scholarly journals Determination of the membrane topology of PORCN, an O-acyl transferase that modifies Wnt signalling proteins

Open Biology ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 200400
Author(s):  
Lisa M. Galli ◽  
Marc O. Anderson ◽  
J. Gabriel Fraley ◽  
Luis Sanchez ◽  
Raymund Bueno ◽  
...  
Keyword(s):  
Homology Modelling ◽  
Three Dimensional ◽  
Cancer Therapeutics ◽  
Membrane Topology ◽  
Dimensional Structure ◽  
Dependent Manner ◽  
Acyl Transferase ◽  
Wnt Proteins ◽  
C Terminus ◽  
Membrane Spanning

Wnt gradients elicit distinct cellular responses, such as proliferation, specification, differentiation and survival in a dose-dependent manner. Porcupine (PORCN), a membrane-bound O-acyl transferase (MBOAT) that resides in the endoplasmic reticulum, catalyses the addition of monounsaturated palmitate to Wnt proteins and is required for Wnt gradient formation and signalling. In humans, PORCN mutations are causal for focal dermal hypoplasia (FDH), an X-linked dominant syndrome characterized by defects in mesodermal and endodermal tissues. PORCN is also an emerging target for cancer therapeutics. Despite the importance of this enzyme, its structure remains poorly understood. Recently, the crystal structure of DltB, an MBOAT family member from bacteria, was solved. In this report, we use experimental data along with homology modelling to DltB to determine the membrane topology of PORCN. Our studies reveal that PORCN has 11 membrane domains, comprising nine transmembrane spanning domains and two reentrant domains. The N-terminus is oriented towards the lumen while the C-terminus is oriented towards the cytosol. Like DltB, PORCN has a funnel-like structure that is encapsulated by multiple membrane-spanning helices. This new model for PORCN topology allows us to map residues that are important for biological activity (and implicated in FDH) onto its three-dimensional structure.

scholarly journals Unravelling the structure of the pneumococcal autolytic lysozyme

2005 ◽  
Vol 391 (1) ◽  
pp. 41-49 ◽  
Author(s):  
Begoña Monterroso ◽  
Consuelo López-Zumel ◽  
José L. García ◽  
José L. Sáiz ◽  
Pedro García ◽  
...  
Keyword(s):  
Homology Modelling ◽  
Tight Binding ◽  
Three Dimensional ◽  
Divergent Evolution ◽  
Glycosyl Hydrolases ◽  
Three Dimensional Model ◽  
Lytic Enzymes ◽  
C Terminus ◽  
Global Fold ◽  
Positively Charged

The LytC lysozyme of Streptococcus pneumoniae forms part of the autolytic system of this important pathogen. This enzyme is composed of a C-terminal CM (catalytic module), belonging to the GH25 family of glycosyl hydrolases, and an N-terminal CBM (choline-binding module), made of eleven homologous repeats, that specifically recognizes the choline residues that are present in pneumococcal teichoic and lipoteichoic acids. This arrangement inverts the general assembly pattern of the major pneumococcal autolysin, LytA, and the lytic enzymes encoded by pneumococcal bacteriophages that place the CBM (made of six repeats) at the C-terminus. In the present paper, a three-dimensional model of LytC built by homology modelling of each module and consistent with spectroscopic and hydrodynamic studies is shown. In addition, the putative catalytic-pair residues are identified. Despite the inversion in the modular arrangement, LytC and the bacteriophage-encoded Cpl-1 lysozyme most probably adopt a similar global fold. However, the distinct choline-binding ability and their substrate-binding surfaces may reflect a divergent evolution directed by the different roles played by them in the host (LytC) or in the bacteriophage (Cpl-1). The tight binding of LytC to the pneumococcal envelope, mediated by the acquisition of additional choline-binding repeats, could facilitate the regulation of the potentially suicidal activity of this autolysin. In contrast, a looser attachment of Cpl-1 to the cell wall and the establishment of more favourable interactions between its highly negatively charged catalytic surface and the positively charged chains of pneumococcal murein could enhance the lytic activity of the parasite-encoded enzyme and therefore liberation of the phage progeny.

scholarly journals Complete In Vitro Assembly of the Reovirus Outer Capsid Produces Highly Infectious Particles Suitable for Genetic Studies of the Receptor-Binding Protein

2001 ◽  
Vol 75 (11) ◽  
pp. 5335-5342 ◽  
Author(s):  
Kartik Chandran ◽  
Xing Zhang ◽  
Norman H. Olson ◽  
Stephen B. Walker ◽  
James D. Chappell ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Cell Attachment ◽  
Molecular Genetic ◽  
Three Dimensional ◽  
Host Cells ◽  
Dimensional Structure ◽  
Dependent Manner ◽  
Viral Attachment ◽  
Structure Assembly

ABSTRACT Mammalian reoviruses, prototype members of theReoviridae family of nonenveloped double-stranded RNA viruses, use at least three proteins—ς1, μ1, and ς3—to enter host cells. ς1, a major determinant of cell tropism, mediates viral attachment to cellular receptors. Studies of ς1 functions in reovirus entry have been restricted by the lack of methodologies to produce infectious virions containing engineered mutations in viral proteins. To mitigate this problem, we produced virion-like particles by “recoating” genome-containing core particles that lacked ς1, μ1, and ς3 with recombinant forms of these proteins in vitro. Image reconstructions from cryoelectron micrographs of the recoated particles revealed that they closely resembled native virions in three-dimensional structure, including features attributable to ς1. The recoated particles bound to and infected cultured cells in a ς1-dependent manner and were approximately 1 million times as infectious as cores and 0.5 times as infectious as native virions. Experiments with recoated particles containing recombinant ς1 from either of two different reovirus strains confirmed that differences in cell attachment and infectivity previously observed between those strains are determined by the ς1 protein. Additional experiments showed that recoated particles containing ς1 proteins with engineered mutations can be used to analyze the effects of such mutations on the roles of particle-bound ς1 in infection. The results demonstrate a powerful new system for molecular genetic dissections of ς1 with respect to its structure, assembly into particles, and roles in entry.

Unliganded GroEL at 2.8 Å: structure and functional implications

1995 ◽  
Vol 348 (1323) ◽  
pp. 113-119 ◽  
Keyword(s):  
Three Dimensional ◽  
Binding Pocket ◽  
Dimensional Structure ◽  
Outer Diameter ◽  
Central Channel ◽  
Crystal Forms ◽  
Monoclinic Form ◽  
C Terminus ◽  
Ligand Free ◽  
Dyad Symmetry

The three-dimensional structure of the E. coli chaperonin, GroEL, has been determined crystallo-graphically and refined to 2.7 Å in two crystal forms: an orthorhombic form from high salt and a monoclinic form from polyethylene glycol. The former is ligand free, the latter is both liganded with ATP analogues and ligand free. These structures provide a structural scaffold upon which to interpret extensive mutagenesis and biochemical studies. GroEL contains two sevenfold rotationally symmetric rings of identical 547-amino acid subunits. The rings are arranged ‘back-to-back’ with exact dyad symmetry to form a stubby cylinder that is 146 Å high with an outer diameter of about 143 Å. The cylinder has a substantial central channel that is unobstructed for the entire length of the cylinder and has a diameter of about 45 Å except for large bulges that lead into a sevenfold symmetric array of elliptical side windows in each ring. Each subunit is composed of three distinct domains: (i) an ‘equatorial’ domain that contains the N- and C-terminus and the ATP-binding pocket, .(ii) an ‘apical domain’ that forms the opening of the central channel and contains poorly ordered segments that mutational studies implicate in binding unfolded polypeptides and GroES, and (iii) an intermediate domain tht connects the other two domains and may serve to transmit allosteric adjustments.

scholarly journals A structural insight into the inhibition of human and Leishmania donovani ornithine decarboxylases by 1-amino-oxy-3-aminopropane

2007 ◽  
Vol 405 (2) ◽  
pp. 261-268 ◽  
Author(s):  
Veronica T. Dufe ◽  
Daniel Ingner ◽  
Olle Heby ◽  
Alex R. Khomutov ◽  
Lo Persson ◽  
...  
Keyword(s):  
Leishmania Donovani ◽  
Homology Modelling ◽  
Critical Role ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Irreversible Inhibitor ◽  
Polyamine Biosynthesis ◽  
Oxime Formation ◽  
Large Conformational Change ◽  
Similar Mode

The critical role of polyamines in key processes such as cell growth, differentiation and macromolecular synthesis makes the enzymes involved in their synthesis potential targets in the treatment of certain types of cancer and parasitic diseases. Here we present a study on the inhibition of human and Leishmania donovani ODC (ornithine decarboxylase), the first committed enzyme in the polyamine biosynthesis pathway, by APA (1-amino-oxy-3-aminopropane). The present study shows APA to be a potent inhibitor of both human and L. donovani ODC with a Ki value of around 1.0 nM. We also show that L. donovani ODC binds the substrate, the co-enzyme pyridoxal 5′-phosphate and the irreversible inhibitor α-difluoromethylornithine (a curative agent of West African sleeping sickness) with less affinity than human ODC. We have also determined the three-dimensional structure of human ODC in complex with APA, which revealed the mode of the inhibitor binding to the enzyme. In contrast with earlier reports, the structure showed no indication of oxime formation between APA and PLP (pyridoxal 5′-phosphate). Homology modelling suggests a similar mode of binding of APA to L. donovani ODC. A comparison of the ODC–APA–PLP structure with earlier ODC structures also shows that the protease-sensitive loop (residues 158–168) undergoes a large conformational change and covers the active site of the protein. The understanding of the structural mode of APA binding may constitute the basis for the development of more specific inhibitors of L. donovani ODC.

scholarly journals Transketolase from Leishmania mexicana has a dual subcellular localization

2004 ◽  
Vol 382 (2) ◽  
pp. 759-767 ◽  
Author(s):  
Nicola J. VEITCH ◽  
Dante A. MAUGERI ◽  
Juan Jose CAZZULO ◽  
Ylva LINDQVIST ◽  
Michael P. BARRETT
Keyword(s):  
Subcellular Localization ◽  
Three Dimensional ◽  
Total Activity ◽  
Pentose Phosphate ◽  
Dimensional Structure ◽  
Leishmania Mexicana ◽  
Gene Encoding ◽  
X Ray Crystallography ◽  
C Terminus ◽  
Targeting Signal

Transketolase has been characterized in Leishmania mexicana. A gene encoding this enzyme was identified and cloned. The gene was expressed in Escherichia coli and the protein was purified and characterized. An apparent Km of 2.75 mM for ribose 5-phosphate was determined. X-ray crystallography was used to determine the three-dimensional structure of the enzyme to a resolution of 2.2 Å (1 Å≡0.1 nm). The C-terminus of the protein contains a type-1 peroxisome-targeting signal, suggestive of a possible glycosomal subcellular localization. Subcellular localization experiments performed with promastigote forms of the parasite revealed that the protein was predominantly cytosolic, although a significant component of the total activity was associated with the glycosomes. Transketolase is thus the first enzyme of the nonoxidative branch of the pentose phosphate pathway whose presence has been demonstrated in a peroxisome-like organelle.

scholarly journals IN SILICO CHARACTERISATION AND HOMOLOGY MODELLING OF TOLL/TOLL LIKE RECEPTORS (TLRS) FROM PENAEID SHRIMPS

2020 ◽  
Vol 67 (1) ◽  
Author(s):  
A. Angela Mercy ◽  
N. Hemamalini ◽  
K. Aruljothi ◽  
B. Chrisolite ◽  
K. Karalmarx
Keyword(s):  
Protein Quality ◽  
Transmembrane Protein ◽  
Homology Modelling ◽  
Three Dimensional ◽  
Chemical Properties ◽  
Receptor Protein ◽  
Dimensional Structure ◽  
Toll Like Receptors ◽  
Physico Chemical ◽  
Penaeid Shrimps

Toll or Toll-like receptors are conserved receptors, which act as the first line of defense against infection by pathogens. To use Toll/Toll like receptors as drug targets, it is essential to understand their physico-chemical properties and three dimensional structures. In the present study, physico-chemical properties and secondary structure of Toll/Toll like receptors from selected species of penaeid shrimps viz., Penaeus chinensis, P. vannamei, P. monodon and P. japonicus were computed using online servers. Three dimensional structure was predicted by homology modelling using different softwares, SWISS-MODEL, Phyre2 and Geno3D softwares were validated using online tools to find the best model for the protein under study. From the physicochemical properties, nature of the Toll/Toll like receptor protein was revealed as acidic, thermostable, hydrophobic and transmembrane protein. Structural analysis indicated the presence of alpha helices and random coils as predominant elements followed by extended stands and beta turns. Three dimensional structures predicted using SWISS-MODEL was validated as extremely good model using Protein Quality Predictor online server.

scholarly journals N- and C-Terminal Cooperation in Rotavirus Enterotoxin: Novel Mechanism of Modulation of the Properties of a Multifunctional Protein by a Structurally and Functionally Overlapping Conformational Domain

2006 ◽  
Vol 80 (1) ◽  
pp. 412-425 ◽  
Author(s):  
M. R. Jagannath ◽  
M. M. Kesavulu ◽  
R. Deepa ◽  
P. Narayan Sastri ◽  
S. Senthil Kumar ◽  
...  
Keyword(s):  
Nonstructural Protein ◽  
Three Dimensional ◽  
Binding Activity ◽  
Dimensional Structure ◽  
Newborn Mouse ◽  
Multifunctional Protein ◽  
C Terminus ◽  
N Terminus ◽  
Α Helix ◽  
Pleiotropic Properties

ABSTRACT Rotavirus NSP4 is a multifunctional endoplasmic reticulum (ER)-resident nonstructural protein with the N terminus anchored in the ER and about 131 amino acids (aa) of the C-terminal tail (CT) oriented in the cytoplasm. Previous studies showed a peptide spanning aa 114 to 135 to induce diarrhea in newborn mouse pups with the 50% diarrheal dose approximately 100-fold higher than that for the full-length protein, suggesting a role for other regions in the protein in potentiating its diarrhea-inducing ability. In this report, employing a large number of methods and deletion and amino acid substitution mutants, we provide evidence for the cooperation between the extreme C terminus and a putative amphipathic α-helix located between aa 73 and 85 (AAH73-85) at the N terminus of ΔN72, a mutant that lacked the N-terminal 72 aa of nonstructural protein 4 (NSP4) from Hg18 and SA11. Cooperation between the two termini appears to generate a unique conformational state, specifically recognized by thioflavin T, that promoted efficient multimerization of the oligomer into high-molecular-mass soluble complexes and dramatically enhanced resistance against trypsin digestion, enterotoxin activity of the diarrhea-inducing region (DIR), and double-layered particle-binding activity of the protein. Mutations in either the C terminus, AAH73-85, or the DIR resulted in severely compromised biological functions, suggesting that the properties of NSP4 are subject to modulation by a single and/or overlapping highly sensitive conformational domain that appears to encompass the entire CT. Our results provide for the first time, in the absence of a three-dimensional structure, a unique conformation-dependent mechanism for understanding the NSP4-mediated pleiotropic properties including virus virulence and morphogenesis.

scholarly journals Deep Homology-Based Protein Contact-Map Prediction

2020 ◽  
Author(s):  
Omer Ronen ◽  
Or Zuk
Keyword(s):  
Fundamental Problem ◽  
Homology Modelling ◽  
Three Dimensional ◽  
Amino Acid Sequences ◽  
Physical Contact ◽  
Dimensional Structure ◽  
Evolutionary Information ◽  
Contact Map ◽  
Sequence Alignments ◽  
Three Dimensional Structure

AbstractPrediction of Proteins’ three dimensional structure and their contact maps from their amino-acid sequences is a fundamental problem in structural computational biology. The structure and contacts shed light on protein function, enhance our basic understanding of their molecular biology and may potentially aid in drug design. In recent years we have seen significant progress in protein contact map prediction from Multiple Sequence Alignments (MSA) of the target protein and its homologous, using signals of co-evolution and applying deep learning methods.Homology modelling is a popular and successful approach, where the structure of a protein is determined using information from known template structures of similar proteins, and has been shown to improve prediction even in cases of low sequence identity. Motivated by these observations, we developed Periscope, a method for homology-assisted contact map prediction using a deep convolutional network. Our method automatically integrates the co-evolutionary information from the MSA, and the physical contact information from the template structures.We apply our method to families of CAMEO and membrane proteins, and show improved prediction accuracy compared to the MSA-only based method RaptorX. Finally, we use our method to improve the subsequent task of predicting the proteins’ three dimensional structure based on the (improved) predicted contact map, and show initial promising results in this task too - our overall accuracy is comparable to the template-based Modeller software, yet the two methods are complementary and succeed on different targets.

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