword(s):

Crystal Structure ◽

Point Group ◽

Intermetallic Phase ◽

Three Dimensional ◽

Crystal Structure Analysis ◽

Ion Milling ◽

Composition And Structure ◽

Unit Cells ◽

Sputter Deposited ◽

Convergent Beam

Convergent beam electron diffraction is a powerful technique for determining the crystal structure of a material in TEM. In this paper we have applied it to the study of the intermetallic phases in the Cu-rich end of the Cu-Zr system. These phases are highly ordered. Their composition and structure has been previously studied by microprobe and x-ray diffraction with sometimes conflicting results.The crystalline phases were obtained by annealing amorphous sputter-deposited Cu-Zr. Specimens were thinned for TEM by ion milling and observed in a Philips EM 400. Due to the large unit cells involved, a small convergence angle of diffraction was used; however, the three-dimensional lattice and symmetry information of convergent beam microdiffraction patterns is still present. The results are as follows:1) 21 at% Zr in Cu: annealed at 500°C for 5 hours. An intermetallic phase, Cu3.6Zr (21.7% Zr), space group P6/m has been proposed near this composition (2). The major phase of our annealed material was hexagonal with a point group determined as 6/m.

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