scholarly journals Structural basis underlying complex assembly and conformational transition of the type I R-M system

2017 ◽  
Vol 114 (42) ◽  
pp. 11151-11156 ◽  
Author(s):  
Yan-Ping Liu ◽  
Qun Tang ◽  
Jie-Zhong Zhang ◽  
Li-Fei Tian ◽  
Pu Gao ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Conformational Transition ◽  
Structural Information ◽  
Structural Basis ◽  
Type I ◽  
Structural Comparison ◽  
Complex Assembly ◽  
Adenosyl Methionine ◽  
Restriction Modification

Type I restriction-modification (R-M) systems are multisubunit enzymes with separate DNA-recognition (S), methylation (M), and restriction (R) subunits. Despite extensive studies spanning five decades, the detailed molecular mechanisms underlying subunit assembly and conformational transition are still unclear due to the lack of high-resolution structural information. Here, we report the atomic structure of a type I MTase complex (2M+1S) bound to DNA and cofactor S-adenosyl methionine in the “open” form. The intermolecular interactions between M and S subunits are mediated by a four-helix bundle motif, which also determines the specificity of the interaction. Structural comparison between open and previously reported low-resolution “closed” structures identifies the huge conformational changes within the MTase complex. Furthermore, biochemical results show that R subunits prefer to load onto the closed form MTase. Based on our results, we proposed an updated model for the complex assembly. The work reported here provides guidelines for future applications in molecular biology.

scholarly journals Structural basis of membrane recognition of Toxoplasma gondii vacuole by Irgb6

2021 ◽  
Vol 5 (1) ◽  
pp. e202101149
Author(s):  
Yumiko Saijo-Hamano ◽  
Aalaa Alrahman Sherif ◽  
Ariel Pradipta ◽  
Miwa Sasai ◽  
Naoki Sakai ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Structural Information ◽  
Parasitophorous Vacuole ◽  
Membrane Binding ◽  
Structural Basis ◽  
In Silico Docking ◽  
Binding Interface ◽  
Dimerization Interface

The p47 immunity-related GTPase (IRG) Irgb6 plays a pioneering role in host defense against Toxoplasma gondii infection. Irgb6 is recruited to the parasitophorous vacuole membrane (PVM) formed by T. gondii and disrupts it. Despite the importance of this process, the molecular mechanisms accounting for PVM recognition by Irgb6 remain elusive because of lack of structural information on Irgb6. Here we report the crystal structures of mouse Irgb6 in the GTP-bound and nucleotide-free forms. Irgb6 exhibits a similar overall architecture to other IRGs in which GTP binding induces conformational changes in both the dimerization interface and the membrane-binding interface. The membrane-binding interface of Irgb6 assumes a unique conformation, composed of N- and C-terminal helical regions forming a phospholipid binding site. In silico docking of phospholipids further revealed membrane-binding residues that were validated through mutagenesis and cell-based assays. Collectively, these data demonstrate a novel structural basis for Irgb6 to recognize T. gondii PVM in a manner distinct from other IRGs.

scholarly journals Structural basis of membrane recognition of Toxoplasma gondii vacuole by Irgb6

2021 ◽  
Author(s):  
Yumiko Saijo Hamano ◽  
Aalaa Alrahman Sherif ◽  
Ariel Pradipta ◽  
Miwa Sasai ◽  
Naoki Sakai ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Structural Information ◽  
Parasitophorous Vacuole ◽  
Membrane Binding ◽  
Structural Basis ◽  
In Silico Docking ◽  
Binding Interface ◽  
Toxoplasma Gondii Infection

The p47 immunity-related GTPase (IRG) Irgb6 plays a pioneering role in host defense against Toxoplasma gondii infection. It is recruited to the parasitophorous vacuole membrane (PVM) formed by T. gondii and disrupts it. Despite the importance of this process, the molecular mechanisms accounting for PVM recognition by Irgb6 remain elusive due to lack of structural information on Irgb6. Here we report the crystal structures of mouse Irgb6 in the GTP-bound and nucleotide-free forms. Irgb6 exhibits a similar overall architecture to other IRGs in which GTP-binding induces conformational changes in both the dimerization interface and the membrane-binding interface. The membrane-binding interface of Irgb6 assumes a unique conformation, composed of N- and C-terminal helical regions forming a phospholipid binding site. In silico docking of phospholipids further revealed membrane binding residues that were validated through mutagenesis and cell-based assays. Collectively, these data demonstrate a novel structural basis for Irgb6 to recognize T. gondii PVM in a manner distinct from other IRGs.

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 inhibition of the type I-F CRISPR–Cas surveillance complex by AcrIF4, AcrIF7 and AcrIF14

2020 ◽  
Author(s):  
Clinton Gabel ◽  
Zhuang Li ◽  
Heng Zhang ◽  
Leifu Chang
Keyword(s):  
Genome Editing ◽  
Conformational Changes ◽  
Structural Basis ◽  
Type I ◽  
Structural Detail ◽  
Immune Systems ◽  
Helical Bundle ◽  
Adaptive Immune ◽  
Target Dna ◽  
Adaptive Immune Systems

Abstract CRISPR–Cas systems are adaptive immune systems in bacteria and archaea to defend against mobile genetic elements (MGEs) and have been repurposed as genome editing tools. Anti-CRISPR (Acr) proteins are produced by MGEs to counteract CRISPR–Cas systems and can be used to regulate genome editing by CRISPR techniques. Here, we report the cryo-EM structures of three type I-F Acr proteins, AcrIF4, AcrIF7 and AcrIF14, bound to the type I-F CRISPR–Cas surveillance complex (the Csy complex) from Pseudomonas aeruginosa. AcrIF4 binds to an unprecedented site on the C-terminal helical bundle of Cas8f subunit, precluding conformational changes required for activation of the Csy complex. AcrIF7 mimics the PAM duplex of target DNA and is bound to the N-terminal DNA vise of Cas8f. Two copies of AcrIF14 bind to the thumb domains of Cas7.4f and Cas7.6f, preventing hybridization between target DNA and the crRNA. Our results reveal structural detail of three AcrIF proteins, each binding to a different site on the Csy complex for inhibiting degradation of MGEs.

scholarly journals Structure and inhibitor binding characterization of oncogenic MLLT1 mutants

2021 ◽  
Author(s):  
Xiaomin Ni ◽  
Allyn T. Londregan ◽  
Dafydd R. Owen ◽  
Stefan Knapp ◽  
Apirat Chaikuad
Keyword(s):  
Conformational Changes ◽  
Structural Basis ◽  
Wild Type ◽  
Structural Comparison ◽  
Inhibitor Binding ◽  
Binding Interface ◽  
Association Behavior ◽  
Self Association ◽  
Potential Strategy

AbstractDysfunction of YEATS-domain-containing MLLT1, an acetyl/acyl-lysine dependent epigenetic reader domain, has been implicated in the development of aggressive cancers. Mutations in the YEATS domain have been recently reported as a cause of MLLT1 aberrant reader function. However, structural basis for the reported alterations in affinity for acetyled/acylated histone has remained elusive. Here, we report the crystal structures of both insertion and substitution present in cancer, revealing significant conformational changes of the YEATS-domain loop 8. Structural comparison demonstrates that such alteration not only altered the binding interface for acetylated/acylated histones, but the sequence alterations in the T1 loop may enable dimeric assembly consistent inducing self-association behavior. Nevertheless, we show that also the MLLT1 mutants can be targeted by developed acetyllysine mimetic inhibitors with affinities similarly to wild type. Our report provides a structural basis for the altered behaviors and potential strategy for targeting oncogenic MLLT1 mutants.

scholarly journals Determinants shaping the nanoscale architecture of the mouse rod outer segment

2021 ◽  
Author(s):  
Matthias Pöge ◽  
Julia Mahamid ◽  
Sanae S Imanishi ◽  
Jürgen M Plitzko ◽  
Krzysztof Palczewski ◽  
...  
Keyword(s):  
Outer Segment ◽  
Molecular Mechanisms ◽  
Structural Information ◽  
Structural Basis ◽  
Radius Of Curvature ◽  
Rod Photoreceptor ◽  
Molecular Architecture ◽  
Structural Determinants ◽  
Rod Outer Segment ◽  
Wide Range

The unique membrane organization of the rod outer segment (ROS), the specialized sensory cilium of rod photoreceptor cells, provides the foundation for phototransduction, the initial step in vision. ROS architecture is characterized by a stack of identically shaped and tightly packed membrane disks loaded with the visual receptor rhodopsin. A wide range of genetic aberrations compromise ROS ultrastructure, impairing photoreceptor viability and function. Yet, the structural basis giving rise to the remarkable long range order of ROS membrane stacks and the molecular mechanisms underlying genetically inherited diseases remain elusive. Here, cryo-electron tomography (cryo-ET) performed on native ROS at molecular resolution provides insights into key structural determinants of ROS membrane architecture. Our data reveal the existence of two molecular connectors/spacers which likely contribute to the nanometer scale precise stacking of the ROS disks. We further show that the extreme radius of curvature at the disk rims is enforced by a continuous supramolecular assembly composed of peripherin-2 (PRPH2) and rod outer segment membrane protein 1 (ROM1) tetramers. We suggest that, together these molecular assemblies constitute the structural basis of the highly specialized ROS functional architecture. Cryo-ET therefore provides novel quantitative and structural information on the molecular architecture in ROS and insights into possible mechanisms underlying pathologies of certain PRPH2 mutations leading to blindness.

Redox- and pH-linked conformational changes in triheme cytochrome PpcA from Geobacter sulfurreducens

2017 ◽  
Vol 474 (2) ◽  
pp. 231-246 ◽  
Author(s):  
Leonor Morgado ◽  
Marta Bruix ◽  
P. Raj Pokkuluri ◽  
Carlos A. Salgueiro ◽  
David L. Turner
Keyword(s):  
Conformational Changes ◽  
Solution Structure ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Axial Ligand ◽  
Geobacter Sulfurreducens ◽  
Cellular Growth ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Natural Habitats

The periplasmic triheme cytochrome PpcA from Geobacter sulfurreducens is highly abundant; it is the likely reservoir of electrons to the outer surface to assist the reduction of extracellular terminal acceptors; these include insoluble metal oxides in natural habitats and electrode surfaces from which electricity can be harvested. A detailed thermodynamic characterization of PpcA showed that it has an important redox-Bohr effect that might implicate the protein in e−/H+ coupling mechanisms to sustain cellular growth. This functional mechanism requires control of both the redox state and the protonation state. In the present study, isotope-labeled PpcA was produced and the three-dimensional structure of PpcA in the oxidized form was determined by NMR. This is the first solution structure of a G. sulfurreducens cytochrome in the oxidized state. The comparison of oxidized and reduced structures revealed that the heme I axial ligand geometry changed and there were other significant changes in the segments near heme I. The pH-linked conformational rearrangements observed in the vicinity of the redox-Bohr center, both in the oxidized and reduced structures, constitute the structural basis for the differences observed in the pKa values of the redox-Bohr center, providing insights into the e−/H+ coupling molecular mechanisms driven by PpcA in G. sulfurreducens.

scholarly journals Determinants shaping the nanoscale architecture of the mouse rod outer segment

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Matthias Pöge ◽  
Julia Mahamid ◽  
Sanae S Imanishi ◽  
Jürgen M Plitzko ◽  
Krzysztof Palczewski ◽  
...  
Keyword(s):  
Outer Segment ◽  
Molecular Mechanisms ◽  
Structural Information ◽  
Structural Basis ◽  
Radius Of Curvature ◽  
Rod Photoreceptor ◽  
Molecular Architecture ◽  
Structural Determinants ◽  
Rod Outer Segment ◽  
Wide Range

The unique membrane organization of the rod outer segment (ROS), the specialized sensory cilium of rod photoreceptor cells, provides the foundation for phototransduction, the initial step in vision. ROS architecture is characterized by a stack of identically shaped and tightly packed membrane disks loaded with the visual receptor rhodopsin. A wide range of genetic aberrations have been reported to compromise ROS ultrastructure, impairing photoreceptor viability and function. Yet, the structural basis giving rise to the remarkably precise arrangement of ROS membrane stacks and the molecular mechanisms underlying genetically inherited diseases remain elusive. Here, cryo-electron tomography (cryo-ET) performed on native ROS at molecular resolution provides insights into key structural determinants of ROS membrane architecture. Our data confirm the existence of two previously observed molecular connectors/spacers which likely contribute to the nanometer-scale precise stacking of the ROS disks. We further provide evidence that the extreme radius of curvature at the disk rims is enforced by a continuous supramolecular assembly composed of peripherin-2 (PRPH2) and rod outer segment membrane protein 1 (ROM1) oligomers. We suggest that together these molecular assemblies constitute the structural basis of the highly specialized ROS functional architecture. Our Cryo-ET data provide novel quantitative and structural information on the molecular architecture in ROS and substantiate previous results on proposed mechanisms underlying pathologies of certain PRPH2 mutations leading to blindness.

scholarly journals An allosteric ligand stabilizes distinct conformations in the M2 muscarinic acetylcholine receptor

2021 ◽  
Author(s):  
Jun Xu ◽  
Harald Hübner ◽  
Yunfei Hu ◽  
Xiaogang Niu ◽  
Peter Gmeiner ◽  
...  
Keyword(s):  
Muscarinic Receptors ◽  
Conformational Changes ◽  
Rational Design ◽  
Molecular Mechanisms ◽  
Structural Information ◽  
Muscarinic Acetylcholine Receptor ◽  
Activation Process ◽  
Allosteric Modulators ◽  
Positive Allosteric Modulator ◽  
M2 Muscarinic Receptor

AbstractAllosteric modulators provide therapeutic advantages over orthosteric drugs. A plethora of allosteric modulators have been identified for several GPCRs, particularly for muscarinic receptors (mAChRs)1,2. To study the molecular mechanisms governing allosteric modulation, we utilized a recently developed NMR system to investigate the conformational changes in the M2 muscarinic receptor (M2R) in response to the positive allosteric modulator (PAM) LY2119620. Our studies provide the first biophysical data showing that LY2119620 can substantially change the structure and dynamics of M2R in both the extracellular and G-protein coupling domains during the activation process. These NMR data suggest that LY2119620 may function by stabilizing distinct sets of conformations not observed in the presence of orthosteric agonists alone, which may account for the different signaling behaviors of the M2R when bound to LY2119620. Our studies provide new structural information for understanding the mechanism of GPCR allostery, and may facilitate the rational design of allosteric therapeutics targeting muscarinic receptors.

scholarly journals Conformational dynamics of androgen receptors bound to agonists and antagonists

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hyo Jin Gim ◽  
Jiyong Park ◽  
Michael E. Jung ◽  
K. N. Houk
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Structural Integrity ◽  
Structural Information ◽  
Close Contact ◽  
Conformational Dynamics ◽  
Principal Component ◽  
Binding Pocket ◽  
Structural Basis ◽  
Accelerated Molecular Dynamics

AbstractThe androgen receptor (AR) is critical in the progression of prostate cancer (PCa). Small molecule antagonists that bind to the ligand binding domain (LBD) of the AR have been successful in treating PCa. However, the structural basis by which the AR antagonists manifest their therapeutic efficacy remains unclear, due to the lack of detailed structural information of the AR bound to the antagonists. We have performed accelerated molecular dynamics (aMD) simulations of LBDs bound to a set of ligands including a natural substrate (dihydrotestosterone), an agonist (RU59063) and three antagonists (bicalutamide, enzalutamide and apalutamide) as well as in the absence of ligand (apo). We show that the binding of AR antagonists at the substrate binding pocket alter the dynamic fluctuations of H12, thereby disrupting the structural integrity of the agonistic conformation of AR. Two antagonists, enzalutamide and apalutamide, induce considerable structural changes to the agonist conformation of LBD, when bound close to H12 of AR LBD. When the antagonists bind to the pocket with different orientations having close contact with H11, no significant conformational changes were observed, suggesting the AR remains in the functionally activated (agonistic) state. The simulations on a drug resistance mutant F876L bound to enzalutamide demonstrated that the mutation stabilizes the agonistic conformation of AR LBD, which compromises the efficacy of the antagonists. Principal component analysis (PCA) of the structural fluctuations shows that the binding of enzalutamide and apalutamide induce conformational fluctuations in the AR, which are markedly different from those caused by the agonist as well as another antagonist, bicalutamide. These fluctuations could only be observed with the use of aMD.

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