scholarly journals The full-length structure of Thermus scotoductus OLD defines the ATP hydrolysis properties and catalytic mechanism of Class 1 OLD family nucleases

2020 ◽  
Vol 48 (5) ◽  
pp. 2762-2776 ◽  
Author(s):  
Carl J Schiltz ◽  
Myfanwy C Adams ◽  
Joshua S Chappie
Keyword(s):  
Dna Cleavage ◽  
Spatial Organization ◽  
Catalytic Mechanism ◽  
Atp Hydrolysis ◽  
Domain Architecture ◽  
Full Length ◽  
Sequence Length ◽  
Atpase Domain ◽  
Dimerization Domain ◽  
Class 1

Abstract OLD family nucleases contain an N-terminal ATPase domain and a C-terminal Toprim domain. Homologs segregate into two classes based on primary sequence length and the presence/absence of a unique UvrD/PcrA/Rep-like helicase gene immediately downstream in the genome. Although we previously defined the catalytic machinery controlling Class 2 nuclease cleavage, degenerate conservation of the C-termini between classes precludes pinpointing the analogous residues in Class 1 enzymes by sequence alignment alone. Our Class 2 structures also provide no information on ATPase domain architecture and ATP hydrolysis. Here we present the full-length structure of the Class 1 OLD nuclease from Thermus scotoductus (Ts) at 2.20 Å resolution, which reveals a dimerization domain inserted into an N-terminal ABC ATPase fold and a C-terminal Toprim domain. Structural homology with genome maintenance proteins identifies conserved residues responsible for Ts OLD ATPase activity. Ts OLD lacks the C-terminal helical domain present in Class 2 OLD homologs yet preserves the spatial organization of the nuclease active site, arguing that OLD proteins use a conserved catalytic mechanism for DNA cleavage. We also demonstrate that mutants perturbing ATP hydrolysis or DNA cleavage in vitro impair P2 OLD-mediated killing of recBC−Escherichia coli hosts, indicating that both the ATPase and nuclease activities are required for OLD function in vivo.

scholarly journals Structural dynamics and catalytic mechanism of ATP13A2 (PARK9) from simulations

2021 ◽  
Author(s):  
Teodora Mateeva ◽  
Marco Klaehn ◽  
Edina Rosta
Keyword(s):  
Barrier Height ◽  
Catalytic Reaction ◽  
Active Site ◽  
Electrostatic Interactions ◽  
Catalytic Mechanism ◽  
Atp Hydrolysis ◽  
Binding Mode ◽  
Full Length ◽  
Dimensional Structure ◽  
Missense Mutations

ATP13A2 is a gene encoding a protein of the P5B subfamily of ATPases and is a PARK gene. Molecular defects of the gene are mainly associated with variations of Parkinson's disease (PD). Despite the established importance of the protein in regulating neuronal integrity, the three-dimensional structure of the protein currently remains unresolved crystallographically. We have modelled the structure and reactivity of the full-length protein in its E1-ATP state. Using Molecular Dynamics (MD), Quantum cluster and Quantum Mechanical/Molecular mechanical (QM/MM) methods, we aimed at describing the main catalytic reaction, leading to the phosphorylation of Asp513. Our MD simulations suggest that two positively charged Mg2+ cations are present at the active site during the catalytic reaction, stabilizing a specific triphosphate binding mode. Using QM/MM calculations, we subsequently calculated the reaction profiles for the phosphate transfer step in the presence of one and two Mg2+ cations. The calculated barrier heights in both cases are found to be around 10.5 and 13.0 kcal mol-1, respectively. We elucidated details of the catalytically competent ATP conformation and the binding mode of the second Mg2+ cofactor. We also examined the role of the conserved Arg686 and Lys859 catalytic residues. We observed that by lowering significantly the barrier height of the ATP hydrolysis reaction, Arg686 had significant effect on the reaction. The removal of Arg686 increased the barrier height for the ATP hydrolysis by ~3.5 kcal mol-1 while the removal of key electrostatic interactions created by Lys859 to the gamma-phosphate and Arg513 destabilizes the reactant state. When missense mutations occur in close proximity to an active site residue, they can interfere with the barrier height of the reaction, which can halt the normal enzymatic rate of the protein. We also identified the main binding pockets in the full-length structure, including the pocket in the transmembrane region, which is likely where ATP13A2 cargo binds.

scholarly journals Domain Architecture of the Catalytic Subunit in the ISW2-Nucleosome Complex

2007 ◽  
Vol 27 (23) ◽  
pp. 8306-8317 ◽  
Author(s):  
Weiwei Dang ◽  
Blaine Bartholomew
Keyword(s):  
Chromatin Remodeling ◽  
Cellular Differentiation ◽  
Atp Hydrolysis ◽  
Domain Architecture ◽  
Catalytic Subunit ◽  
Atpase Domain ◽  
Entry Site ◽  
Physical Connection ◽  
C Terminus ◽  
First Time

ABSTRACT ATP-dependent chromatin remodeling has an important role in the regulation of cellular differentiation and development. For the first time, a topological view of one of these complexes has been revealed, by mapping the interactions of the catalytic subunit Isw2 with nucleosomal and extranucleosomal DNA in the complex with all four subunits of ISW2 bound to nucleosomes. Different domains of Isw2 were shown to interact with the nucleosome near the dyad axis, another near the entry site of the nucleosome, and another with extranucleosomal DNA. The conserved DEXD or ATPase domain was found to contact the superhelical location 2 (SHL2) of the nucleosome, providing a direct physical connection of ATP hydrolysis with this region of nucleosomes. The C terminus of Isw2, comprising the SLIDE (SANT-like domain) and HAND domains, was found to be associated with extranucleosomal DNA and the entry site of nucleosomes. It is thus proposed that the C-terminal domains of Isw2 are involved in anchoring the complex to nucleosomes through their interactions with linker DNA and that they facilitate the movement of DNA along the surface of nucleosomes.

scholarly journals Staring at the Naked Goddess: Unraveling the Structure and Reactivity of Artemis Endonuclease Interacting with a DNA Double Strand

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3986
Author(s):  
Cécilia Hognon ◽  
Antonio Monari
Keyword(s):  
Dna Cleavage ◽  
Catalytic Mechanism ◽  
Dna Lesions ◽  
Cleavage Reaction ◽  
Dynamic Simulations ◽  
Important Target ◽  
System Adaptation ◽  
Dna Strands ◽  
Dna Strand ◽  
Dna Substrate

Artemis is an endonuclease responsible for breaking hairpin DNA strands during immune system adaptation and maturation as well as the processing of potentially toxic DNA lesions. Thus, Artemis may be an important target in the development of anticancer therapy, both for the sensitization of radiotherapy and for immunotherapy. Despite its importance, its structure has been resolved only recently, and important questions concerning the arrangement of its active center, the interaction with the DNA substrate, and the catalytic mechanism remain unanswered. In this contribution, by performing extensive molecular dynamic simulations, both classically and at the hybrid quantum mechanics/molecular mechanics level, we evidenced the stable interaction modes of Artemis with a model DNA strand. We also analyzed the catalytic cycle providing the free energy profile and key transition states for the DNA cleavage reaction.

scholarly journals Human Miro Proteins Act as NTP Hydrolases through a Novel, Non-Canonical Catalytic Mechanism

2018 ◽  
Vol 19 (12) ◽  
pp. 3839 ◽  
Author(s):  
Daniel Peters ◽  
Laura Kay ◽  
Jeyanthy Eswaran ◽  
Jeremy Lakey ◽  
Meera Soundararajan
Keyword(s):  
Catalytic Mechanism ◽  
Catalytic Domain ◽  
Domain Architecture ◽  
Subcellular Localisation ◽  
Cellular Processes ◽  
Calcium Sensing ◽  
And Function ◽  
Arginine Finger ◽  
Catalytic Functions ◽  
Unique Domain

Mitochondria are highly dynamic organelles that play a central role in multiple cellular processes, including energy metabolism, calcium homeostasis and apoptosis. Miro proteins (Miros) are “atypical” Ras superfamily GTPases that display unique domain architecture and subcellular localisation regulating mitochondrial transport, autophagy and calcium sensing. Here, we present systematic catalytic domain characterisation and structural analyses of human Miros. Despite lacking key conserved catalytic residues (equivalent to Ras Y32, T35, G60 and Q61), the Miro N-terminal GTPase domains display GTPase activity. Surprisingly, the C-terminal GTPase domains previously assumed to be “relic” domains were also active. Moreover, Miros show substrate promiscuity and function as NTPases. Molecular docking and structural analyses of Miros revealed unusual features in the Switch I and II regions, facilitating promiscuous substrate binding and suggesting the usage of a novel hydrolytic mechanism. The key substitution in position 13 in the Miros leads us to suggest the existence of an “internal arginine finger”, allowing an unusual catalytic mechanism that does not require GAP protein. Together, the data presented here indicate novel catalytic functions of human Miro atypical GTPases through altered catalytic mechanisms.

scholarly journals A new insight into the zinc-dependent DNA-cleavage by the colicin E7 nuclease: a crystallographic and computational study

Metallomics ◽  
2014 ◽  
Vol 6 (11) ◽  
pp. 2090-2099 ◽  
Author(s):  
Anikó Czene ◽  
Eszter Tóth ◽  
Eszter Németh ◽  
Harm Otten ◽  
Jens-Christian N. Poulsen ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Dna Cleavage ◽  
Catalytic Mechanism ◽  
Computational Study ◽  
Colicin E7 ◽  
Insight Into

The crystal structure of a colicin E7 metallonuclease mutant complemented by QM/MM calculations suggests an alternative catalytic mechanism of Zn2+-containing HNH nucleases.

scholarly journals Structural characterization of transient interactions in DNA mismatch repair

2014 ◽  
Vol 70 (a1) ◽  
pp. C418-C418
Author(s):  
Monica Pillon ◽  
Vignesh Babu ◽  
Mark Sutton ◽  
Alba Guarne
Keyword(s):  
Complex Formation ◽  
Mismatch Repair ◽  
Structural Characterization ◽  
Dna Mismatch Repair ◽  
Full Length ◽  
Endonuclease Activity ◽  
Dimerization Domain ◽  
Dna Mismatch ◽  
Replication Errors

DNA mismatch repair (MMR) is a conserved pathway that safeguards genome integrity by correcting replication errors. The initiation of MMR is orchestrated by two proteins –MutS and MutL. MutS detects replication errors and recruits MutL, a key regulator in coordinating downstream MMR events. The processivity clamp, typically known to tether the replicative polymerase to DNA during DNA synthesis, also has a role in several steps in MMR. We have previously shown that MutL transiently interacts with the clamp and that this complex is important for MMR in vivo. The role of the clamp in eukaryotes and most bacteria is believed to license MutL endonuclease activity. In bacterial organisms where MutL does not have endonuclease activity, such as in Escherichia coli, the clamp also interacts with MutL and this interaction is also important for MMR activity. However, the transient nature of this complex prevents its functional and structural characterization. Here, we develop a method to stabilize the E. coli MutL-clamp complex by engineering a disulfide bond at the known protein complex interface and characterize its structure using small angle X-ray scattering (SAXS). MutL binds the clamp through a consensus motif found in its dimerization domain. Using this domain (MutL-CTD) we monitor complex formation with the clamp. We observe two complexes using SAXS. In one complex the MutL-CTD occupies a single hydrophobic cleft of the clamp, while the other occupies both hydrophobic clefts simultaneously. To identify the physiological complex, we used the full length MutL protein to impose further constraints. Analysis of complex formation suggests that full length MutL binds a single cleft on the clamp. Altogether, our data reveals how MutL interacts with the clamp in the early steps of MMR and this approach could be implemented to structurally characterize other transient complexes, an aspect of structural biology that is largely unexplored.

scholarly journals The interplay of DNA binding, ATP hydrolysis and helicase activities of the archaeal MCM helicase

2011 ◽  
Vol 436 (2) ◽  
pp. 409-414 ◽  
Author(s):  
Li Phing Liew ◽  
Stephen D. Bell
Keyword(s):  
Dna Binding ◽  
Active Site ◽  
Atp Hydrolysis ◽  
Sulfolobus Solfataricus ◽  
Active Form ◽  
Dna Helicase ◽  
Atpase Domain ◽  
Minichromosome Maintenance ◽  
Conserved Residues ◽  
Mcm Helicase

The MCM (minichromosome maintenance) proteins of archaea are widely believed to be the replicative DNA helicase of these organisms. Most archaea possess a single MCM orthologue that forms homo-multimeric assemblies with a single hexamer believed to be the active form. In the present study we characterize the roles of highly conserved residues in the ATPase domain of the MCM of the hyperthermophilic archaeon Sulfolobus solfataricus. Our results identify a potential conduit for communicating DNA-binding information to the ATPase active site.

scholarly journals Hindering the Strand Passage Reaction of Human Topoisomerase IIα without Disturbing DNA Cleavage, ATP Hydrolysis, or the Operation of the N-terminal Clamp

2004 ◽  
Vol 279 (27) ◽  
pp. 28093-28099 ◽  
Author(s):  
Vibe H. Oestergaard ◽  
Laura Giangiacomo ◽  
Lotte Bjergbaek ◽  
Birgitta R. Knudsen ◽  
Anni H. Andersen
Keyword(s):  
Dna Cleavage ◽  
Atp Hydrolysis ◽  
Topoisomerase Iiα ◽  
Strand Passage

Molecular cloning and characterization of an ADP-ribosylation factor 6 gene (ptARF6) from Pisolithus tinctorius

2016 ◽  
Vol 62 (5) ◽  
pp. 383-393
Author(s):  
Liling Wang ◽  
Haibo Li ◽  
Yifeng Zhou ◽  
Yuchuan Qin ◽  
Yanbin Wang ◽  
...  
Keyword(s):  
Domain Architecture ◽  
Phospholipid Composition ◽  
Pisolithus Tinctorius ◽  
Full Length ◽  
White Rot ◽  
Bioinformatics Analyses ◽  
Reading Frame ◽  
Adp Ribosylation ◽  
Real Time Quantitative Pcr ◽  
Adp Ribosylation Factor

ADP-ribosylation factor 6 (ARF6) is an evolutionarily conserved molecule that has an essential function in intracellular trafficking and organelle structure. To better understand its role during presymbiosis between plant roots and compatible filamentous fungi, the full-length cDNA sequence of ARF6 from Pisolithus tinctorius was cloned and a variety of bioinformatics analyses performed. The full-length sequence was 849 bp long and contained a 549 bp open reading frame encoding a protein of 182 amino acids. A phylogenetic analysis showed that ptARF6 was the ortholog of the ADP ribosylation factor 6/GTPase SAR1 gene from the white-rot basidiomycete Trametes versicolor. A domain architecture analysis of the ARF6 protein revealed a repeat region, which is a common feature of ARF6 in other species. Recombinant ARF6 protein was expressed with an N-terminal 6×His tag and purified using Ni2+-NTA affinity chromatography. The molecular mass of the recombinant protein was estimated by SDS–PAGE to be 25 kDa. The recombinant ARF6 protein bound strongly to 18:1 and 18:2 phosphatidic acids. Thus, ARF6 may participate in the signaling pathways involved in membrane phospholipid composition. The intracellular distribution of ptADP6 in HEK239T cells also indicates that ptADP6 may function not only in plasma membrane events but also in endosomal membranes events. Real-time quantitative PCR revealed that the differential expression of ptARF6 was associated with the presymbiotic stage. ptARF6 may be induced by presymbiosis during the regulation of mycorrhizal formation.

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