Functional Characterization of Primordial Protein Repair Enzyme M38 Metallo-Peptidase From Fervidobacterium islandicum AW-1

The NA23_RS08100 gene of Fervidobacterium islandicum AW-1 encodes a keratin-degrading β-aspartyl peptidase (FiBAP) that is highly expressed under starvation conditions. Herein, we expressed the gene in Escherichia coli, purified the recombinant enzyme to homogeneity, and investigated its function. The 318 kDa recombinant FiBAP enzyme exhibited maximal activity at 80°C and pH 7.0 in the presence of Zn2+. Size-exclusion chromatography revealed that the native enzyme is an octamer comprising a tetramer of dimers; this was further supported by determination of its crystal structure at 2.6 Å resolution. Consistently, the structure of FiBAP revealed three additional salt bridges in each dimer, involving 12 ionic interactions that might contribute to its high thermostability. In addition, the co-crystal structure containing the substrate analog N-carbobenzoxy-β-Asp-Leu at 2.7 Å resolution revealed binuclear Zn2+-mediated substrate binding, suggesting that FiBAP is a hyperthermophilic type-I IadA, in accordance with sequence-based phylogenetic analysis. Indeed, complementation of a Leu auxotrophic E. coli mutant strain (ΔiadA and ΔleuB) with FiBAP enabled the mutant strain to grow on isoAsp-Leu peptides. Remarkably, LC-MS/MS analysis of soluble keratin hydrolysates revealed that FiBAP not only cleaves the C-terminus of isoAsp residues but also has a relatively broad substrate specificity toward α-peptide bonds. Moreover, heat shock-induced protein aggregates retarded bacterial growth, but expression of BAP alleviated the growth defect by degrading damaged proteins. Taken together, these results suggest that the viability of hyperthermophiles under stressful conditions may rely on the activity of BAP within cellular protein repair systems.

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Crystal and solution structures of fragments of the human leucocyte common antigen-related protein

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798320003885 ◽

2020 ◽

Vol 76 (5) ◽

pp. 406-417

Author(s):

Joachim Vilstrup ◽

Amanda Simonsen ◽

Thea Birkefeldt ◽

Dorthe Strandbygård ◽

Jeppe Lyngsø ◽

...

Keyword(s):

Crystal Structure ◽

Size Exclusion ◽

Receptor Protein ◽

Type I ◽

Common Antigen ◽

Heparin Binding ◽

Related Protein ◽

Saxs Data ◽

Exclusion Chromatography ◽

The Individual

Leucocyte common antigen-related protein (LAR) is a post-synaptic type I transmembrane receptor protein that is important for neuronal functionality and is genetically coupled to neuronal disorders such as attention deficit hyperactivity disorder (ADHD). To understand the molecular function of LAR, structural and biochemical studies of protein fragments derived from the ectodomain of human LAR have been performed. The crystal structure of a fragment encompassing the first four FNIII domains (LARFN1–4) showed a characteristic L shape. SAXS data suggested limited flexibility within LARFN1–4, while rigid-body refinement of the SAXS data using the X-ray-derived atomic model showed a smaller angle between the domains defining the L shape compared with the crystal structure. The capabilities of the individual LAR fragments to interact with heparin was examined using microscale thermophoresis and heparin-affinity chromatography. The results showed that the three N-terminal immunoglobulin domains (LARIg1–3) and the four C-terminal FNIII domains (LARFN5–8) both bound heparin, while LARFN1–4 did not. The low-molecular-weight heparin drug Innohep induced a shift in hydrodynamic volume as assessed by size-exclusion chromatography of LARIg1–3 and LARFN5–8, while the chemically defined pentameric heparin drug Arixtra did not. Together, the presented results suggest the presence of an additional heparin-binding site in human LAR.

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Faculty Opinions recommendation of An RNA cap (nucleoside-2'-O-)-methyltransferase in the flavivirus RNA polymerase NS5: crystal structure and functional characterization.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1007868.99105 ◽

2002 ◽

Author(s):

Peter Colman

Keyword(s):

Crystal Structure ◽

Rna Polymerase ◽

Functional Characterization

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Exploring the taxonomical and functional profile of As Burgas hot spring focusing on thermostable β-galactosidases

Scientific Reports ◽

10.1038/s41598-020-80489-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

María-Eugenia DeCastro ◽

Michael P. Doane ◽

Elizabeth Ann Dinsdale ◽

Esther Rodríguez-Belmonte ◽

María-Isabel González-Siso

Keyword(s):

Microbial Community ◽

Hot Spring ◽

Functional Characterization ◽

Tca Cycle ◽

Maximal Activity ◽

Sulfur Cycle ◽

E Coli ◽

Complex Microbial Community ◽

Relationship Of ◽

The Relationship

AbstractIn the present study we investigate the microbial community inhabiting As Burgas geothermal spring, located in Ourense (Galicia, Spain). The approximately 23 Gbp of Illumina sequences generated for each replicate revealed a complex microbial community dominated by Bacteria in which Proteobacteria and Aquificae were the two prevalent phyla. An association between the two most prevalent genera, Thermus and Hydrogenobacter, was suggested by the relationship of their metabolism. The high relative abundance of sequences involved in the Calvin–Benson cycle and the reductive TCA cycle unveils the dominance of an autotrophic population. Important pathways from the nitrogen and sulfur cycle are potentially taking place in As Burgas hot spring. In the assembled reads, two complete ORFs matching GH2 beta-galactosidases were found. To assess their functional characterization, the two ORFs were cloned and overexpressed in E. coli. The pTsbg enzyme had activity towards o-Nitrophenyl-β-d-galactopyranoside (ONPG) and p-Nitrophenyl-β-d-fucopyranoside, with high thermal stability and showing maximal activity at 85 °C and pH 6, nevertheless the enzyme failed to hydrolyze lactose. The other enzyme, Tsbg, was unable to hydrolyze even ONPG or lactose. This finding highlights the challenge of finding novel active enzymes based only on their sequence.

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Crystal structure of magnesium chromium vanadate Mg2CrV3O11, a member of the A2BV3O11 vanadate family

Powder Diffraction ◽

10.1154/1.2770746 ◽

2007 ◽

Vol 22 (3) ◽

pp. 246-252 ◽

Cited By ~ 4

Author(s):

A. Worsztynowicz ◽

S. M. Kaczmarek ◽

W. Paszkowicz ◽

R. Minikayev

Keyword(s):

Crystal Structure ◽

Rietveld Method ◽

Ion Pairs ◽

Exchange Constant ◽

Type I ◽

Type Ii ◽

Paramagnetic Resonance ◽

Interatomic Distances ◽

Electron Paramagnetic ◽

Full Occupancy

The crystal structure of recently discovered chromium (III) dimagnesium trivanadate (V) Mg2CrV3O11 was refined using the Rietveld method. The crystal system of Mg2CrV3O11 is triclinic with space group P1− (Mg1.7Zn0.3GaV3O11 type) and lattice parameters a=6.4057(1) Å, b=6.8111(1) Å, c=10.0640(2) Å, α=97.523(1)°, β=103.351(1)°, γ=101.750(1)°, and Z=2. The characteristic feature of compounds in the A2BV3O11 (A=Mg, Zn and B=Ga, Fe, Cr) family is a strong tendency to share the octahedral M(1) and M(2) sites by both divalent A and trivalent B atoms, and the bipyramidal M(3) sites occupied by divalent A ions. In the present refinement, the only constraint assuming full occupancy of the M(1), M(2), and M(3) sites leads to the following Cr/(Cr+Mg) ratios: 0.70(2) at M(1), 0.24(2) at M(2), and 0.03(2) at M(3). These occupancies are discussed and compared to those of isotypic compounds. The values of interatomic distances are found to be comparable with those reported by R. D. Shannon in 1976. Electron paramagnetic resonance has been also analyzed. Two absorption lines with g≈2.0 (type I) and g≈1.98 (type II) have been recorded in the EPR spectra, and attributed to V4+ ions and Cr3+–Cr3+ ion pairs, respectively. The exchange constant J between Cr3+ ions has been calculated.

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Intrinsic mechanical properties of the extracellular matrix affect the behavior of pre-osteoblastic MC3T3-E1 cells

AJP Cell Physiology ◽

10.1152/ajpcell.00455.2005 ◽

2006 ◽

Vol 290 (6) ◽

pp. C1640-C1650 ◽

Cited By ~ 177

Author(s):

Chirag B. Khatiwala ◽

Shelly R. Peyton ◽

Andrew J. Putnam

Keyword(s):

Mechanical Properties ◽

Type I Collagen ◽

Focal Adhesions ◽

Microscopic Analysis ◽

Maximal Activity ◽

Type I ◽

Cell Functions ◽

Collagen Density ◽

Cells Cultured ◽

In Cells

Mechanical cues present in the ECM have been hypothesized to provide instructive signals that dictate cell behavior. We probed this hypothesis in osteoblastic cells by culturing MC3T3-E1 cells on the surface of type I collagen-modified hydrogels with tunable mechanical properties and assessed their proliferation, migration, and differentiation. On gels functionalized with a low type I collagen density, MC3T3-E1 cells cultured on polystyrene proliferated twice as fast as those cultured on the softest substrate. Quantitative time-lapse video microscopic analysis revealed random motility speeds were significantly retarded on the softest substrate (0.25 ± 0.01 μm/min), in contrast to maximum speeds on polystyrene substrates (0.42 ± 0.04 μm/min). On gels functionalized with a high type I collagen density, migration speed exhibited a biphasic dependence on ECM compliance, with maximum speeds (0.34 ± 0.02 μm/min) observed on gels of intermediate stiffness, whereas minimum speeds (0.24 ± 0.03 μm/min) occurred on both the softest and most rigid (i.e., polystyrene) substrates. Immature focal contacts and a poorly organized actin cytoskeleton were observed in cells cultured on the softest substrates, whereas those on more rigid substrates assembled mature focal adhesions and robust actin stress fibers. In parallel, focal adhesion kinase (FAK) activity (assessed by detecting pY397-FAK) was influenced by compliance, with maximal activity occurring in cells cultured on polystyrene. Finally, mineral deposition by the MC3T3-E1 cells was also affected by ECM compliance, leading to the conclusion that altering ECM mechanical properties may influence a variety of MC3T3-E1 cell functions, and perhaps ultimately, their differentiated phenotype.

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Synthesis, Crystal Structure, and Physical Properties of the Type-I Clathrate Ba8−δNix□ySi46–x–y

Inorganic Chemistry ◽

10.1021/ic2027626 ◽

2012 ◽

Vol 51 (8) ◽

pp. 4730-4741 ◽

Cited By ~ 23

Author(s):

U. Aydemir ◽

C. Candolfi ◽

A. Ormeci ◽

H. Borrmann ◽

U. Burkhardt ◽

...

Keyword(s):

Crystal Structure ◽

Physical Properties ◽

Type I

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Functional Characterization of a Redundant Plasmodium TRAP Family Invasin, TRAP-Like Protein, by Aldolase Binding and a Genetic Complementation Test

Eukaryotic Cell ◽

10.1128/ec.00089-08 ◽

2008 ◽

Vol 7 (6) ◽

pp. 1062-1070 ◽

Cited By ~ 39

Author(s):

Kirsten Heiss ◽

Hui Nie ◽

Sumit Kumar ◽

Thomas M. Daly ◽

Lawrence W. Bergman ◽

...

Keyword(s):

Functional Characterization ◽

Cell Entry ◽

Host Cells ◽

Complementation Test ◽

Type I ◽

Cycle Progression ◽

Targeted Gene Disruption ◽

Specific Host ◽

Host Cell Entry

ABSTRACT Efficient and specific host cell entry is of exquisite importance for intracellular pathogens. Parasites of the phylum Apicomplexa are highly motile and actively enter host cells. These functions are mediated by type I transmembrane invasins of the TRAP family that link an extracellular recognition event to the parasite actin-myosin motor machinery. We systematically tested potential parasite invasins for binding to the actin bridging molecule aldolase and complementation of the vital cytoplasmic domain of the sporozoite invasin TRAP. We show that the ookinete invasin CTRP and a novel, structurally related protein, termed TRAP-like protein (TLP), are functional members of the TRAP family. Although TLP is expressed in invasive stages, targeted gene disruption revealed a nonvital role during life cycle progression. This is the first genetic analysis of TLP, encoding a redundant TRAP family invasin, in the malaria parasite.

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