Specific Inhibition of VanZ-Mediated Resistance to Lipoglycopeptide Antibiotics

Teicoplanin is a natural lipoglycopeptide antibiotic with a similar activity spectrum as vancomycin; however, it has with the added benefit to the patient of low cytotoxicity. Both teicoplanin and vancomycin antibiotics are actively used in medical practice in the prophylaxis and treatment of severe life-threatening infections caused by gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, Enterococcus faecium and Clostridium difficile. The expression of vancomycin Z (vanZ), encoded either in the vancomycin A (vanA) glycopeptide antibiotic resistance gene cluster or in the genomes of E. faecium, as well as Streptococcus pneumoniae and C. difficile, was shown to specifically compromise the antibiotic efficiency through the inhibition of teicoplanin binding to the bacterial surface. However, the exact mechanisms of this action and protein structure remain unknown. In this study, the three-dimensional structure of VanZ from E. faecium EnGen0191 was predicted by using the I-TASSER web server. Based on the VanZ structure, a benzimidazole based ligand was predicted to bind to the VanZ by molecular docking. Importantly, this new ligand, named G3K, was further confirmed to specifically inhibit VanZ-mediated resistance to teicoplanin in vivo.

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Structure of the Lectin Mannose 6-Phosphate Receptor Homology (MRH) Domain of Glucosidase II, an Enzyme That Regulates Glycoprotein Folding Quality Control in the Endoplasmic Reticulum

Journal of Biological Chemistry ◽

10.1074/jbc.m113.450239 ◽

2013 ◽

Vol 288 (23) ◽

pp. 16460-16475 ◽

Cited By ~ 21

Author(s):

Linda J. Olson ◽

Ramiro Orsi ◽

Solana G. Alculumbre ◽

Francis C. Peterson ◽

Ivan D. Stigliano ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Three Dimensional ◽

Binding Pocket ◽

Dimensional Structure ◽

Mannose Residue ◽

Glucosidase Ii ◽

Mannose 6 Phosphate ◽

First Time ◽

Conserved Residue

Here we report for the first time the three-dimensional structure of a mannose 6-phosphate receptor homology (MRH) domain present in a protein with enzymatic activity, glucosidase II (GII). GII is involved in glycoprotein folding in the endoplasmic reticulum. GII removes the two innermost glucose residues from the Glc3Man9GlcNAc2 transferred to nascent proteins and the glucose added by UDP-Glc:glycoprotein glucosyltransferase. GII is composed of a catalytic GIIα subunit and a regulatory GIIβ subunit. GIIβ participates in the endoplasmic reticulum localization of GIIα and mediates in vivo enhancement of N-glycan trimming by GII through its C-terminal MRH domain. We determined the structure of a functional GIIβ MRH domain by NMR spectroscopy. It adopts a β-barrel fold similar to that of other MRH domains, but its binding pocket is the most shallow known to date as it accommodates a single mannose residue. In addition, we identified a conserved residue outside the binding pocket (Trp-409) present in GIIβ but not in other MRHs that influences GII glucose trimming activity.

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Protein Folding: Search for Basic Physical Models

The Scientific World JOURNAL ◽

10.1100/tsw.2003.50 ◽

2003 ◽

Vol 3 ◽

pp. 623-635 ◽

Cited By ~ 3

Author(s):

Ivan Y. Torshin ◽

Robert W. Harrison

Keyword(s):

Protein Folding ◽

Tertiary Structure ◽

Three Dimensional ◽

Physical Models ◽

Dimensional Structure ◽

Computational Techniques ◽

Linear Sequence ◽

Physical Forces

How a unique three-dimensional structure is rapidly formed from the linear sequence of a polypeptide is one of the important questions in contemporary science. Apart from biological context ofin vivoprotein folding (which has been studied only for a few proteins), the roles of the fundamental physical forces in thein vitrofolding remain largely unstudied. Despite a degree of success in using descriptions based on statistical and/or thermodynamic approaches, few of the current models explicitly include more basic physical forces (such as electrostatics and Van Der Waals forces). Moreover, the present-day models rarely take into account that the protein folding is, essentially, a rapid process that produces a highly specific architecture. This review considers several physical models that may provide more direct links between sequence and tertiary structure in terms of the physical forces. In particular, elaboration of such simple models is likely to produce extremely effective computational techniques with value for modern genomics.

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Computational Protein Stabilization Can Affect Folding Energy Landscapes and Lead to Domain-Swapped Dimers

10.26434/chemrxiv.13634021.v1 ◽

2021 ◽

Author(s):

Klara Markova ◽

Antonin Kunka ◽

Klaudia Chmelova ◽

Martin Havlasek ◽

Petra Babkova ◽

...

Keyword(s):

Protein Folding ◽

Protein Design ◽

Energy Landscape ◽

Single Point ◽

Three Dimensional ◽

Protein Stabilization ◽

Dimensional Structure ◽

Evolutionary Analysis ◽

Folding Energy

The functionality of a protein depends on its unique three-dimensional structure, which is a result of the folding process when the nascent polypeptide follows a funnel-like energy landscape to reach a global energy minimum. Computer-encoded algorithms are increasingly employed to stabilize native proteins for use in research and biotechnology applications. Here, we reveal a unique example where the computational stabilization of a monomeric α/β-hydrolase enzyme (Tm = 73.5°C; ΔTm > 23°C) affected the protein folding energy landscape. Introduction of eleven single-point stabilizing mutations based on force field calculations and evolutionary analysis yielded catalytically active domain-swapped intermediates trapped in local energy minima. Crystallographic structures revealed that these stabilizing mutations target cryptic hinge regions and newly introduced secondary interfaces, where they make extensive non-covalent interactions between the intertwined misfolded protomers. The existence of domain-swapped dimers in a solution is further confirmed experimentally by data obtained from SAXS and crosslinking mass spectrometry. Unfolding experiments showed that the domain-swapped dimers can be irreversibly converted into native-like monomers, suggesting that the domain-swapping occurs exclusively in vivo. Our findings uncovered hidden protein-folding consequences of computational protein design, which need to be taken into account when applying a rational stabilization to proteins of biological and pharmaceutical interest.

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Computational Protein Stabilization Can Affect Folding Energy Landscapes and Lead to Domain-Swapped Dimers

10.26434/chemrxiv.13634021 ◽

2021 ◽

Author(s):

Klara Markova ◽

Antonin Kunka ◽

Klaudia Chmelova ◽

Martin Havlasek ◽

Petra Babkova ◽

...

Keyword(s):

Protein Folding ◽

Protein Design ◽

Energy Landscape ◽

Single Point ◽

Three Dimensional ◽

Protein Stabilization ◽

Dimensional Structure ◽

Evolutionary Analysis ◽

Folding Energy

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Three-dimensional Printing of Customized Bioresorbable Airway Stents

10.1101/2020.09.12.294751 ◽

2020 ◽

Author(s):

Nevena Paunović ◽

Yinyin Bao ◽

Fergal Brian Coulter ◽

Kunal Masania ◽

Anna Karoline Geks ◽

...

Keyword(s):

Three Dimensional ◽

Standard Of Care ◽

Time Frame ◽

Psychological Burden ◽

Three Dimensional Printing ◽

Life Threatening ◽

Reasonable Time Frame ◽

Printed Materials ◽

Airway Stents

AbstractCentral airway obstruction is a life-threatening disorder causing a high physical and psychological burden to patients due to severe breathlessness and impaired quality of life. Standard-of-care airway stents are silicone tubes, which cause immediate relief, but are prone to migration, especially in growing patients, and require additional surgeries to be removed, which may cause further tissue damage. Customized airway stents with tailorable bioresorbability that can be produced in a reasonable time frame would be highly needed in the management of this disorder. Here, we report poly(D,L-lactide-co-ε-caprolactone) methacrylate blends-based biomedical inks and their use for the rapid fabrication of customized and bioresorbable airway stents. The 3D printed materials are cytocompatible and exhibit silicone-like mechanical properties with suitable biodegradability. In vivo studies in healthy rabbits confirmed biocompatibility and showed that the stents stayed in place for 7 weeks after which they became radiographically invisible. The developed biomedical inks open promising perspectives for the rapid manufacturing of the customized medical devices for which high precision, tuneable elasticity and predictable degradation are sought-after.

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METHODS FOR INDICATION OF NONCULTURATED VIABLE MICROORGANISMS WHEN CONTROL OF CRITICAL POINTS OF LIVESTOCK, POULTRY AND FOOD PRODUCTION TECHNOLOGY

Problems of Veterinary Sanitation, Hygiene and Ecology ◽

10.36871/vet.san.hyg.ecol.202104010 ◽

2021 ◽

Vol 1 (4) ◽

pp. 441-447

Author(s):

Ekaterina M. Lenchenko ◽

◽

Damir I. Udavliev ◽

Inna B. Pavlova ◽

◽

...

Keyword(s):

Cell Wall ◽

Luminescence Spectrum ◽

Three Dimensional ◽

Yeast Cells ◽

Dimensional Structure ◽

Heterogeneous Structure ◽

Bacterial Cells ◽

Microscopic Fungi ◽

Gram Positive Bacteria ◽

Red Luminescence

The results of morphometric and densitometric parameters biofilms are presented, effective methods of detecting uncultivated viable microorganisms isolated from a representative sample of objects of veterinary and sanitary supervision are tested and selected. Optical, luminescent and scanning electron microscopy revealed the formation of a three-dimensional structure biofilms in the form a dense network consisting of gram-negative and gram-positive bacteria, yeast cells, hyphal and pseudohyphalic forms, surrounded by an intercellular polymer matrix. The presence hyphae of microscopic fungi causes an increase in the number of cells adhered to the substrate, microcolonies were formed from bacteria and yeast cells of microscopic fungi. The pathogenesis of the syndrome of overgrowth of microorganisms is provided by the presence of various dissociative variants, the dispersion of uncultivated bacterial cells, which gain advantages in the hyperagregation of the architectonics of heterogeneous biofilms. Multilayer membranes, vesicles, cells with a defective cell wall, spheroplasts, protoplasts, L-shapes, needle-like and giant structures, and revertant cells were identified. The dynamics of changes in the viable structures microorganisms was characterized by alternating periods of decrease and increase in the intensity of biofilm formation. When detecting the viability of microorganisms in the composition biofilms, viable and non-viable cells were differentiated – a green luminescence spectrum and a red luminescence spectrum, respectively. The dissociation of the population caused an increase in the concentration of R-dissociant cells with a higher growth rate, cell lysis was detected after 48–72 h of cultivation, a change in the ratio phenotypic forms was observed – the M-dissociant was predominant. The study of the heterogeneous structure of the population, without disturbing the natural architectonics of biofilms, revealed direct correlations (r = 0,89) between morphometric (≥90 % of the field of view) and densitometric parameters (OD). The efficiency of a nutrient medium containing pancreatic hydrolyzate, mannitol, L-asparagine and glycerol was established for the repair of the cell wall, the reversal of L-forms of microorganisms.

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Antibodies against Thrombospondin-Related Anonymous Protein Do Not Inhibit Plasmodium Sporozoite Infectivity In Vivo

Infection and Immunity ◽

10.1128/iai.68.6.3667-3673.2000 ◽

2000 ◽

Vol 68 (6) ◽

pp. 3667-3673 ◽

Cited By ~ 79

Author(s):

Soren Gantt ◽

Cathrine Persson ◽

Keith Rose ◽

Ashley J. Birkett ◽

Ruben Abagyan ◽

...

Keyword(s):

Metal Ion ◽

Competitive Inhibition ◽

Polyclonal Antibodies ◽

Three Dimensional ◽

Calcium Ionophore ◽

Plasmodium Yoelii ◽

Gliding Motility ◽

Vaccine Antigen ◽

Dimensional Structure

ABSTRACT Thrombospondin-related anonymous protein (TRAP), a candidate malaria vaccine antigen, is required for Plasmodiumsporozoite gliding motility and cell invasion. For the first time, the ability of antibodies against TRAP to inhibit sporozoite infectivity in vivo is evaluated in detail. TRAP contains an A-domain, a well-characterized adhesive motif found in integrins. We modeled here a three-dimensional structure of the TRAP A-domain of Plasmodium yoelii and located regions surrounding the MIDAS (metal ion-dependent adhesion site), the presumed business end of the domain. Mice were immunized with constructs containing these A-domain regions but were not protected from sporozoite challenge. Furthermore, monoclonal and rabbit polyclonal antibodies against the A-domain, the conserved N terminus, and the repeat region of TRAP had no effect on the gliding motility or sporozoite infectivity to mice. TRAP is located in micronemes, secretory organelles of apicomplexan parasites. Accordingly, the antibodies tested here stained cytoplasmic TRAP brightly by immunofluorescence. However, very little TRAP could be detected on the surface of sporozoites. In contrast, a dramatic relocalization of TRAP onto the parasite surface occurred when sporozoites were treated with calcium ionophore. This likely mimics the release of TRAP from micronemes when a sporozoite contacts its target cell in vivo. Contact with hepatoma cells in culture also appeared to induce the release of TRAP onto the surface of sporozoites. If large amounts of TRAP are released in close proximity to its cellular receptor(s), effective competitive inhibition by antibodies may be difficult to achieve.

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Role of YidC in folding of polytopic membrane proteins

The Journal of Cell Biology ◽

10.1083/jcb.200402067 ◽

2004 ◽

Vol 165 (1) ◽

pp. 53-62 ◽

Cited By ~ 143

Author(s):

Shushi Nagamori ◽

Irina N. Smirnova ◽

H. Ronald Kaback

Keyword(s):

Membrane Proteins ◽

Three Dimensional ◽

In Vitro Transcription ◽

Dimensional Structure ◽

Lipid Phase ◽

Lactose Permease ◽

Primary Role ◽

Protein Insertion

YidC of Echerichia coli, a member of the conserved Alb3/Oxa1/YidC family, is postulated to be important for biogenesis of membrane proteins. Here, we use as a model the lactose permease (LacY), a membrane transport protein with a known three-dimensional structure, to determine whether YidC plays a role in polytopic membrane protein insertion and/or folding. Experiments in vivo and with an in vitro transcription/translation/insertion system demonstrate that YidC is not necessary for insertion per se, but plays an important role in folding of LacY. By using the in vitro system and two monoclonal antibodies directed against conformational epitopes, LacY is shown to bind the antibodies poorly in YidC-depleted membranes. Moreover, LacY also folds improperly in proteoliposomes prepared without YidC. However, when the proteoliposomes are supplemented with purified YidC, LacY folds correctly. The results indicate that YidC plays a primary role in folding of LacY into its final tertiary conformation via an interaction that likely occurs transiently during insertion into the lipid phase of the membrane.

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Insights into the molecular basis of sperm–egg recognition in mammals

Reproduction ◽

10.1530/rep.1.00181 ◽

2004 ◽

Vol 127 (4) ◽

pp. 417-422 ◽

Cited By ~ 92

Author(s):

Tanya Hoodbhoy ◽

Jurrien Dean

Keyword(s):

Zona Pellucida ◽

Three Dimensional ◽

Explanatory Models ◽

Dimensional Structure ◽

Side Chain ◽

Egg Recognition ◽

Three Dimensional Structure ◽

Sperm Binding ◽

Single Protein

The zona pellucida surrounding the egg and pre-implantation embryo is required for in vivo fertility and early development. Explanatory models of sperm–egg recognition need to take into account the ability of sperm to bind to ovulated eggs, but not to two-cell embryos. For the last two decades, investigators have sought to identify an individual protein or carbohydrate side chain as the ‘sperm receptor’. However, recent genetic data in mice are more consistent with the three-dimensional structure of the zona pellucida, rather than a single protein (or carbohydrate), determining sperm binding. The mouse and human zonae pellucidae contain three glycoproteins (ZP1, ZP2, ZP3) and, following fertilization, ZP2 is proteolytically cleaved. The replacement of endogenous mouse proteins with human ZP2, ZP3 or both does not alter taxon specificity of sperm binding or prevent fertility. Surprisingly, human ZP2 is not cleaved following fertilization and intact ZP2 correlates with persistent sperm binding to two-cell embryos. Taken together, these data support a model in which the cleavage status of ZP2 modulates the three-dimensional structure of the zona pellucida and determines whether sperm bind (uncleaved) or do not (cleaved).

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Analysis of the cross-reactivity and of the 1.5 Å crystal structure of the Malassezia sympodialis Mala s 6 allergen, a member of the cyclophilin pan-allergen family

Biochemical Journal ◽

10.1042/bj20051708 ◽

2006 ◽

Vol 396 (1) ◽

pp. 41-49 ◽

Cited By ~ 52

Author(s):

Andreas G. Glaser ◽

Andreas Limacher ◽

Sabine Flückiger ◽

Annika Scheynius ◽

Leonardo Scapozza ◽

...

Keyword(s):

Crystal Structure ◽

Three Dimensional ◽

Cross Reactivity ◽

Dimensional Structure ◽

Cellular Functions ◽

Human Proteins ◽

Ige Mediated ◽

Unit Cell Dimensions

Cyclophilins constitute a family of proteins involved in many essential cellular functions. They have also been identified as a panallergen family able to elicit IgE-mediated hypersensitivity reactions. Moreover, it has been shown that human cyclophilins are recognized by serum IgE from patients sensitized to environmental cyclophilins. IgE-mediated autoreactivity to self-antigens that have similarity to environmental allergens is often observed in atopic disorders. Therefore comparison of the crystal structure of human proteins with similarity to allergens should allow the identification of structural similarities to rationally explain autoreactivity. A new cyclophilin from Aspergillus fumigatus (Asp f 27) has been cloned, expressed and showed to exhibit cross-reactivity in vitro and in vivo. The three-dimensional structure of cyclophilin from the yeast Malassezia sympodialis (Mala s 6) has been determined at 1.5 Å (1 Å=0.1 nm) by X-ray diffraction. Crystals belong to space group P41212 with unit cell dimensions of a=b=71.99 Å and c=106.18 Å. The structure was solved by molecular replacement using the structure of human cyclophilin A as the search model. The refined structure includes all 162 amino acids of Mala s 6, an active-site-bound Ala-Pro dipeptide and 173 water molecules, with a crystallographic R- and free R-factor of 14.3% and 14.9% respectively. The overall structure consists of an eight-stranded antiparallel β-barrel and two α-helices covering the top and bottom of the barrel, typical for cyclophilins. We identified conserved solvent-exposed residues in the fungal and human structures that are potentially involved in the IgE-mediated cross-reactivity.

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