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 The inactive C-terminal cassette of the dual-cassette RNA helicase BRR2 both stimulates and inhibits the activity of the N-terminal helicase unit

2019 ◽  
Vol 295 (7) ◽  
pp. 2097-2112 ◽  
Author(s):  
Karen Vester ◽  
Karine F. Santos ◽  
Benno Kuropka ◽  
Christoph Weise ◽  
Markus C. Wahl
Keyword(s):  
Crystal Structure ◽  
Rna Splicing ◽  
Molecular Mechanisms ◽  
Atp Hydrolysis ◽  
Rna Helicase ◽  
Disulfide Bridges ◽  
Relative Positioning ◽  
Distinct Subgroup ◽  
Terminal Unit ◽  
Rna Unwinding

The RNA helicase bad response to refrigeration 2 homolog (BRR2) is required for the activation of the spliceosome before the first catalytic step of RNA 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 pseudoenzyme 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 at 2.4 Å resolution compared with the crystal structure of BRR2 alone. Likewise, inspection of BRR2 structures within spliceosomal complexes revealed that the cassettes occupy different relative positions and engage in different intercassette contacts during different splicing stages. Engineered disulfide bridges that locked the cassettes in two different relative orientations had opposite effects on the RNA-unwinding activity of the N-terminal cassette, with one configuration enhancing and the other configuration inhibiting RNA unwinding compared with the unconstrained protein. Moreover, we found that differences in relative positioning of the cassettes strongly influence RNA-stimulated ATP hydrolysis by the N-terminal cassette. Our results indicate that the inactive C-terminal cassette of BRR2 can both positively and negatively affect the activity of the N-terminal helicase unit from a distance.

scholarly journals Motility and microtubule depolymerization mechanisms of the Kinesin-8 motor, KIF19A

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Doudou Wang ◽  
Ryo Nitta ◽  
Manatsu Morikawa ◽  
Hiroaki Yajima ◽  
Shigeyuki Inoue ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Electron Microscopy ◽  
Molecular Mechanisms ◽  
Atp Hydrolysis ◽  
Dual Function ◽  
Microtubule Depolymerization ◽  
Multiple Strategies ◽  
Cryo Electron Microscopy ◽  
Cilium Length ◽  
Dual Functions

The kinesin-8 motor, KIF19A, accumulates at cilia tips and controls cilium length. Defective KIF19A leads to hydrocephalus and female infertility because of abnormally elongated cilia. Uniquely among kinesins, KIF19A possesses the dual functions of motility along ciliary microtubules and depolymerization of microtubules. To elucidate the molecular mechanisms of these functions we solved the crystal structure of its motor domain and determined its cryo-electron microscopy structure complexed with a microtubule. The features of KIF19A that enable its dual function are clustered on its microtubule-binding side. Unexpectedly, a destabilized switch II coordinates with a destabilized L8 to enable KIF19A to adjust to both straight and curved microtubule protofilaments. The basic clusters of L2 and L12 tether the microtubule. The long L2 with a characteristic acidic-hydrophobic-basic sequence effectively stabilizes the curved conformation of microtubule ends. Hence, KIF19A utilizes multiple strategies to accomplish the dual functions of motility and microtubule depolymerization by ATP hydrolysis.

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 Crystal structure of yeast initiation factor 4A, a DEAD-box RNA helicase

2000 ◽  
Vol 97 (24) ◽  
pp. 13080-13085 ◽  
Author(s):  
J. M. Caruthers ◽  
E. R. Johnson ◽  
D. B. McKay
Keyword(s):  
Crystal Structure ◽  
Rna Helicase ◽  
Initiation Factor ◽  
Dead Box

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.

scholarly journals Conformational communication mediates the reset step in t6A biosynthesis

2019 ◽  
Vol 47 (12) ◽  
pp. 6551-6567 ◽  
Author(s):  
Amit Luthra ◽  
Naduni Paranagama ◽  
William Swinehart ◽  
Susan Bayooz ◽  
Phuc Phan ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Molecular Docking ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Translational Fidelity ◽  
Substrate Analog ◽  
Binding Cavity ◽  
Saxs Analysis

Abstract The universally conserved N6-threonylcarbamoyladenosine (t6A) modification of tRNA is essential for translational fidelity. In bacteria, t6A biosynthesis starts with the TsaC/TsaC2-catalyzed synthesis of the intermediate threonylcarbamoyl adenylate (TC–AMP), followed by transfer of the threonylcarbamoyl (TC) moiety to adenine-37 of tRNA by the TC-transfer complex comprised of TsaB, TsaD and TsaE subunits and possessing an ATPase activity required for multi-turnover of the t6A cycle. We report a 2.5-Å crystal structure of the T. maritima TC-transfer complex (TmTsaB2D2E2) bound to Mg2+-ATP in the ATPase site, and substrate analog carboxy-AMP in the TC-transfer site. Site directed mutagenesis results show that residues in the conserved Switch I and Switch II motifs of TsaE mediate the ATP hydrolysis-driven reactivation/reset step of the t6A cycle. Further, SAXS analysis of the TmTsaB2D2-tRNA complex in solution reveals bound tRNA lodged in the TsaE binding cavity, confirming our previous biochemical data. Based on the crystal structure and molecular docking of TC–AMP and adenine-37 in the TC-transfer site, we propose a model for the mechanism of TC transfer by this universal biosynthetic system.

scholarly journals The Dynamics of P Granule Liquid Droplets Are Regulated by the Caenorhabditis elegans Germline RNA Helicase GLH-1 via Its ATP Hydrolysis Cycle

Genetics ◽  
2020 ◽  
Vol 215 (2) ◽  
pp. 421-434 ◽  
Author(s):  
Wenjun Chen ◽  
Yabing Hu ◽  
Charles F. Lang ◽  
Jordan S. Brown ◽  
Sierra Schwabach ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Cell Fate ◽  
Rna Binding ◽  
Atp Hydrolysis ◽  
Rna Binding Proteins ◽  
Rna Helicase ◽  
Liquid Droplets ◽  
P Granules ◽  
Link Type ◽  
Show Evidence

P granules are phase-separated liquid droplets that play important roles in the maintenance of germ cell fate in Caenorhabditis elegans. Both the localization and formation of P granules are highly dynamic, but mechanisms that regulate such processes remain poorly understood. Here, we show evidence that the VASA-like germline RNA helicase GLH-1 couples distinct steps of its ATPase hydrolysis cycle to control the formation and disassembly of P granules. In addition, we found that the phenylalanine-glycine-glycine repeats in GLH-1 promote its localization at the perinucleus. Proteomic analyses of the GLH-1 complex with a GLH-1 mutation that interferes with P granule disassembly revealed transient interactions of GLH-1 with several Argonautes and RNA-binding proteins. Finally, we found that defects in recruiting the P granule component PRG-1 to perinuclear foci in the adult germline correlate with the fertility defects observed in various GLH-1 mutants. Together, our results highlight the versatile roles of an RNA helicase in controlling the formation of liquid droplets in space and time.

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.

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