scholarly journals Solution Structure of the dATP-Inactivated Class I Ribonucleotide Reductase From Leeuwenhoekiella blandensis by SAXS and Cryo-Electron Microscopy

2021 ◽  
Vol 8 ◽  
Author(s):  
Mahmudul Hasan ◽  
Ipsita Banerjee ◽  
Inna Rozman Grinberg ◽  
Britt-Marie Sjöberg ◽  
Derek T. Logan
Keyword(s):  
Ribonucleotide Reductase ◽  
Solution Structure ◽  
Three Dimensional ◽  
Class I ◽  
Structural Basis ◽  
Α Subunit ◽  
Cryo Electron Microscopy ◽  
N Terminus ◽  
Small Domain ◽  
Essential Enzyme

The essential enzyme ribonucleotide reductase (RNR) is highly regulated both at the level of overall activity and substrate specificity. Studies of class I, aerobic RNRs have shown that overall activity is downregulated by the binding of dATP to a small domain known as the ATP-cone often found at the N-terminus of RNR subunits, causing oligomerization that prevents formation of a necessary α2β2 complex between the catalytic (α2) and radical generating (β2) subunits. In some relatively rare organisms with RNRs of the subclass NrdAi, the ATP-cone is found at the N-terminus of the β subunit rather than more commonly the α subunit. Binding of dATP to the ATP-cone in β results in formation of an unusual β4 tetramer. However, the structural basis for how the formation of the active complex is hindered by such oligomerization has not been studied. Here we analyse the low-resolution three-dimensional structures of the separate subunits of an RNR from subclass NrdAi, as well as the α4β4 octamer that forms in the presence of dATP. The results reveal a type of oligomer not previously seen for any class of RNR and suggest a mechanism for how binding of dATP to the ATP-cone switches off catalysis by sterically preventing formation of the asymmetrical α2β2 complex.

scholarly journals Structural Basis for Superoxide Activation of Flavobacterium johnsoniae Class I Ribonucleotide Reductase and for Radical Initiation by Its Dimanganese Cofactor

Biochemistry ◽  
2018 ◽  
Vol 57 (18) ◽  
pp. 2679-2693 ◽  
Author(s):  
Hannah R. Rose ◽  
Manas K. Ghosh ◽  
Ailiena O. Maggiolo ◽  
Christopher J. Pollock ◽  
Elizabeth J. Blaesi ◽  
...  
Keyword(s):  
Ribonucleotide Reductase ◽  
Class I ◽  
Structural Basis ◽  
Radical Initiation ◽  
Flavobacterium Johnsoniae

scholarly journals A Small Domain in the N Terminus of the Regulatory α-Subunit Kv2.3 Modulates Kv2.1 Potassium Channel Gating

1999 ◽  
Vol 19 (16) ◽  
pp. 6865-6873 ◽  
Author(s):  
Marı́a Dolores Chiara ◽  
Francisco Monje ◽  
Antonio Castellano ◽  
José López-Barneo
Keyword(s):  
Potassium Channel ◽  
Channel Gating ◽  
Α Subunit ◽  
N Terminus ◽  
Small Domain ◽  
Potassium Channel Gating

scholarly journals A peptide mimic of an antigenic loop of α-human chorionic gonadotropin hormone: solution structure and interaction with a llama VHH domain

2002 ◽  
Vol 366 (2) ◽  
pp. 415-422 ◽  
Author(s):  
Gilles FERRAT ◽  
Jean-Guillaume RENISIO ◽  
Xavier MORELLI ◽  
Jerry SLOOTSTRA ◽  
Rob MELOEN ◽  
...  
Keyword(s):  
Chemical Shift ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Solution Structure ◽  
Three Dimensional ◽  
Conformational Exchange ◽  
Dimensional Structure ◽  
Single Chain ◽  
Α Subunit ◽  
Human Chorionic Gonadotropin Hormone

The X-ray structure of a ternary complex between human chorionic gonadotropin hormone (hCG) and two Fvs recognizing its α and β subunits has been recently determined. The Fvs recognize the elongated hCG molecule by its two ends, one being the Leu-12–Cys-29 loop of the α subunit. We have designed and synthesized a 17-amino-acid peptide (named PepH14) derived from the sequence of this antigenic loop with the purpose of mimicking its three-dimensional structure and its affinity for antibodies. We have determined the solution structure of PepH14 by homonuclear NMR spectroscopy and derived distance restraints. Comparison of this structure with that of the corresponding antigenic loop of α-hCG reveals strong conformational similarities. In particular, the two pairs of residues that establish crucial contacts with the Fv fragment share the same conformation in PepH14 and in the authentic hormone loop. We propose a three-dimensional model of interaction of PepH14 with a llama VHH (VHH-H14) fragment cloned from a single-chain llama immunoglobulin raised against α-hCG. This model has been constrained by the chemical shift variations of the H14 1HN and 15N resonances monitored upon binding with PepH14. Mapping of the backbone chemical shift variations on the VHH structure determined by NMR indicates that PepH14 binds to VHH-H14 and forms a complex using the three complementary determining regions (CDRs). They define a shallow groove encompassing residues Thr-31, Ala-56, Tyr-59 and Trp-104 which have been shown to be in conformational exchange [Renisio, Pérez, Czisch, Guenneugues, Bornet, Frenken, Cambillau and Darbon (2002) Proteins 47, 546–555] and also Phe-37 and Ala-50. This groove is close to the hydrophobic interface area observed between VH and VL domains in Fvs from classical antibodies, which explains the rather lateral binding of PepH14 on the VHH.

scholarly journals Purification and Crystallization Reveal Two Types of Interactions of the Fusion Protein Homotrimer of Semliki Forest Virus

2004 ◽  
Vol 78 (7) ◽  
pp. 3514-3523 ◽  
Author(s):  
Don L. Gibbons ◽  
Brigid Reilly ◽  
Anna Ahn ◽  
Marie-Christine Vaney ◽  
Armelle Vigouroux ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Semliki Forest Virus ◽  
Three Dimensional ◽  
Class Ii ◽  
Class I ◽  
Structural Basis ◽  
Viral Fusion ◽  
Purification Method ◽  
Viral Fusion Proteins

ABSTRACT The fusion proteins of the alphaviruses and flaviviruses have a similar native structure and convert to a highly stable homotrimer conformation during the fusion of the viral and target membranes. The properties of the alpha- and flavivirus fusion proteins distinguish them from the class I viral fusion proteins, such as influenza virus hemagglutinin, and establish them as the first members of the class II fusion proteins. Understanding how this new class carries out membrane fusion will require analysis of the structural basis for both the interaction of the protein subunits within the homotrimer and their interaction with the viral and target membranes. To this end we report a purification method for the E1 ectodomain homotrimer from the alphavirus Semliki Forest virus. The purified protein is trimeric, detergent soluble, retains the characteristic stability of the starting homotrimer, and is free of lipid and other contaminants. In contrast to the postfusion structures that have been determined for the class I proteins, the E1 homotrimer contains the fusion peptide region responsible for interaction with target membranes. This E1 trimer preparation is an excellent candidate for structural studies of the class II viral fusion proteins, and we report conditions that generate three-dimensional crystals suitable for analysis by X-ray diffraction. Determination of the structure will provide our first high-resolution views of both the low-pH-induced trimeric conformation and the target membrane-interacting region of the alphavirus fusion protein.

scholarly journals Metal-free class Ie ribonucleotide reductase from pathogens initiates catalysis with a tyrosine-derived dihydroxyphenylalanine radical

2018 ◽  
Vol 115 (40) ◽  
pp. 10022-10027 ◽  
Author(s):  
Elizabeth J. Blaesi ◽  
Gavin M. Palowitch ◽  
Kai Hu ◽  
Amelia J. Kim ◽  
Hannah R. Rose ◽  
...  
Keyword(s):  
Ribonucleotide Reductase ◽  
Metal Ion ◽  
Pathogenic Bacteria ◽  
Cysteine Residue ◽  
Class I ◽  
Tyrosyl Radical ◽  
Human Pathogens ◽  
Α Subunit ◽  
Metal Free ◽  
Iron Deprivation

All cells obtain 2′-deoxyribonucleotides for DNA synthesis through the activity of a ribonucleotide reductase (RNR). The class I RNRs found in humans and pathogenic bacteria differ in (i) use of Fe(II), Mn(II), or both for activation of the dinuclear-metallocofactor subunit, β; (ii) reaction of the reduced dimetal center with dioxygen or superoxide for this activation; (iii) requirement (or lack thereof) for a flavoprotein activase, NrdI, to provide the superoxide from O2; and (iv) use of either a stable tyrosyl radical or a high-valent dimetal cluster to initiate each turnover by oxidizing a cysteine residue in the α subunit to a radical (Cys•). The use of manganese by bacterial class I, subclass b-d RNRs, which contrasts with the exclusive use of iron by the eukaryotic Ia enzymes, appears to be a countermeasure of certain pathogens against iron deprivation imposed by their hosts. Here, we report a metal-free type of class I RNR (subclass e) from two human pathogens. The Cys• in its α subunit is generated by a stable, tyrosine-derived dihydroxyphenylalanine radical (DOPA•) in β. The three-electron oxidation producing DOPA• occurs inEscherichia colionly if the β is coexpressed with the NrdI activase encoded adjacently in the pathogen genome. The independence of this new RNR from transition metals, or the requirement for a single metal ion only transiently for activation, may afford the pathogens an even more potent countermeasure against transition metal-directed innate immunity.

scholarly journals Domain-swapped Dimerization of the Second PDZ Domain of ZO2 May Provide a Structural Basis for the Polymerization of Claudins

2007 ◽  
Vol 282 (49) ◽  
pp. 35988-35999 ◽  
Author(s):  
Jiawen Wu ◽  
Yinshan Yang ◽  
Jiahai Zhang ◽  
Peng Ji ◽  
Wenjing Du ◽  
...  
Keyword(s):  
Solution Structure ◽  
Pdz Domain ◽  
Three Dimensional ◽  
Structural Basis ◽  
Pdz Domains ◽  
Zonula Occludens ◽  
Associated Proteins ◽  
High Conservation

Zonula occludens proteins (ZOs), including ZO1/2/3, are tight junction-associated proteins. Each of them contains three PDZ domains. It has been demonstrated that ZO1 can form either homodimers or heterodimers with ZO2 or ZO3 through the second PDZ domain. However, the underlying structural basis is not well understood. In this study, the solution structure of the second PDZ domain of ZO2 (ZO2-PDZ2) was determined using NMR spectroscopy. The results revealed a novel dimerization mode for PDZ domains via three-dimensional domain swapping, which can be generalized to homodimers of ZO1-PDZ2 or ZO3-PDZ2 and heterodimers of ZO1-PDZ2/ZO2-PDZ2 or ZO1-PDZ2/ZO3-PDZ2 due to high conservation between PDZ2 domains in ZO proteins. Furthermore, GST pulldown experiments and immunoprecipitation studies demonstrated that interactions between ZO1-PDZ2 and ZO2-PDZ2 and their self-associations indeed exist both in vitro and in vivo. Chemical cross-linking and dynamic laser light scattering experiments revealed that both ZO1-PDZ2 and ZO2-PDZ2 can form oligomers in solution. This PDZ domain-mediated oligomerization of ZOs may provide a structural basis for the polymerization of claudins, namely the formation of tight junctions.

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 Structural basis of ribosomal RNA transcription regulation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yeonoh Shin ◽  
M. Zuhaib Qayyum ◽  
Danil Pupov ◽  
Daria Esyunina ◽  
Andrey Kulbachinskiy ◽  
...  
Keyword(s):  
Complex Formation ◽  
Ribosomal Rna ◽  
Structural Basis ◽  
Α Subunit ◽  
Promoter Escape ◽  
Cryo Electron Microscopy ◽  
Promoter Recognition ◽  
Carboxyl Terminal ◽  
Open Complex Formation ◽  
Template Dna

AbstractRibosomal RNA (rRNA) is most highly expressed in rapidly growing bacteria and is drastically downregulated under stress conditions by the global transcriptional regulator DksA and the alarmone ppGpp. Here, we determined cryo-electron microscopy structures of the Escherichia coli RNA polymerase (RNAP) σ70 holoenzyme during rRNA promoter recognition with and without DksA/ppGpp. RNAP contacts the UP element using dimerized α subunit carboxyl-terminal domains and scrunches the template DNA with the σ finger and β’ lid to select the transcription start site favorable for rapid promoter escape. Promoter binding induces conformational change of σ domain 2 that opens a gate for DNA loading and ejects σ1.1 from the RNAP cleft to facilitate open complex formation. DksA/ppGpp binding also opens the DNA loading gate, which is not coupled to σ1.1 ejection and impedes open complex formation. These results provide a molecular basis for the exceptionally active rRNA transcription and its vulnerability to DksA/ppGpp.

scholarly journals Molecular and Structural Basis of the Proteasome α Subunit Assembly Mechanism Mediated by the Proteasome-Assembling Chaperone PAC3-PAC4 Heterodimer

2019 ◽  
Vol 20 (9) ◽  
pp. 2231 ◽  
Author(s):  
Tadashi Satoh ◽  
Maho Yagi-Utsumi ◽  
Kenta Okamoto ◽  
Eiji Kurimoto ◽  
Keiji Tanaka ◽  
...  
Keyword(s):  
Three Dimensional ◽  
26S Proteasome ◽  
Structural Basis ◽  
Three Dimensional Model ◽  
Homologous Proteins ◽  
Α Subunit ◽  
Subunit Assembly ◽  
Selective Degradation ◽  
Assembly Mechanism ◽  
Potential Basis

The 26S proteasome is critical for the selective degradation of proteins in eukaryotic cells. This enzyme complex is composed of approximately 70 subunits, including the structurally homologous proteins α1–α7, which combine to form heptameric rings. The correct arrangement of these α subunits is essential for the function of the proteasome, but their assembly does not occur autonomously. Assembly of the α subunit is assisted by several chaperones, including the PAC3-PAC4 heterodimer. In this study we showed that the PAC3-PAC4 heterodimer functions as a molecular matchmaker, stabilizing the α4-α5-α6 subcomplex during the assembly of the α-ring. We solved a 0.96-Å atomic resolution crystal structure for a PAC3 homodimer which, in conjunction with nuclear magnetic resonance (NMR) data, highlighted the mobility of the loop comprised of residues 51 to 61. Based on these structural and dynamic data, we created a three-dimensional model of the PAC3-4/α4/α5/α6 quintet complex, and used this model to investigate the molecular and structural basis of the mechanism of proteasome α subunit assembly, as mediated by the PAC3-PAC4 heterodimeric chaperone. Our results provide a potential basis for the development of selective inhibitors against proteasome biogenesis.

Structural basis of bacterial transcription activation

Science ◽  
2017 ◽  
Vol 358 (6365) ◽  
pp. 947-951 ◽  
Author(s):  
Bin Liu ◽  
Chuan Hong ◽  
Rick K. Huang ◽  
Zhiheng Yu ◽  
Thomas A. Steitz
Keyword(s):  
High Resolution ◽  
De Novo ◽  
Transcription Activation ◽  
Class I ◽  
Receptor Protein ◽  
Structural Basis ◽  
Complete Class ◽  
Bacterial Transcription ◽  
Cryo Electron Microscopy ◽  
Promoter Dna

In bacteria, the activation of gene transcription at many promoters is simple and only involves a single activator. The cyclic adenosine 3′,5′-monophosphate receptor protein (CAP), a classic activator, is able to activate transcription independently through two different mechanisms. Understanding the class I mechanism requires an intact transcription activation complex (TAC) structure at a high resolution. Here we report a high-resolution cryo–electron microscopy structure of an intact Escherichia coli class I TAC containing a CAP dimer, a σ70–RNA polymerase (RNAP) holoenzyme, a complete class I CAP-dependent promoter DNA, and a de novo synthesized RNA oligonucleotide. The structure shows how CAP wraps the upstream DNA and how the interactions recruit RNAP. Our study provides a structural basis for understanding how activators activate transcription through the class I recruitment mechanism.

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