scholarly journals Characterization of microbial antifreeze protein with intermediate activity suggests that a bound-water network is essential for hyperactivity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
N. M.-Mofiz Uddin Khan ◽  
Tatsuya Arai ◽  
Sakae Tsuda ◽  
Hidemasa Kondo
Keyword(s):  
Crystal Structure ◽  
Basal Plane ◽  
Molecular Mechanisms ◽  
Bound Water ◽  
Hydrophobic Hydration ◽  
Water Network ◽  
Molecular Scaffold ◽  
Intermediate Activity ◽  
Mold Fungus ◽  
Antifreeze Activity

AbstractAntifreeze proteins (AFPs) inhibit ice growth by adsorbing onto specific ice planes. Microbial AFPs show diverse antifreeze activity and ice plane specificity, while sharing a common molecular scaffold. To probe the molecular mechanisms responsible for AFP activity, we here characterized the antifreeze activity and crystal structure of TisAFP7 from the snow mold fungus Typhula ishikariensis. TisAFP7 exhibited intermediate activity, with the ability to bind the basal plane, compared with a hyperactive isoform TisAFP8 and a moderately active isoform TisAFP6. Analysis of the TisAFP7 crystal structure revealed a bound-water network arranged in a zigzag pattern on the surface of the protein’s ice-binding site (IBS). While the three AFP isoforms shared the water network pattern, the network on TisAFP7 IBS was not extensive, which was likely related to its intermediate activity. Analysis of the TisAFP7 crystal structure also revealed the presence of additional water molecules that form a ring-like network surrounding the hydrophobic side chain of a crucial IBS phenylalanine, which might be responsible for the increased adsorption of AFP molecule onto the basal plane. Based on these observations, we propose that the extended water network and hydrophobic hydration at IBS together determine the TisAFP activity.

scholarly journals Characterization of microbial antifreeze protein with intermediate activity suggests that a bound-water network is essential for hyperactivity

2020 ◽  
Author(s):  
N. M.-Mofiz Khan ◽  
Tatsuya Arai ◽  
Sakae Tsuda ◽  
Hidemasa Kondo
Keyword(s):  
Crystal Structure ◽  
Basal Plane ◽  
Molecular Mechanisms ◽  
Bound Water ◽  
Hydrophobic Hydration ◽  
Water Network ◽  
Molecular Scaffold ◽  
Intermediate Activity ◽  
Mold Fungus ◽  
Antifreeze Activity

Abstract Antifreeze proteins (AFPs) inhibit ice growth by adsorbing onto a specific ice plane. Microbial AFPs show diverse antifreeze activity and ice plane specificity, while sharing a common molecular scaffold. To probe the molecular mechanisms responsible for AFP activity, we here characterized the antifreeze activity and crystal structure of TisAFP7 from the snow mold fungus Typhula ishikariensis. TisAFP7 exhibited intermediate activity, with the ability to bind ice basal plane, compared with a hyperactive isoform TisAFP8 and a moderately active isoform TisAFP6. Analysis of the TisAFP7 crystal structure revealed a bound-water network arranged in a zigzag pattern on the surface of the protein’s ice-binding site (IBS). While the three AFP isoforms shared the water network pattern, the network on TisAFP7 IBS was not extensive, which was likely related to its intermediate activity. Analysis of the TisAFP7 crystal structure also revealed the presence of additional water molecules that form a ring-like network surrounding the hydrophobic side chain of a crucial IBS phenylalanine, which might be responsible for the increased adsorption of AFP molecule onto the basal plane. Based on these observations, we propose that the extended water network and hydrophobic hydration at IBS together determine the TisAFP activity.

scholarly journals Elucidating the Molecular Mechanisms for the Interaction of Water with Polyethylene Glycol-Based Hydrogels: Influence of Ionic Strength and Gel Network Structure

Polymers ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 845
Author(s):  
Xin Yang ◽  
Bronwin Dargaville ◽  
Dietmar Hutmacher
Keyword(s):  
Polyethylene Glycol ◽  
Ionic Strength ◽  
Molecular Mechanisms ◽  
Repeat Unit ◽  
Bound Water ◽  
Weight Fraction ◽  
Swelling Ratio ◽  
Polyethylene Glycol Diacrylate ◽  
Glycol Diacrylate ◽  
Hydrogel System

The interaction of water within synthetic and natural hydrogel systems is of fundamental importance in biomaterial science. A systematic study is presented on the swelling behavior and states of water for a polyethylene glycol-diacrylate (PEGDA)-based model neutral hydrogel system that goes beyond previous studies reported in the literature. Hydrogels with different network structures are crosslinked and swollen in different combinations of water and phosphate-buffered saline (PBS). Network variables, polyethylene glycol (PEG) molecular weight (MW), and weight fraction are positively correlated with swelling ratio, while “non-freezable bound water” content decreases with PEG MW. The presence of ions has the greatest influence on equilibrium water and “freezable” and “non-freezable” water, with all hydrogel formulations showing a decreased swelling ratio and increased bound water as ionic strength increases. Similarly, the number of “non-freezable bound water” molecules, calculated from DSC data, is greatest—up to six molecules per PEG repeat unit—for gels swollen in PBS. Fundamentally, the balance of osmotic pressure and non-covalent bonding is a major factor within the molecular structure of the hydrogel system. The proposed model explains the dynamic interaction of water within hydrogels in an osmotic environment. This study will point toward a better understanding of the molecular nature of the water interface in hydrogels.

scholarly journals Positive and negative intra-molecular modulation in a dual-cassette RNA helicase

2019 ◽  
Author(s):  
Karen Vester ◽  
Karine F. Santos ◽  
Benno Kuropka ◽  
Christoph Weise ◽  
Markus C. Wahl
Keyword(s):  
Crystal Structure ◽  
Molecular Mechanisms ◽  
Atp Hydrolysis ◽  
Rna Helicase ◽  
Negative Influence ◽  
Disulfide Bridges ◽  
Relative Positioning ◽  
Distinct Subgroup ◽  
Terminal Unit ◽  
Molecular Modulation

ABSTRACTRNA helicase Brr2 is required for the activation of the spliceosome prior to the first catalytic step of splicing. Brr2 represents a distinct subgroup of Ski2-like nucleic acid helicases whose members comprise tandem helicase cassettes. Only the N-terminal cassette of Brr2 is an active ATPase and can unwind substrate RNAs. The C-terminal cassette represents a pseudo-enzyme that can stimulate RNA-related activities of the N-terminal cassette. However, the molecular mechanisms, by which the C-terminal cassette modulates the activities of the N-terminal unit remain elusive. Here, we show that N- and C-terminal cassettes adopt vastly different relative orientations in a crystal structure of Brr2 in complex with an activating domain of the spliceosomal Prp8 protein as compared to the crystal structure of isolated Brr2. Likewise, the cassettes occupy different relative positions and engage in different inter-cassette contacts during different stages of splicing. Engineered disulfide bridges that lock the cassettes in two different relative orientations have opposite effects on RNA-related activities of the N-terminal cassette compared to the unrestrained protein. Moreover, different relative positioning of the cassettes strongly influences ATP hydrolysis by the N-terminal cassette. Our results demonstrate that the inactive C-terminal cassette of Brr2 can exert both positive and negative influence on the active N-terminal helicase unit from a distance.

scholarly journals Crystal structure of (2-formylphenolato-κ2O,O′)oxido(2-{[(2-oxidoethyl)imino]methyl}phenolato-κ3O,N,O′)vanadium(V)

2015 ◽  
Vol 71 (5) ◽  
pp. m104-m105
Author(s):  
Sowmianarayanan Parimala ◽  
Parasuraman Selvam
Keyword(s):  
Crystal Structure ◽  
Metal Atom ◽  
Basal Plane ◽  
Octahedral Geometry ◽  
Distorted Octahedral Geometry ◽  
Interplanar Angle ◽  
Vanadyl Complex ◽  
Distorted Octahedral ◽  
Benzene Rings ◽  
Axial Bond

In the unsymmetrical title vanadyl complex, [V(C9H9NO2)(C7H5O2)O], one of the ligands (2-formylphenol) is disordered over two sets of sites, with an occupancy ratio of 0.55 (2):0.45 (2). The metal atom is hexacoordinated, with a distorted octahedral geometry. The vanadyl O atom (which subtends the shortest V—O bond) occupies one of the apical positions and the remaining axial bond (the longest in the polyhedron) is provided by the (disordered) formyl O atoms. The basal plane is defined by the two phenoxide O atoms, the iminoalcoholic O and the imino N atom. The planes of the two benzene rings are almost perpendicular to each other, subtending an interplanar angle of 84.1 (2)° between the major parts. The crystal structure features weak C—H...O and C—H...π interactions, forming a lateral arrangement of adjacent molecules.

Crystal structure of the C-terminal domain of envelope protein VP37 from white spot syndrome virus reveals sulphate binding sites responsible for heparin binding

2021 ◽  
Vol 102 (6) ◽  
Author(s):  
Wasusit Somsoros ◽  
Takeshi Sangawa ◽  
Katsuki Takebe ◽  
Jakrada Attarataya ◽  
Kanokpan Wongprasert ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Sites ◽  
Envelope Protein ◽  
Molecular Mechanisms ◽  
White Spot Syndrome Virus ◽  
Significant Loss ◽  
White Spot ◽  
Heparin Binding ◽  
Terminal Domain ◽  
White Spot Syndrome

White spot syndrome virus (WSSV) is the most virulent pathogen causing high mortality and economic loss in shrimp aquaculture and various crustaceans. Therefore, the understanding of molecular mechanisms of WSSV infection is important to develop effective therapeutics to control the spread of this viral disease. In a previous study, we found that VP37 could bind with shrimp haemocytes through the interaction between its C-terminal domain and heparin-like molecules on the shrimp cells, and this interaction can also be inhibited by sulphated galactan. In this study, we present the crystal structure of C-terminal domain of VP37 from WSSV at a resolution of 2.51 Å. The crystal structure contains an eight-stranded β-barrel fold with an antiparallel arrangement and reveals a trimeric assembly. Moreover, there are two sulphate binding sites found in the position corresponding to R213 and K257. In order to determine whether these sulphate binding sites are involved in binding of VP37 to heparin, mutagenesis was performed to replace these residues with alanine (R213A and K257A), and the Surface Plasmon Resonance (SPR) system was used to study the interaction of each mutated VP37 with heparin. The results showed that mutants R213A and K257A exhibited a significant loss in heparin binding activity. These findings indicated that the sites of R213 and K257 on the C-terminal domain of envelope protein VP37 are essential for binding to sulphate molecules of heparin. This study provides further insight into the structure of C-terminal domain of VP37 and it is anticipated that the structure of VP37 might be used as a guideline for development of antivirus agent targeting on the VP37 protein.

Abstract 304: Crystal Structure Of Fak/mef2 Complex Reveals The Role Of Fak In The Regulation Of Mef2 Transcription Factor.

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Alisson C Cardoso ◽  
Ana H Pereira ◽  
Andre L Ambrosio ◽  
Silvio R Consonni ◽  
Sandra M Dias ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mechanical Stress ◽  
Focal Adhesion ◽  
Molecular Mechanisms ◽  
Structural Information ◽  
Mechanistic Model ◽  
Focal Adhesions ◽  
Saxs Data ◽  
X Ray ◽  
Gene Assay

Members of MEF2 (Myocyte Enhancer Factor 2) family of transcription factors are major regulators of cardiac development and homeostasis. Their functions are regulated at several levels, including the association with a variety of protein partners. We have previously shown that FAK (Focal Adhesion Kinase) regulates the stretch-induced activation of MEF2 in cardiomyocytes. But, the molecular mechanisms, involved in this process, are unclear. Here, we integrated biochemical, imaging and structural analyses to characterize a novel interaction between MEF2 and FAK. An association between MEF2 and FAK was detected by co-immunoprecipitation in the extracts of stretched cardiomyocytes (10%, 60Hz, 2 hours). MEF2 and FAK staining were co-localized in the nuclei of stretched cells. Pull down assays indicated that the Focal Adhesion Targeting (FAT) domain is sufficient to confer FAK interaction with MEF2. Gene reporter assays indicated that the interaction with FAK enhances the MEF2C transcriptional activity in cultured cardiomyocytes. Also, we present a 2.9-Å X-ray crystal structure for the FAK_FAT domain bound to MEF2C (1-95), comprised by the MADS box/MEF2 domain. The structural information, when used in combination with biochemical studies, small-angle X-ray scattering (SAXS) data and reporter gene assay, lead to a mechanistic model describing how FAK binds to MEF2C and stimulates its transcription function in cardiomyocytes. We further validated this model by showing that the binding of FAK to MEF2C is essential for the hypertrophy of cardiomyocyte in response to mechanical stress. Our results present FAK as a new positive regulator of MEF2, implicated in the fine control of the signal transduction between focal adhesions and the nucleus of cardiac myocytes during mechanical stress.

Crystal Structure and Magnetic Properties of (2,2'-Dipyridyl)-(2-acetylphenolato)copper(II) Perchlorate

2002 ◽  
Vol 57 (10) ◽  
pp. 1129-1132 ◽  
Author(s):  
A. Elmali ◽  
Y. Elerman ◽  
I. Svoboda
Keyword(s):  
Crystal Structure ◽  
Magnetic Properties ◽  
Magnetic Susceptibility ◽  
Magnetic Moment ◽  
Basal Plane ◽  
Effective Magnetic Moment ◽  
Mixed Ligand ◽  
Magnetic Susceptibility Data ◽  
Susceptibility Data ◽  
Dimeric Unit

The mixed-ligand dinuclear complex (2,2'-dipyridyl)-(2-acetylphenolato)copper(II) perchlorate was synthesized and its crystal structures determined. The structure consists of a dimeric unit involving a planar Cu2O2 group. The coordination sphere of the Cu atom can be described as an alongated octahedron where the basal plane is formed by the two N atoms of the 2,2'-dipyridyl molecule and the two O atoms of the acetophenon anion. Two apical Cu - O contacts complete the 4+2 coordination of the Cu atoms. They correspond to one of the O atoms of the perchlorate anion and to the O atom of the second unit. Magnetic susceptibility data obey the Curie-Weiss law with θ = -8.1(2) K. The decreasing of the effective magnetic moment from 1.94(8) μB at 300 K to 1.86(8) μB at 70 K and the negative Weiss constant indicate weak antiferromagnetic interactions between the two copper atoms in the dimeric units.

scholarly journals The dissociation mechanism of processive cellulases

2019 ◽  
Vol 116 (46) ◽  
pp. 23061-23067 ◽  
Author(s):  
Josh V. Vermaas ◽  
Riin Kont ◽  
Gregg T. Beckham ◽  
Michael F. Crowley ◽  
Mikael Gudmundsson ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Molecular Mechanisms ◽  
Soluble Sugars ◽  
Dissociation Rate ◽  
Replica Exchange ◽  
Wild Type ◽  
Replica Exchange Molecular Dynamics ◽  
Hamiltonian Replica Exchange ◽  
Biochemical Measurements ◽  
Rate Limiting

Cellulase enzymes deconstruct recalcitrant cellulose into soluble sugars, making them a biocatalyst of biotechnological interest for use in the nascent lignocellulosic bioeconomy. Cellobiohydrolases (CBHs) are cellulases capable of liberating many sugar molecules in a processive manner without dissociating from the substrate. Within the complete processive cycle of CBHs, dissociation from the cellulose substrate is rate limiting, but the molecular mechanism of this step is unknown. Here, we present a direct comparison of potential molecular mechanisms for dissociation via Hamiltonian replica exchange molecular dynamics of the model fungal CBH, Trichoderma reesei Cel7A. Computational rate estimates indicate that stepwise cellulose dethreading from the binding tunnel is 4 orders of magnitude faster than a clamshell mechanism, in which the substrate-enclosing loops open and release the substrate without reversing. We also present the crystal structure of a disulfide variant that covalently links substrate-enclosing loops on either side of the substrate-binding tunnel, which constitutes a CBH that can only dissociate via stepwise dethreading. Biochemical measurements indicate that this variant has a dissociation rate constant essentially equivalent to the wild type, implying that dethreading is likely the predominant mechanism for dissociation.

scholarly journals Structural and functional studies of Arabidopsis thaliana legumain beta reveal isoform specific mechanisms of activation and substrate recognition

2020 ◽  
Vol 295 (37) ◽  
pp. 13047-13064 ◽  
Author(s):  
Elfriede Dall ◽  
Florian B. Zauner ◽  
Wai Tuck Soh ◽  
Fatih Demir ◽  
Sven O. Dahms ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Arabidopsis Thaliana ◽  
Cyclic Peptides ◽  
Molecular Mechanisms ◽  
Functional Studies ◽  
Plant Characteristic ◽  
Activation Mechanisms ◽  
Binding Pockets ◽  
First Time ◽  
Autocatalytic Activation

The vacuolar cysteine protease legumain plays important functions in seed maturation and plant programmed cell death. Because of their dual protease and ligase activity, plant legumains have become of particular biotechnological interest, e.g. for the synthesis of cyclic peptides for drug design or for protein engineering. However, the molecular mechanisms behind their dual protease and ligase activities are still poorly understood, limiting their applications. Here, we present the crystal structure of Arabidopsis thaliana legumain isoform β (AtLEGβ) in its zymogen state. Combining structural and biochemical experiments, we show for the first time that plant legumains encode distinct, isoform-specific activation mechanisms. Whereas the autocatalytic activation of isoform γ (AtLEGγ) is controlled by the latency-conferring dimer state, the activation of the monomeric AtLEGβ is concentration independent. Additionally, in AtLEGβ the plant-characteristic two-chain intermediate state is stabilized by hydrophobic rather than ionic interactions, as in AtLEGγ, resulting in significantly different pH stability profiles. The crystal structure of AtLEGβ revealed unrestricted nonprime substrate binding pockets, consistent with the broad substrate specificity, as determined by degradomic assays. Further to its protease activity, we show that AtLEGβ exhibits a true peptide ligase activity. Whereas cleavage-dependent transpeptidase activity has been reported for other plant legumains, AtLEGβ is the first example of a plant legumain capable of linking free termini. The discovery of these isoform-specific differences will allow us to identify and rationally design efficient ligases with application in biotechnology and drug development.

scholarly journals Crystal structure of {2,2′-[ethylenebis(nitrilomethanylylidene)]diphenolato-κ4O,N,N′,O′}oxidovanadium(IV) methanol monosolvate

2014 ◽  
Vol 70 (11) ◽  
pp. m380-m381
Author(s):  
Rachel E. Hsuan ◽  
Jemma E. Hughes ◽  
Thomas H. Miller ◽  
Nabila Shaikh ◽  
Phoebe H. M. Cunningham ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bond ◽  
Hydrogen Bonds ◽  
Basal Plane ◽  
Solvent Molecule ◽  
Vanadium Atom ◽  
Asymmetric Unit ◽  
Extended Structure ◽  
Independent Molecules ◽  
Ethylenediamine Ligand

Two independent molecules of the title solvated complex, [V(C16H14N2O2)O]·CH3OH, also known as [N,N′-bis(salicylidene)ethylenediamine]oxidovanadium(IV) or vanadyl salen, crystallize in the asymmetric unit. Each disordered methanol solvent molecule [occupancy ratios 0.678 (4):0.322 (4) and 0.750 (5):0.250 (5)] is linked to a [N,N′-bis(salicylidene)ethylenediamine]oxidovanadium(IV) molecule by an O—H...O hydrogen bond and to others by C—H...O hydrogen bonds. The resulting extended structure consists of a bilayer of molecules parallel to theabplane. Despite the fact that solvates are common in complexes derived from substituted analogues of theN,N′-bis(salicylidene)ethylenediamine ligand, the title solvate is the first one of [N,N′-bis(salicylidene)ethylenediamine]oxidovanadium(IV) to be structurally characterized. The two vanadyl species have very similar internal geometries, which are best characterized as distorted square-based pyramidal with the vanadium atom displaced from the N2O2basal plane by 0.5966 (9) Å in the direction of the doubly-bonded oxide ligand.

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