scholarly journals Structural basis for DEAH-helicase activation by G-patch proteins

2020 ◽  
Vol 117 (13) ◽  
pp. 7159-7170 ◽  
Author(s):  
Michael K. Studer ◽  
Lazar Ivanović ◽  
Marco E. Weber ◽  
Sabrina Marti ◽  
Stefanie Jonas
Keyword(s):  
Conformational Changes ◽  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Adenosine Diphosphate ◽  
Structural Basis ◽  
Rna Helicases ◽  
Catalytic Core ◽  
Terminal Domains

RNA helicases of the DEAH/RHA family are involved in many essential cellular processes, such as splicing or ribosome biogenesis, where they remodel large RNA–protein complexes to facilitate transitions to the next intermediate. DEAH helicases couple adenosine triphosphate (ATP) hydrolysis to conformational changes of their catalytic core. This movement results in translocation along RNA, which is held in place by auxiliary C-terminal domains. The activity of DEAH proteins is strongly enhanced by the large and diverse class of G-patch activators. Despite their central roles in RNA metabolism, insight into the molecular basis of G-patch–mediated helicase activation is missing. Here, we have solved the structure of human helicase DHX15/Prp43, which has a dual role in splicing and ribosome assembly, in complex with the G-patch motif of the ribosome biogenesis factor NKRF. The G-patch motif binds in an extended conformation across the helicase surface. It tethers the catalytic core to the flexibly attached C-terminal domains, thereby fixing a conformation that is compatible with RNA binding. Structures in the presence or absence of adenosine diphosphate (ADP) suggest that motions of the catalytic core, which are required for ATP binding, are still permitted. Concomitantly, RNA affinity, helicase, and ATPase activity of DHX15 are increased when G-patch is bound. Mutations that detach one end of the tether but maintain overall binding severely impair this enhancement. Collectively, our data suggest that the G-patch motif acts like a flexible brace between dynamic portions of DHX15 that restricts excessive domain motions but maintains sufficient flexibility for catalysis.

scholarly journals Structural insights into the mechanism of the DEAH-box RNA helicase Prp43

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Marcel J Tauchert ◽  
Jean-Baptiste Fourmann ◽  
Reinhard Lührmann ◽  
Ralf Ficner
Keyword(s):  
Conformational Changes ◽  
Bound States ◽  
Rna Binding ◽  
Atp Hydrolysis ◽  
Bound State ◽  
Rna Helicases ◽  
Large Rearrangements ◽  
Rna Complex ◽  
Structural Insights ◽  
Terminal Domains

The DEAH-box helicase Prp43 is a key player in pre-mRNA splicing as well as the maturation of rRNAs. The exact modus operandi of Prp43 and of all other spliceosomal DEAH-box RNA helicases is still elusive. Here, we report crystal structures of Prp43 complexes in different functional states and the analysis of structure-based mutants providing insights into the unwinding and loading mechanism of RNAs. The Prp43•ATP-analog•RNA complex shows the localization of the RNA inside a tunnel formed by the two RecA-like and C-terminal domains. In the ATP-bound state this tunnel can be transformed into a groove prone for RNA binding by large rearrangements of the C-terminal domains. Several conformational changes between the ATP- and ADP-bound states explain the coupling of ATP hydrolysis to RNA translocation, mainly mediated by a β-turn of the RecA1 domain containing the newly identified RF motif. This mechanism is clearly different to those of other RNA helicases.

scholarly journals Regulation of DEAH-box RNA helicases by G-patch proteins

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Katherine E. Bohnsack ◽  
Ralf Ficner ◽  
Markus T. Bohnsack ◽  
Stefanie Jonas
Keyword(s):  
Ribosome Biogenesis ◽  
Cellular Localization ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Adaptor Proteins ◽  
Innate Immune ◽  
Telomere Maintenance ◽  
Rna Helicases ◽  
Mechanistic Basis ◽  
Rna Protein Complexes

Abstract RNA helicases of the DEAH/RHA family form a large and conserved class of enzymes that remodel RNA protein complexes (RNPs) by translocating along the RNA. Driven by ATP hydrolysis, they exert force to dissociate hybridized RNAs, dislocate bound proteins or unwind secondary structure elements in RNAs. The sub-cellular localization of DEAH-helicases and their concomitant association with different pathways in RNA metabolism, such as pre-mRNA splicing or ribosome biogenesis, can be guided by cofactor proteins that specifically recruit and simultaneously activate them. Here we review the mode of action of a large class of DEAH-specific adaptor proteins of the G-patch family. Defined only by their eponymous short glycine-rich motif, which is sufficient for helicase binding and stimulation, this family encompasses an immensely varied array of domain compositions and is linked to an equally diverse set of functions. G-patch proteins are conserved throughout eukaryotes and are even encoded within retroviruses. They are involved in mRNA, rRNA and snoRNA maturation, telomere maintenance and the innate immune response. Only recently was the structural and mechanistic basis for their helicase enhancing activity determined. We summarize the molecular and functional details of G-patch-mediated helicase regulation in their associated pathways and their involvement in human diseases.

scholarly journals Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

2021 ◽  
Author(s):  
Hauke S. Hillen ◽  
Elena Lavdovskaia ◽  
Franziska Nadler ◽  
Elisa Hanitsch ◽  
Andreas Linden ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Large Subunit ◽  
Structural Basis ◽  
Catalytic Core ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
Translation Systems

Ribosome biogenesis is an essential process that requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. In particular, maturation of the peptidyl transferase center (PTC), the catalytic core of the ribosome, is mediated by universally conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial ribosomal large subunit (mtLSU) using a combination of endogenous complex purification, in vitro reconstitution and cryo-electron microscopy (cryo-EM). Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Subsequent addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch by releasing MTERF4-NSUN4 and GTPBP5 accompanied by the progression to a near-mature PTC state. In addition, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results define the molecular basis of dynamic GTPase-mediated PTC maturation during mitochondrial ribosome biogenesis and provide a framework for understanding step-wise progression of PTC folding as a critical quality control checkpoint in all translation systems.

scholarly journals Dissecting the conformation of glycans and their interactions with proteins

2020 ◽  
Vol 27 (1) ◽  
Author(s):  
Sheng-Hung Wang ◽  
Tsai-Jung Wu ◽  
Chien-Wei Lee ◽  
John Yu
Keyword(s):  
Conformational Changes ◽  
Molecular Interactions ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Unmet Need ◽  
Protein Molecule ◽  
Structural Basis ◽  
Glycan Structure ◽  
Chronic Obstructive ◽  
Technology Platforms

Abstract The use of in silico strategies to develop the structural basis for a rational optimization of glycan-protein interactions remains a great challenge. This problem derives, in part, from the lack of technologies to quantitatively and qualitatively assess the complex assembling between a glycan and the targeted protein molecule. Since there is an unmet need for developing new sugar-targeted therapeutics, many investigators are searching for technology platforms to elucidate various types of molecular interactions within glycan-protein complexes and aid in the development of glycan-targeted therapies. Here we discuss three important technology platforms commonly used in the assessment of the complex assembly of glycosylated biomolecules, such as glycoproteins or glycosphingolipids: Biacore analysis, molecular docking, and molecular dynamics simulations. We will also discuss the structural investigation of glycosylated biomolecules, including conformational changes of glycans and their impact on molecular interactions within the glycan-protein complex. For glycoproteins, secreted protein acidic and rich in cysteine (SPARC), which is associated with various lung disorders, such as chronic obstructive pulmonary disease (COPD) and lung cancer, will be taken as an example showing that the core fucosylation of N-glycan in SPARC regulates protein-binding affinity with extracellular matrix collagen. For glycosphingolipids (GSLs), Globo H ceramide, an important tumor-associated GSL which is being actively investigated as a target for new cancer immunotherapies, will be used to demonstrate how glycan structure plays a significant role in enhancing angiogenesis in tumor microenvironments.

scholarly journals Assembly and cryo-EM structures of RNA-specific measles virus nucleocapsids provide mechanistic insight into paramyxoviral replication

2019 ◽  
Vol 116 (10) ◽  
pp. 4256-4264 ◽  
Author(s):  
Ambroise Desfosses ◽  
Sigrid Milles ◽  
Malene Ringkjøbing Jensen ◽  
Serafima Guseva ◽  
Jacques-Philippe Colletier ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Measles Virus ◽  
Rna Binding ◽  
Structural Basis ◽  
Particle Assembly ◽  
Interaction Sites ◽  
Assembly Kinetics ◽  
High Resolution Structure ◽  
Binding Groove

Assembly of paramyxoviral nucleocapsids on the RNA genome is an essential step in the viral cycle. The structural basis of this process has remained obscure due to the inability to control encapsidation. We used a recently developed approach to assemble measles virus nucleocapsid-like particles on specific sequences of RNA hexamers (poly-Adenine and viral genomic 5′) in vitro, and determined their cryoelectron microscopy maps to 3.3-Å resolution. The structures unambiguously determine 5′ and 3′ binding sites and thereby the binding-register of viral genomic RNA within nucleocapsids. This observation reveals that the 3′ end of the genome is largely exposed in fully assembled measles nucleocapsids. In particular, the final three nucleotides of the genome are rendered accessible to the RNA-dependent RNA polymerase complex, possibly enabling efficient RNA processing. The structures also reveal local and global conformational changes in the nucleoprotein upon assembly, in particular involving helix α6 and helix α13 that form edges of the RNA binding groove. Disorder is observed in the bound RNA, localized at one of the two backbone conformational switch sites. The high-resolution structure allowed us to identify putative nucleobase interaction sites in the RNA-binding groove, whose impact on assembly kinetics was measured using real-time NMR. Mutation of one of these sites, R195, whose sidechain stabilizes both backbone and base of a bound nucleic acid, is thereby shown to be essential for nucleocapsid-like particle assembly.

scholarly journals Structural basis for inhibition of the AAA-ATPase Drg1 by diazaborine

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Michael Prattes ◽  
Irina Grishkovskaya ◽  
Victor-Valentin Hodirnau ◽  
Ingrid Rössler ◽  
Isabella Klein ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Atp Hydrolysis ◽  
Ribosomal Subunit ◽  
Covalent Bond ◽  
Resistance Mechanisms ◽  
Drug Binding ◽  
Structural Basis ◽  
Aaa Atpase ◽  
Maturation Factor ◽  
Key Factor

AbstractThe hexameric AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis and initiates cytoplasmic maturation of the large ribosomal subunit by releasing the shuttling maturation factor Rlp24. Drg1 monomers contain two AAA-domains (D1 and D2) that act in a concerted manner. Rlp24 release is inhibited by the drug diazaborine which blocks ATP hydrolysis in D2. The mode of inhibition was unknown. Here we show the first cryo-EM structure of Drg1 revealing the inhibitory mechanism. Diazaborine forms a covalent bond to the 2′-OH of the nucleotide in D2, explaining its specificity for this site. As a consequence, the D2 domain is locked in a rigid, inactive state, stalling the whole Drg1 hexamer. Resistance mechanisms identified include abolished drug binding and altered positioning of the nucleotide. Our results suggest nucleotide-modifying compounds as potential novel inhibitors for AAA-ATPases.

scholarly journals Biological and Structural Basis for Aha1 Regulation of Hsp90 ATPase Activity in Maintaining Proteostasis in the Human Disease Cystic Fibrosis

2010 ◽  
Vol 21 (6) ◽  
pp. 871-884 ◽  
Author(s):  
Atanas V. Koulov ◽  
Paul LaPointe ◽  
Bingwen Lu ◽  
Abbas Razvi ◽  
Judith Coppinger ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Atpase Activity ◽  
Protein Misfolding ◽  
Atp Hydrolysis ◽  
Structural Basis ◽  
Inherited Disease ◽  
Hsp90 Chaperone ◽  
Terminal Domains

The activator of Hsp90 ATPase 1, Aha1, has been shown to participate in the Hsp90 chaperone cycle by stimulating the low intrinsic ATPase activity of Hsp90. To elucidate the structural basis for ATPase stimulation of human Hsp90 by human Aha1, we have developed novel mass spectrometry approaches that demonstrate that the N- and C-terminal domains of Aha1 cooperatively bind across the dimer interface of Hsp90 to modulate the ATP hydrolysis cycle and client activity in vivo. Mutations in both the N- and C-terminal domains of Aha1 impair its ability to bind Hsp90 and stimulate its ATPase activity in vitro and impair in vivo the ability of the Hsp90 system to modulate the folding and trafficking of wild-type and variant (ΔF508) cystic fibrosis transmembrane conductance regulator (CFTR) responsible for the inherited disease cystic fibrosis (CF). We now propose a general model for the role of Aha1 in the Hsp90 ATPase cycle in proteostasis whereby Aha1 regulates the dwell time of Hsp90 with client. We suggest that Aha1 activity integrates chaperone function with client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions, thereby protecting transient folding intermediates in vivo that could contribute to protein misfolding systems disorders such as CF when destabilized.

scholarly journals The mechanism of ATP-dependent RNA unwinding by DEAD box proteins

2009 ◽  
Vol 390 (12) ◽  
Author(s):  
Manuel Hilbert ◽  
Anne R. Karow ◽  
Dagmar Klostermeier
Keyword(s):  
Rna Binding ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Current Data ◽  
Protein Activity ◽  
Closed Conformation ◽  
Dead Box ◽  
Rna Duplexes ◽  
The Dead ◽  
Rna Unwinding

Abstract DEAD box proteins catalyze the ATP-dependent unwinding of double-stranded RNA (dsRNA). In addition, they facilitate protein displacement and remodeling of RNA or RNA/protein complexes. Their hallmark feature is local destabilization of RNA duplexes. Here, we summarize current data on the DEAD box protein mechanism and present a model for RNA unwinding that integrates recent data on the effect of ATP analogs and mutations on DEAD box protein activity. DEAD box proteins share a conserved helicase core with two flexibly linked RecA-like domains that contain all helicase signature motifs. Variable flanking regions contribute to substrate binding and modulate activity. In the presence of ATP and RNA, the helicase core adopts a compact, closed conformation with extensive interdomain contacts and high affinity for RNA. In the closed conformation, the RecA-like domains form a catalytic site for ATP hydrolysis and a continuous RNA binding site. A kink in the backbone of the bound RNA locally destabilizes the duplex. Rearrangement of this initial complex generates a hydrolysis- and unwinding-competent state. From this complex, the first RNA strand can dissociate. After ATP hydrolysis and phosphate release, the DEAD box protein returns to a low-affinity state for RNA. Dissociation of the second RNA strand and reopening of the cleft in the helicase core allow for further catalytic cycles.

scholarly journals Searching for kinesin's mechanical amplifier

2000 ◽  
Vol 355 (1396) ◽  
pp. 449-457 ◽  
Author(s):  
Ronald D. Vale ◽  
Ryan Case ◽  
Elena Sablin ◽  
Cindy Hart ◽  
Robert Fletterick
Keyword(s):  
Amino Acids ◽  
Conformational Changes ◽  
Common Ancestor ◽  
Atp Hydrolysis ◽  
Power Stroke ◽  
Catalytic Core ◽  
Present Evidence ◽  
Directional Motion ◽  
Mechanical Element ◽  
Mechanical Elements

Kinesin, a microtubule–based motor, and myosin, an actin–based motor, share a similar core structure, indicating that they arose from a common ancestor. However, kinesin lacks the long lever–arm domain that is believed to drive the myosin power stroke. Here, we present evidence that a much smaller region of ca . 10–40 amino acids serves as a mechanical element for kinesin motor proteins. These ‘neck regions’ are class conserved and have distinct structures in plus–end and minus–end–directed kinesin motors. Mutagenesis studies also indicate that the neck regions are involved in coupling ATP hydrolysis and energy into directional motion along the microtubule. We suggest that the kinesin necks drive motion by undergoing a conformational change in which they detach and re–dock onto the catalytic core during the ATPase cycle. Thus, kinesin and myosin have evolved unique mechanical elements that amplify small, nucleotide–dependent conformational changes that occur in their similar catalytic cores.

scholarly journals The crystal structure of human DEAH-box RNA helicase 15 reveals a domain organization of the mammalian DEAH/RHA family

2017 ◽  
Vol 73 (6) ◽  
pp. 347-355 ◽  
Author(s):  
Karin Murakami ◽  
Kenji Nakano ◽  
Toshiyuki Shimizu ◽  
Umeharu Ohto
Keyword(s):  
Crystal Structure ◽  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Structural Information ◽  
Rna Helicase ◽  
Structural Features ◽  
Common Mechanism ◽  
Compact Structure ◽  
Catalytic Core ◽  
Immune Sensing

DEAH-box RNA helicase 15 (DHX15) plays important roles in RNA metabolism, including in splicing and in ribosome biogenesis. In addition, mammalian DHX15 also mediates the innate immune sensing of viral RNA. However, structural information on this protein is not available, although the structure of the fungal orthologue of this protein, Prp43, has been elucidated. Here, the crystal structure of the ADP-bound form of human DHX15 is reported at a resolution of 2.0 Å. This is the first structure to be revealed of a member of the mammalian DEAH-box RNA helicase (DEAH/RHA) family in a nearly complete form, including the catalytic core consisting of the two N-terminal RecA domains and the C-terminal regulatory domains (CTD). The ADP-bound form of DHX15 displayed a compact structure, in which the RecA domains made extensive contacts with the CTD. Notably, a potential RNA-binding site was found on the surface of a RecA domain with positive electrostatic potential. Almost all structural features were conserved between the fungal Prp43 and the human DHX15, suggesting that they share a fundamentally common mechanism of action and providing a better understanding of the specific mammalian functions of DHX15.

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