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

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.

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Conformation and membrane interaction studies of the potent antimicrobial and anticancer peptide palustrin-Ca

10.1101/2021.07.22.453375 ◽

2021 ◽

Author(s):

Patrick Brendan Timmons ◽

Chandralal M Hewage

Keyword(s):

Sodium Dodecyl ◽

Three Dimensional ◽

Dynamics Simulation ◽

Peptide Sequence ◽

Helical Conformation ◽

Dimensional Structure ◽

Amino Acid Residues ◽

Anticancer Activities ◽

American Bullfrog ◽

Α Helix

Palustrin-Ca (GFLDIIKDTGKEFAVKILNNLKCKLAGGCPP) is a host defense peptide with potent antimicrobial and anticancer activities, first isolated from the skin of the American bullfrog Lithobates catesbeianus. The peptide is 31 amino acid residues long, cationic and amphipathic. Two-dimensional NMR spectroscopy was employed to characterise its three-dimensional structure in a 50/50% water/2,2,2-trifluoroethanol-d3 mixture. The structure is defined by an α-helix that spans between Ile6-Ala26, and a cyclic disulphide bridged domain at the C-terminal end of the peptide sequence, between residues 23 and 29. A molecular dynamics simulation was employed to model the peptide's interactions with sodium dodecyl sulphate micelles, a widely used bacterial membrane-mimicking environment. Throughout the simulation, the peptide was found to maintain its α-helical conformation between residues Ile6-Ala26, while adopting a position parallel to the surface to micelle, which is energetically-favourable due to many hydrophobic and electrostatic contacts with the micelle.

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Structural characterization of a Thorarchaeota profilin indicates eukaryotic-like features but with an extended N-terminus

10.1101/2021.08.06.455371 ◽

2021 ◽

Author(s):

Celestine N Chi ◽

Ravi Teja Inturi ◽

Sandra Martinez Lara ◽

Mahmoud Darweesh

Keyword(s):

Common Ancestor ◽

Three Dimensional ◽

Eukaryotic Cell ◽

Dimensional Structure ◽

Resonance Spectroscopy ◽

Last Eukaryotic Common Ancestor ◽

Rigid Core ◽

N Terminus ◽

Metagenomics Analysis

The emergence of the first eukaryotic cell was preceded by evolutionary events which are still highly debatable. Recently, comprehensive metagenomics analysis has uncovered that the Asgard super-phylum is the closest yet known archaea host of eukaryotes. However, it remains to be established if a large number of eukaryotic signature proteins predicated to be encoded by the Asgard super-phylum are functional at least, in the context of a eukaryotic cell. Here, we determined the three-dimensional structure of profilin from Thorarchaeota by nuclear magnetic resonance spectroscopy and show that this profilin has a rigid core with a flexible N-terminus which was previously implicated in polyproline binding. In addition, we also show that thorProfilin co-localizes with eukaryotic actin in cultured HeLa cells. This finding reaffirm the notion that Asgardean encoded proteins possess eukaryotic-like characteristics and strengthen likely existence of a complex cytoskeleton already in a last eukaryotic common ancestor

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Improved scFv Anti-LOX-1 Binding Activity by Fusion with LOX-1-Binding Peptides

BioMed Research International ◽

10.1155/2017/8946935 ◽

2017 ◽

Vol 2017 ◽

pp. 1-9

Author(s):

Wei Hu ◽

Qiuhong Xie ◽

Hongyu Xiang

Keyword(s):

Targeted Delivery ◽

Structural Characteristics ◽

Low Density Lipoprotein ◽

Three Dimensional ◽

Density Lipoprotein ◽

Binding Activity ◽

Single Chain ◽

C Terminus ◽

Density Lipoprotein Receptor ◽

Therapeutic Molecules

The oxidized low-density lipoprotein receptor-1 (LOX-1) targeted single-chain variable fragment (scFvs) is a promising molecule for the targeted delivery of imaging and therapeutic molecules of atherosclerotic diseases; however, its applications are limited by the inherent low antigen affinity. In this study, the three-dimensional (3D) model of the anti-LOX-1 scFv was constructed and its docking with the LOX-1 protein was developed. To improve the LOX-1-binding activity, the anti-LOX-1 scFv was designed to fuse with one of three LOX-1-binding heptapeptides, LTPATAI, FQTPPQL, and LSIPPKA, at its N-terminus and C-terminus and in the linker region, which have different LOX-1-binding interfaces with the anti-LOX-1 scFv analyzed by an array of computational approaches. These scFv/peptide fusions were constructed, successfully expressed in Brevibacillus choshinensis hosts, and purified by a two-step column purification process. The antigen binding activity, structural characteristics, thermal stability, and stability in serum of these fusion proteins were examined. Results showed that the scFv with N-terminal fusing peptides proteins demonstrated increased LOX-1-binding activity without decrease in stability. These findings will help increase the application efficacy of LOX-1 targeting scFv in LOX-1-based therapy.

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Evolutionary conservation of the intrinsic disorder-based Radical-Induced Cell Death1 hub interactome

Scientific Reports ◽

10.1038/s41598-019-55385-3 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Lise Friis Christensen ◽

Lasse Staby ◽

Katrine Bugge ◽

Charlotte O’Shea ◽

Birthe B. Kragelund ◽

...

Keyword(s):

Transcription Factor ◽

Stress Responses ◽

Plant Stress ◽

Three Dimensional ◽

Intrinsic Disorder ◽

Dimensional Structure ◽

Linear Motif ◽

Intrinsically Disordered ◽

Helix Formation ◽

Α Helix

AbstractRadical-Induced Cell Death1 (RCD1) functions as a cellular hub interacting with intrinsically disordered transcription factor regions, which lack a well-defined three-dimensional structure, to regulate plant stress. Here, we address the molecular evolution of the RCD1-interactome. Using bioinformatics, its history was traced back more than 480 million years to the emergence of land plants with the RCD1-binding short linear motif (SLiM) identified from mosses to flowering plants. SLiM variants were biophysically verified to be functional and to depend on the same RCD1 residues as the DREB2A transcription factor. Based on this, numerous additional members may be assigned to the RCD1-interactome. Conservation was further strengthened by similar intrinsic disorder profiles of the transcription factor homologs. The unique structural plasticity of the RCD1-interactome, with RCD1-binding induced α-helix formation in DREB2A, but not detectable in ANAC046 or ANAC013, is apparently conserved. Thermodynamic analysis also indicated conservation with interchangeability between Arabidopsis and soybean RCD1 and DREB2A, although with fine-tuned co-evolved binding interfaces. Interruption of conservation was observed, as moss DREB2 lacked the SLiM, likely reflecting differences in plant stress responses. This whole-interactome study uncovers principles of the evolution of SLiM:hub-interactions, such as conservation of α-helix propensities, which may be paradigmatic for disorder-based interactomes in eukaryotes.

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Ribokinase fromLeishmania donovani: purification, characterization and X-ray crystallographic analysis

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x18000109 ◽

2018 ◽

Vol 74 (2) ◽

pp. 99-104 ◽

Cited By ~ 1

Author(s):

Santhosh Gatreddi ◽

Sayanna Are ◽

Insaf Ahmed Qureshi

Keyword(s):

Functional Characterization ◽

Three Dimensional ◽

Protozoan Parasite ◽

Crystallographic Analysis ◽

Size Exclusion ◽

Dimensional Structure ◽

Diffusion Method ◽

Gene Encoding ◽

Cell Parameters ◽

Α Helix

Leishmaniais an auxotrophic protozoan parasite which acquires D-ribose by transporting it from the host cell and also by the hydrolysis of nucleosides. The enzyme ribokinase (RK) catalyzes the first step of ribose metabolism by phosphorylating D-ribose using ATP to produce D-ribose-5-phosphate. To understand its structure and function, the gene encoding RK fromL. donovaniwas cloned, expressed and purified using affinity and size-exclusion chromatography. Circular-dichroism spectroscopy of the purified protein showed comparatively more α-helix in the secondary-structure content, and thermal unfolding revealed theTmto be 317.2 K. Kinetic parameters were obtained by functional characterization ofL. donovaniRK, and theKmvalues for ribose and ATP were found to be 296 ± 36 and 116 ± 9.0 µM, respectively. Crystals obtained by the hanging-drop vapour-diffusion method diffracted to 1.95 Å resolution and belonged to the hexagonal space groupP61, with unit-cell parametersa=b= 100.25,c= 126.77 Å. Analysis of the crystal content indicated the presence of two protomers in the asymmetric unit, with a Matthews coefficient (VM) of 2.45 Å3 Da−1and 49.8% solvent content. Further study revealed that human counterpart of this protein could be used as a template to determine the first three-dimensional structure of the RK from trypanosomatid parasites.

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Zona Pellucida Proteins, Fibrils, and Matrix

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-011520-105310 ◽

2020 ◽

Vol 89 (1) ◽

pp. 695-715

Author(s):

Eveline S. Litscher ◽

Paul M. Wassarman

Keyword(s):

Extracellular Matrix ◽

Zona Pellucida ◽

Three Dimensional ◽

Structural Features ◽

Biological Properties ◽

Preimplantation Development ◽

Dimensional Structure ◽

Three Dimensional Structure ◽

Early Embryos ◽

N Terminus

The zona pellucida (ZP) is an extracellular matrix that surrounds all mammalian oocytes, eggs, and early embryos and plays vital roles during oogenesis, fertilization, and preimplantation development. The ZP is composed of three or four glycosylated proteins, ZP1–4, that are synthesized, processed, secreted, and assembled into long, cross-linked fibrils by growing oocytes. ZP proteins have an immunoglobulin-like three-dimensional structure and a ZP domain that consists of two subdomains, ZP-N and ZP-C, with ZP-N of ZP2 and ZP3 required for fibril assembly. A ZP2–ZP3 dimer is located periodically along ZP fibrils that are cross-linked by ZP1, a protein with a proline-rich N terminus. Fibrils in the inner and outer regions of the ZP are oriented perpendicular and parallel to the oolemma, respectively, giving the ZP a multilayered appearance. Upon fertilization of eggs, modification of ZP2 and ZP3 results in changes in the ZP's physical and biological properties that have important consequences. Certain structural features of ZP proteins suggest that they may be amyloid-like proteins.

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Structural modeling and role of HAX-1 as a positive allosteric modulator of human serine protease HtrA2

Biochemical Journal ◽

10.1042/bcj20190569 ◽

2019 ◽

Vol 476 (20) ◽

pp. 2965-2980

Author(s):

Lalith K. Chaganti ◽

Shubhankar Dutta ◽

Raja Reddy Kuppili ◽

Mriganka Mandal ◽

Kakoli Bose

Keyword(s):

Pdz Domain ◽

Limited Proteolysis ◽

Three Dimensional ◽

Dimensional Structure ◽

Multifunctional Protein ◽

Promising Therapeutic Target ◽

Wide Range ◽

Structural Details ◽

Structural Architecture ◽

First Time

Abstract HAX-1, a multifunctional protein involved in cell proliferation, calcium homeostasis, and regulation of apoptosis, is a promising therapeutic target. It regulates apoptosis through multiple pathways, understanding of which is limited by the obscurity of its structural details and its intricate interaction with its cellular partners. Therefore, using computational modeling, biochemical, functional enzymology and spectroscopic tools, we predicted the structure of HAX-1 as well as delineated its interaction with one of it pro-apoptotic partner, HtrA2. In this study, three-dimensional structure of HAX-1 was predicted by threading and ab initio tools that were validated using limited proteolysis and fluorescence quenching studies. Our pull-down studies distinctly demonstrate that the interaction of HtrA2 with HAX-1 is directly through its protease domain and not via the conventional PDZ domain. Enzymology studies further depicted that HAX-1 acts as an allosteric activator of HtrA2. This ‘allosteric regulation’ offers promising opportunities for the specific control and functional modulation of a wide range of biological processes associated with HtrA2. Hence, this study for the first time dissects the structural architecture of HAX-1 and elucidates its role in PDZ-independent activation of HtrA2.

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Altered affinity of insulin-like growth factor II (IGF-II) for receptors and IGF-binding proteins, resulting from limited modifications of the IGF-II molecule

Biochemical Journal ◽

10.1042/bj2780249 ◽

1991 ◽

Vol 278 (1) ◽

pp. 249-254 ◽

Cited By ~ 7

Author(s):

Y Oh ◽

M W Beukers ◽

H M Pham ◽

P A Smanik ◽

M C Smith ◽

...

Keyword(s):

Amino Acids ◽

Three Dimensional ◽

Dimensional Structure ◽

Type I ◽

Type Ii ◽

Factor Ii ◽

N Terminus ◽

Severe Impairment ◽

Igf Binding ◽

Terminal Amino

The binding affinities of seven analogues of recombinant human insulin-like growth factor II (hIGF-II) were characterized for the IGF type-I and type-II receptors and insulin receptors, as well as for IGF-binding protein (IGFBP)-1, IGFBP-2, IGFPB-3 and human serum IGFBPs. A switch of two of the three cysteine bridges in hIGF-II, 9-47 and 46-51 to 9-46 and 47-51, severely impaired the binding of this analogue to all receptors and to the IGFBPs. The affinities for the IGF type-I receptor and the IGFBPs were decreased over 100-fold, while the binding to the insulin receptor and the IGF type-II receptor was less affected, with a 6-10-fold decrease in affinity. Slight modifications of the N-terminus had only minor effects upon the binding of hIGF-II to the IGFBPs or to the receptors. Deletion of both the N-terminal amino acid and the two C-terminal amino acids resulted in moderate decreases in affinity, with a 60% decrease in affinity for IGFBP-1 and the IGF type-I receptor. Acetylation of the N-terminus of Ala1 and the epsilon-nitrogen of Lys65 decreased the affinity, by 60-90%, of hIGF-II for all of the IGFBPs and receptors. The experiments involving acetylation of IGF-II or switching of its cysteine bridges indicated that these modifications (no substitution, deletion or addition of any of the 67 amino acids of hIGF-II) may lead to a severe impairment of the binding affinity of IGF-II for both the IGFBPs and the receptors. Acetylation of the epsilon-nitrogen of Lys65, which causes a charge change, or alteration of the three-dimensional structure, as shown by the cysteine bridge switch, lead to a severe impairment of the binding affinity for the binding proteins and for the receptors. In general, care should be taken with the synthesis of analogues and the interpretation of resulting binding data, since affinity alterations ascribed to amino acid changes may instead be caused by alterations of the charge or the three-dimensional structure of the protein.

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Unliganded GroEL at 2.8 Å: structure and functional implications

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1995.0052 ◽

1995 ◽

Vol 348 (1323) ◽

pp. 113-119 ◽

Cited By ~ 10

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.

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Solution structure of the strawberry allergen Fra a 1

Bioscience Reports ◽

10.1042/bsr20120058 ◽

2012 ◽

Vol 32 (6) ◽

pp. 567-575 ◽

Cited By ~ 14

Author(s):

Christian Seutter von Loetzen ◽

Kristian Schweimer ◽

Wilfried Schwab ◽

Paul Rösch ◽

Olivia Hartl-Spiegelhauer

Keyword(s):

Solution Structure ◽

Three Dimensional ◽

Protein Family ◽

Dimensional Structure ◽

Hydrophobic Pocket ◽

Pigment Synthesis ◽

Family Protein ◽

Pr10 Protein ◽

Α Helix

The PR10 family protein Fra a 1E from strawberry (Fragaria x ananassa) is down-regulated in white strawberry mutants, and transient RNAi (RNA interference)-mediated silencing experiments confirmed that Fra a 1 is involved in fruit pigment synthesis. In the present study, we determined the solution structure of Fra a 1E. The protein fold is identical with that of other members of the PR10 protein family and consists of a seven-stranded antiparallel β-sheet, two short V-shaped α-helices and a long C-terminal α-helix that encompass a hydrophobic pocket. Whereas Fra a 1E contains the glycine-rich loop that is highly conserved throughout the protein family, the volume of the hydrophobic pocket and the size of its entrance are much larger than expected. The three-dimensional structure may shed some light on its physiological function and may help to further understand the role of PR10 proteins in plants.

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