scholarly journals Ubiquitin-Mimicking Peptides Transfer Differentiates by E1 and E2 Enzymes

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Bo Jin ◽  
Jiayue Wang ◽  
Xiangnan Liu ◽  
Shuai Fang ◽  
Bo Jiang ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Short Peptides ◽  
Carrier Protein ◽  
Conserved Motif ◽  
Protein Databases ◽  
C Terminus ◽  
E2 Enzymes ◽  
Related Pathway ◽  
Mimicking Peptides

Ubiquitin and ubiquitin like proteins (UBLs) play key roles in eukaryotes. These proteins are attached to their target proteins through an E1-E2-E3 cascade and modify the functions of these proteins. Since the discovery of ubiquitin, several UBLs have been identified, including Nedd8, SUMO, ISG15, and Atg8. Ubiquitin and UBLs share a similar three-dimensional structure: β-grasp fold and an X-X-[R/A/E/K]-X-X-[G/X]-G motif at the C-terminus. We have previously reported that ubiquitin, Nedd8, and SUMO mimicking peptides which all contain the conserved motif X-X-[R/A/E/K]-X-X-[G/X]-G still retained their reactivity toward their corresponding E1, E2, and E3 enzymes. In our current study, we investigated whether such C-terminal peptides could still be transferred onto related pathway enzymes to probe the function of these enzymes when they are fused with a protein. By bioinformatic search of protein databases, we selected eight proteins carrying the X-X-[R/A/E/K]-X-X-[G/X]-G motif at the C-terminus of the β-grasp fold. We synthesized the C-terminal sequences of these candidates as short peptides and found that three of them showed significant reactivity with the ubiquitin E1 enzyme Ube1. We next fused the three reactive short peptides to three different protein frames, including their respective native protein frames, a ubiquitin frame and a peptidyl carrier protein (PCP) frame, and measured the reactivities of these peptide-fused proteins with Ube1. Peptide-fused proteins on ubiquitin and PCP frames showed obvious reactivity with Ube1. However, when we measured E2 UbcH7 transfer, we found that the PCP-peptide fusions lost their reactivity with UbcH7. Taken together, these results suggested that the recognition of E2 enzymes with peptide-fused proteins depended not only on the C-terminal sequences of the ubiquitin-mimicking peptides, but also on the overall structures of the protein frames.

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 The Three-Dimensional Structure of the Biotin Carboxylase-Biotin Carboxyl Carrier Protein Complex of E. coli Acetyl-CoA Carboxylase

Structure ◽  
2013 ◽  
Vol 21 (4) ◽  
pp. 650-657 ◽  
Author(s):  
Tyler C. Broussard ◽  
Matthew J. Kobe ◽  
Svetlana Pakhomova ◽  
David B. Neau ◽  
Amanda E. Price ◽  
...  
Keyword(s):  
Protein Complex ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Carrier Protein ◽  
Biotin Carboxylase ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Biotin Carboxyl Carrier Protein ◽  
Three Dimensional Structure ◽  
E Coli

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 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 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 Crystallization and preliminary crystallographic characterization of the PAS domains of EAG and ELK potassium channels

2010 ◽  
Vol 66 (9) ◽  
pp. 1056-1059 ◽  
Author(s):  
Ricardo Adaixo ◽  
João Henrique Morais-Cabral
Keyword(s):  
Potassium Channels ◽  
Preliminary Analysis ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
C Terminus ◽  
Acid Protein ◽  
Low Degree ◽  
Regulatory Functions ◽  
Amino Acid Protein

Per–Arnt–Sim (PAS) domains are ubiquitous in nature; they are ∼130-amino-acid protein domains that adopt a fairly conserved three-dimensional structure despite their low degree of sequence homology. These domains constitute the N-terminus or, less frequently, the C-terminus of a number of proteins, where they exert regulatory functions. PAS-containing proteins generally display two or more copies of this motif. In this work, the crystallization and preliminary analysis of the PAS domains of two eukaryotic potassium channels from the ether-à-go-go (EAG) family are reported.

Structure of the prolyl-acyl carrier protein oxidase involved in the biosynthesis of the cyanotoxin anatoxin-a

2013 ◽  
Vol 69 (12) ◽  
pp. 2340-2352 ◽  
Author(s):  
Karine Moncoq ◽  
Leslie Regad ◽  
Stéphane Mann ◽  
Annick Méjean ◽  
Olivier Ploux
Keyword(s):  
Molecular Oxygen ◽  
Acyl Carrier Protein ◽  
Three Dimensional ◽  
Accessible Surface Area ◽  
Polyketide Synthases ◽  
Site Directed Mutagenesis ◽  
Solvent Accessible Surface Area ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Carrier Protein

Anatoxin-a and homoanatoxin-a are two potent cyanobacterial neurotoxins biosynthesized from L-proline by a short pathway involving polyketide synthases. Proline is first loaded onto AnaD, an acyl carrier protein, and prolyl-AnaD is then oxidized to 1-pyrroline-5-carboxyl-AnaD by a flavoprotein, AnaB. Three polyketide synthases then transform this imine into anatoxin-a or homoanatoxin-a. AnaB was crystallized in its holo form and its three-dimensional structure was determined by X-ray diffraction at 2.8 Å resolution. AnaB is a homotetramer and its fold is very similar to that of the acyl-CoA dehydrogenases (ACADs). The active-site base of AnaB, Glu244, superimposed very well with that of human isovaleryl-CoA dehydrogenase, confirming previous site-directed mutagenesis experiments and mechanistic proposals. The substrate-binding site of AnaB is small and is likely to be fitted for the pyrrolidine ring of proline. However, in contrast to ACADs, which use an electron-transport protein, AnaB uses molecular oxygen as the electron acceptor, as in acyl-CoA oxidases. Calculation of the solvent-accessible surface area around the FAD in AnaB and in several homologues showed that it is significantly larger in AnaB than in its homologues. A protonated histidine near the FAD in AnaB is likely to participate in oxygen activation. Furthermore, an array of water molecules detected in the AnaB structure suggests a possible path for molecular oxygen towards FAD. This is consistent with AnaB being an oxidase rather than a dehydrogenase. The structure of AnaB is the first to be described for a prolyl-ACP oxidase and it will contribute to defining the structural basis responsible for oxygen reactivity in flavoenzymes.

scholarly journals Structure of theBacillus anthracisdTDP-L-rhamnose-biosynthetic enzyme glucose-1-phosphate thymidylyltransferase (RfbA)

2017 ◽  
Vol 73 (11) ◽  
pp. 621-628 ◽  
Author(s):  
Jackson Baumgartner ◽  
Jesi Lee ◽  
Andrei S. Halavaty ◽  
George Minasov ◽  
Wayne F. Anderson ◽  
...  
Keyword(s):  
Drug Targets ◽  
Quaternary Structure ◽  
Structural Characteristics ◽  
Enzymatic Reaction ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Site Formation ◽  
Allosteric Site ◽  
C Terminus ◽  
Potential Drug Targets

L-Rhamnose is a ubiquitous bacterial cell-wall component. The biosynthetic pathway for its precursor dTDP-L-rhamnose is not present in humans, which makes the enzymes of the pathway potential drug targets. In this study, the three-dimensional structure of the first protein of this pathway, glucose-1-phosphate thymidylyltransferase (RfbA), fromBacillus anthraciswas determined. In other organisms this enzyme is referred to as RmlA. RfbA was co-crystallized with the products of the enzymatic reaction, dTDP-α-D-glucose and pyrophosphate, and its structure was determined at 2.3 Å resolution. This is the first reported thymidylyltransferase structure from a Gram-positive bacterium. RfbA shares overall structural characteristics with known RmlA homologs. However, RfbA exhibits a shorter sequence at its C-terminus, which results in the absence of three α-helices involved in allosteric site formation. Consequently, RfbA was observed to exhibit a quaternary structure that is unique among currently reported glucose-1-phosphate thymidylyltransferase bacterial homologs. These structural analyses suggest that RfbA may not be allosterically regulated in some organisms and is structurally distinct from other RmlA homologs.

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