scholarly journals Structural basis for inhibition of an archaeal CRISPR–Cas type I-D large subunit by an anti-CRISPR protein

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
M. Cemre Manav ◽  
Lan B. Van ◽  
Jinzhong Lin ◽  
Anders Fuglsang ◽  
Xu Peng ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Functional State ◽  
Dna Cleavage ◽  
Large Subunit ◽  
Domain Architecture ◽  
Structural Basis ◽  
Type I ◽  
Helicase Activity ◽  
Nuclease Domain ◽  
Sulfolobus Islandicus

AbstractA hallmark of type I CRISPR–Cas systems is the presence of Cas3, which contains both the nuclease and helicase activities required for DNA cleavage during interference. In subtype I-D systems, however, the histidine-aspartate (HD) nuclease domain is encoded as part of a Cas10-like large effector complex subunit and the helicase activity in a separate Cas3’ subunit, but the functional and mechanistic consequences of this organisation are not currently understood. Here we show that the Sulfolobus islandicus type I-D Cas10d large subunit exhibits an unusual domain architecture consisting of a Cas3-like HD nuclease domain fused to a degenerate polymerase fold and a C-terminal domain structurally similar to Cas11. Crystal structures of Cas10d both in isolation and bound to S. islandicus rod-shaped virus 3 AcrID1 reveal that the anti-CRISPR protein sequesters the large subunit in a non-functional state unable to form a cleavage-competent effector complex. The architecture of Cas10d suggests that the type I-D effector complex is similar to those found in type III CRISPR–Cas systems and that this feature is specifically exploited by phages for anti-CRISPR defence.

scholarly journals The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e2887 ◽  
Author(s):  
Vitali Bialevich ◽  
Dhiraj Sinha ◽  
Katsiaryna Shamayeva ◽  
Alena Guzanova ◽  
David Řeha ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Dna Cleavage ◽  
Contact Region ◽  
Large Subunit ◽  
Helicase Domain ◽  
Type I ◽  
Dna Translocation ◽  
Helical Domain ◽  
Restriction Modification

Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types: HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR. Because the multiple functions are integrated in this large subunit of 1,038 residues, a large number of interdomain contacts might be expected. The crystal structure of EcoR124I HsdR reveals a surprisingly sparse number of contacts between helicase domain 2 and the C-terminal helical domain that is thought to be involved in assembly with HsdM. Only two potential hydrogen-bonding contacts are found in a very small contact region. In the present work, the relevance of these two potential hydrogen-bonding interactions for the multiple activities of EcoR124I is evaluated by analysing mutant enzymes usingin vivoandin vitroexperiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results indicate that the helical C-terminal domain is involved in the DNA translocation, cleavage, and ATPase activities of HsdR, and a role in controlling those activities is suggested.

scholarly journals The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation

2017 ◽  
Author(s):  
Vitali Bialevich ◽  
Dhiraj Sinha ◽  
Katsiaryna Shamayeva ◽  
Alena Guzanova ◽  
David Řeha ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Dna Cleavage ◽  
Contact Region ◽  
Large Subunit ◽  
Helicase Domain ◽  
Type I ◽  
Dna Translocation ◽  
Helical Domain ◽  
Restriction Modification

Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types, HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR. Because the multiple functions are integrated in this large subunit of 1038 residues, a large number of interdomain contacts might be expected. The crystal structure of EcoR124I HsdR reveals a surprisingly sparse number of contacts between helicase domain 2 and the C-terminal helical domain that is thought to be involved in assembly with HsdM. Only two potential hydrogen-bonding contacts are found in a very small contact region. In the present work, the relevance of these two potential hydrogen-bonding interactions for the multiple activities of EcoR124I is evaluated by analysing mutant enzymes using in vivo and in vitro experiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results indicate that the helical C-terminal domain is involved in the DNA translocation, cleavage, and ATPase activities of HsdR, and a role in controlling those activities is suggested.

scholarly journals The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation

2017 ◽  
Author(s):  
Vitali Bialevich ◽  
Dhiraj Sinha ◽  
Katsiaryna Shamayeva ◽  
Alena Guzanova ◽  
David Řeha ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Dna Cleavage ◽  
Contact Region ◽  
Large Subunit ◽  
Helicase Domain ◽  
Type I ◽  
Dna Translocation ◽  
Helical Domain ◽  
Restriction Modification

Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types, HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR. Because the multiple functions are integrated in this large subunit of 1038 residues, a large number of interdomain contacts might be expected. The crystal structure of EcoR124I HsdR reveals a surprisingly sparse number of contacts between helicase domain 2 and the C-terminal helical domain that is thought to be involved in assembly with HsdM. Only two potential hydrogen-bonding contacts are found in a very small contact region. In the present work, the relevance of these two potential hydrogen-bonding interactions for the multiple activities of EcoR124I is evaluated by analysing mutant enzymes using in vivo and in vitro experiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results indicate that the helical C-terminal domain is involved in the DNA translocation, cleavage, and ATPase activities of HsdR, and a role in controlling those activities is suggested.

Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

2019 ◽  
Vol 476 (21) ◽  
pp. 3227-3240 ◽  
Author(s):  
Shanshan Wang ◽  
Yanxiang Zhao ◽  
Long Yi ◽  
Minghe Shen ◽  
Chao Wang ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Magnaporthe Oryzae ◽  
Structural Information ◽  
Complex Structure ◽  
Critical Role ◽  
Catalytic Process ◽  
Structural Basis ◽  
Salt Bridges ◽  
Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

scholarly journals Structural basis for ALK2/BMPR2 receptor complex signaling through kinase domain oligomerization

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Christopher Agnew ◽  
Pelin Ayaz ◽  
Risa Kashima ◽  
Hanna S. Loving ◽  
Prajakta Ghatpande ◽  
...  
Keyword(s):  
Md Simulations ◽  
Kinase Domain ◽  
Structural Basis ◽  
Type I ◽  
Type Ii ◽  
Exchange Mass Spectrometry ◽  
Receptor Complexes ◽  
Bmp Receptors ◽  
Morphogenetic Protein ◽  
Smad Proteins

AbstractUpon ligand binding, bone morphogenetic protein (BMP) receptors form active tetrameric complexes, comprised of two type I and two type II receptors, which then transmit signals to SMAD proteins. The link between receptor tetramerization and the mechanism of kinase activation, however, has not been elucidated. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, combined with analysis of SMAD signaling, we show that the kinase domain of the type I receptor ALK2 and type II receptor BMPR2 form a heterodimeric complex via their C-terminal lobes. Formation of this dimer is essential for ligand-induced receptor signaling and is targeted by mutations in BMPR2 in patients with pulmonary arterial hypertension (PAH). We further show that the type I/type II kinase domain heterodimer serves as the scaffold for assembly of the active tetrameric receptor complexes to enable phosphorylation of the GS domain and activation of SMADs.

scholarly journals Crystal structures of aconitase X enzymes from bacteria and archaea provide insights into the molecular evolution of the aconitase superfamily

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Seiya Watanabe ◽  
Yohsuke Murase ◽  
Yasunori Watanabe ◽  
Yasuhiro Sakurai ◽  
Kunihiko Tajima
Keyword(s):  
Molecular Evolution ◽  
Agrobacterium Tumefaciens ◽  
Crystal Structures ◽  
Epr Spectroscopy ◽  
Binding Sites ◽  
Active Sites ◽  
Type I ◽  
Type Ii ◽  
Thermococcus Kodakarensis ◽  
Evolutionary Scenario

AbstractAconitase superfamily members catalyze the homologous isomerization of specific substrates by sequential dehydration and hydration and contain a [4Fe-4S] cluster. However, monomeric and heterodimeric types of function unknown aconitase X (AcnX) have recently been characterized as a cis-3-hydroxy-L-proline dehydratase (AcnXType-I) and mevalonate 5-phosphate dehydratase (AcnXType-II), respectively. We herein elucidated the crystal structures of AcnXType-I from Agrobacterium tumefaciens (AtAcnX) and AcnXType-II from Thermococcus kodakarensis (TkAcnX) without a ligand and in complex with substrates. AtAcnX and TkAcnX contained the [2Fe-2S] and [3Fe-4S] clusters, respectively, conforming to UV and EPR spectroscopy analyses. The binding sites of the [Fe-S] cluster and substrate were clearlydifferent from those that were completely conserved in other aconitase enzymes; however, theoverall structural frameworks and locations of active sites were partially similar to each other.These results provide novel insights into the evolutionary scenario of the aconitase superfamilybased on the recruitment hypothesis.

Crystal Structures of the Human and Fungal Cytosolic Leucyl-tRNA Synthetase Editing Domains: A Structural Basis for the Rational Design of Antifungal Benzoxaboroles

2009 ◽  
Vol 390 (2) ◽  
pp. 196-207 ◽  
Author(s):  
Elena Seiradake ◽  
Weimin Mao ◽  
Vincent Hernandez ◽  
Stephen J. Baker ◽  
Jacob J. Plattner ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Rational Design ◽  
Trna Synthetase ◽  
Structural Basis

scholarly journals Structural basis of adhesive binding by desmocollins and desmogleins

2016 ◽  
Vol 113 (26) ◽  
pp. 7160-7165 ◽  
Author(s):  
Oliver J. Harrison ◽  
Julia Brasch ◽  
Gorka Lasso ◽  
Phinikoula S. Katsamba ◽  
Goran Ahlsen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Crystal Structures ◽  
Structural Basis ◽  
Cellular Interactions ◽  
Opposite Charge ◽  
Charged Amino Acids ◽  
Structural Framework ◽  
Classical Cadherins ◽  
Coated Bead

Desmosomes are intercellular adhesive junctions that impart strength to vertebrate tissues. Their dense, ordered intercellular attachments are formed by desmogleins (Dsgs) and desmocollins (Dscs), but the nature of trans-cellular interactions between these specialized cadherins is unclear. Here, using solution biophysics and coated-bead aggregation experiments, we demonstrate family-wise heterophilic specificity: All Dsgs form adhesive dimers with all Dscs, with affinities characteristic of each Dsg:Dsc pair. Crystal structures of ectodomains from Dsg2 and Dsg3 and from Dsc1 and Dsc2 show binding through a strand-swap mechanism similar to that of homophilic classical cadherins. However, conserved charged amino acids inhibit Dsg:Dsg and Dsc:Dsc interactions by same-charge repulsion and promote heterophilic Dsg:Dsc interactions through opposite-charge attraction. These findings show that Dsg:Dsc heterodimers represent the fundamental adhesive unit of desmosomes and provide a structural framework for understanding desmosome assembly.

scholarly journals Structural Insight into African Swine Fever Virus A179L-Mediated Inhibition of Apoptosis

2017 ◽  
Vol 91 (6) ◽  
Author(s):  
Suresh Banjara ◽  
Sofia Caria ◽  
Linda K. Dixon ◽  
Mark G. Hinds ◽  
Marc Kvansakul
Keyword(s):  
Crystal Structures ◽  
African Swine Fever Virus ◽  
Three Dimensional ◽  
African Swine Fever ◽  
Fever Virus ◽  
Structural Basis ◽  
Dna Virus ◽  
Content Type ◽  
Antiapoptotic Proteins ◽  
Apoptosis Inhibition

ABSTRACT Programmed cell death is a tightly controlled process critical for the removal of damaged or infected cells. Pro- and antiapoptotic proteins of the Bcl-2 family are pivotal mediators of this process. African swine fever virus (ASFV) is a large DNA virus, the only member of the Asfarviridae family, and harbors A179L, a putative Bcl-2 like protein. A179L has been shown to bind to several proapoptotic Bcl-2 proteins; however, the hierarchy of binding and the structural basis for apoptosis inhibition are currently not understood. We systematically evaluated the ability of A179L to bind proapoptotic Bcl-2 family members and show that A179L is the first antiapoptotic Bcl-2 protein to bind to all major death-inducing mammalian Bcl-2 proteins. We then defined the structural basis for apoptosis inhibition of A179L by determining the crystal structures of A179L bound to both Bid and Bax BH3 motifs. Our findings provide a mechanistic understanding for the potent antiapoptotic activity of A179L by identifying it as the first panprodeath Bcl-2 binder and serve as a platform for more-detailed investigations into the role of A179L during ASFV infection. IMPORTANCE Numerous viruses have acquired strategies to subvert apoptosis by encoding proteins capable of sequestering proapoptotic host proteins. African swine fever virus (ASFV), a large DNA virus and the only member of the Asfarviridae family, encodes the protein A179L, which functions to prevent apoptosis. We show that A179L is unusual among antiapoptotic Bcl-2 proteins in being able to physically bind to all core death-inducing mammalian Bcl-2 proteins. Currently, little is known regarding the molecular interactions between A179L and the proapoptotic Bcl-2 members. Using the crystal structures of A179L bound to two of the identified proapoptotic Bcl-2 proteins, Bid and Bax, we now provide a three-dimensional (3D) view of how A179L sequesters host proapoptotic proteins, which is crucial for subverting premature host cell apoptosis.

Sign in / Sign up

Export Citation Format

Share Document