scholarly journals Large Terminase Conformational Change Induced by Connector Binding in Bacteriophage T7

2013 ◽  
Vol 288 (23) ◽  
pp. 16998-17007 ◽  
Author(s):  
María I. Daudén ◽  
Jaime Martín-Benito ◽  
Juan C. Sánchez-Ferrero ◽  
Mar Pulido-Cid ◽  
José M. Valpuesta ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Bacteriophage T7 ◽  
Crystallographic Structure ◽  
Dna Translocation ◽  
Phage T4 ◽  
Subunit Rotation ◽  
Molecular Bases ◽  
Mechanical Movement ◽  
Large Terminase ◽  
Shed Light

During bacteriophage morphogenesis DNA is translocated into a preformed prohead by the complex formed by the portal protein, or connector, plus the terminase, which are located at an especial prohead vertex. The terminase is a powerful motor that converts ATP hydrolysis into mechanical movement of the DNA. Here, we have determined the structure of the T7 large terminase by electron microscopy. The five terminase subunits assemble in a toroid that encloses a channel wide enough to accommodate dsDNA. The structure of the complete connector-terminase complex is also reported, revealing the coupling between the terminase and the connector forming a continuous channel. The structure of the terminase assembled into the complex showed a different conformation when compared with the isolated terminase pentamer. To understand in molecular terms the terminase morphological change, we generated the terminase atomic model based on the crystallographic structure of its phage T4 counterpart. The docking of the threaded model in both terminase conformations showed that the transition between the two states can be achieved by rigid body subunit rotation in the pentameric assembly. The existence of two terminase conformations and its possible relation to the sequential DNA translocation may shed light into the molecular bases of the packaging mechanism of bacteriophage T7.

scholarly journals Assisted assembly of bacteriophage T7 core components for genome translocation across the bacterial envelope

2021 ◽  
Vol 118 (34) ◽  
pp. e2026719118
Author(s):  
Mar Pérez-Ruiz ◽  
Mar Pulido-Cid ◽  
Juan Román Luque-Ortega ◽  
José María Valpuesta ◽  
Ana Cuervo ◽  
...  
Keyword(s):  
Bacteriophage T7 ◽  
Dna Translocation ◽  
Oligomeric State ◽  
Viral Dna ◽  
The Core ◽  
Cryo Electron Microscopy ◽  
Core Proteins ◽  
Core Components ◽  
Bacterial Cytoplasm ◽  
Bacterial Peptidoglycan

In most bacteriophages, genome transport across bacterial envelopes is carried out by the tail machinery. In viruses of the Podoviridae family, in which the tail is not long enough to traverse the bacterial wall, it has been postulated that viral core proteins assembled inside the viral head are translocated and reassembled into a tube within the periplasm that extends the tail channel. Bacteriophage T7 infects Escherichia coli, and despite extensive studies, the precise mechanism by which its genome is translocated remains unknown. Using cryo-electron microscopy, we have resolved the structure of two different assemblies of the T7 DNA translocation complex composed of the core proteins gp15 and gp16. Gp15 alone forms a partially folded hexamer, which is further assembled upon interaction with gp16 into a tubular structure, forming a channel that could allow DNA passage. The structure of the gp15–gp16 complex also shows the location within gp16 of a canonical transglycosylase motif involved in the degradation of the bacterial peptidoglycan layer. This complex docks well in the tail extension structure found in the periplasm of T7-infected bacteria and matches the sixfold symmetry of the phage tail. In such cases, gp15 and gp16 that are initially present in the T7 capsid eightfold-symmetric core would change their oligomeric state upon reassembly in the periplasm. Altogether, these results allow us to propose a model for the assembly of the core translocation complex in the periplasm, which furthers understanding of the molecular mechanism involved in the release of T7 viral DNA into the bacterial cytoplasm.

scholarly journals Bacteriophage T7 DNA Translocation during Infection is Bolstered by the Host ATP Synthase Complex

2017 ◽  
Vol 112 (3) ◽  
pp. 335a
Author(s):  
Dustin R. Morado ◽  
Chunyan Wang ◽  
Bo Hu ◽  
Ian Molineux ◽  
Jun Liu
Keyword(s):  
Atp Synthase ◽  
Bacteriophage T7 ◽  
Dna Translocation

High affinity Ca2+–Mg2+ ATPase in the distal tubule of the mouse kidney

1987 ◽  
Vol 65 (10) ◽  
pp. 2093-2098 ◽  
Author(s):  
Michèle G. Brunette ◽  
Sylvie Blouin ◽  
Meathan Chan
Keyword(s):  
Proximal Tubule ◽  
Kinetic Parameters ◽  
Atp Hydrolysis ◽  
Limit Of Detection ◽  
Distal Tubule ◽  
Mouse Kidney ◽  
Distal Nephron ◽  
Low Concentrations ◽  
Distal Tubules ◽  
Shed Light

The purpose of this study was to investigate whether Ca2+–Mg2+ ATPase in the distal tubule (where calcium transport is active, against a gradient, and hormone dependent) presents some characteristics different from those observed in the proximal tubule, and whether these characteristics are likely to shed light on the respective roles of this enzyme at the two sites of the nephron. The Ca2+- and Mg2+-dependent ATP hydrolysis was measured in microdissected segments of the distal nephron, the kinetic parameters were determined, and the influence of magnesium upon the sensitivity to calcium was examined. Results were compared with those obtained in the proximal tubule, and in purified membranes as reported by others. In the distal tubule, low concentrations of Mg2+ (< 10−7 M) did not influence ATP hydrolysis. At concentrations above 10−7 M, Mg2+ increased ATP hydrolysis according to Michaelis kinetics (apparent Km = 11.3 ± 2.4 μM, Vmax = 219 ± 26 pmol∙mm−1∙20 min−1). The addition of 1 μM Ca2+ decreased the apparent Km for Mg2+ and the Vmax for Mg2+. Similar results were obtained in the proximal tubule. At low Mg2+ concentrations, Ca2+ also stimulated ATP hydrolysis according to Michaelis kinetics with an apparent Km value for Ca2+ of 0.18 ± 0.06 and 0.10 ± 0.03 μM Ca2+ (ns) and a Vmax of 101 ± 12 and 89 ± 9 pmol∙mm−1∙20 min−1 (ns) in the distal and proximal tubules, respectively. In the two segments, the addition of Mg2+ strongly decreased the sensitivity to 1 μM Ca2+ so that at 1 mM Mg2+, the Ca2+-dependent ATPase activity was at the limit of detection. In conclusion, the kinetic parameters of the Ca2+- and Mg2+-dependent ATP hydrolysis were similar at the two sites of the nephron, and were also similar to those reported for the enzyme present in purified basolateral membranes. The nonadditive effect of the two cations Ca2+ and Mg2+ suggests that the two ATPase activities belong to the same enzyme, and this enzyme is the same in the proximal and distal tubules. Differences in Ca2+ transport characteristics should be attributed to factors other than variations in the nature of the Ca2+–Mg2+ ATPase.

scholarly journals Potency of SARS-CoV-2 on Ocular Tissues

2021 ◽  
Author(s):  
Saliha Durak ◽  
Hande Eda Sutova ◽  
Abuzer Alp Yetisgin ◽  
Ozlem Kutlu ◽  
Sibel Cetinel
Keyword(s):  
Defense Mechanism ◽  
Nasolacrimal Duct ◽  
Cell Entry ◽  
Ocular Involvement ◽  
Ocular Tissues ◽  
Disease Outcomes ◽  
The World ◽  
Tract Involvement ◽  
Molecular Bases ◽  
Shed Light

The current COVID-19 pandemic has affected more than 100 million people and resulted in morbidity and mortality around the world. Even though the disease caused by SARS-CoV-2 is characterized by respiratory tract involvement, previous and recent data also indicates ocular manifestation. Not surprisingly, cell entry point of the virus, ACE2 receptor, is widely expressed in ocular tissues ranging from conjunctiva to retina. Despite the sensibility of ocular tissues, the sophisticated defense mechanism of the eye might eliminate viral transmission. Nevertheless, the potential of systemic transmission through the nasolacrimal duct may not be eliminated. In the case of ocular involvement, the disease outcomes might be as treatable as conjunctivitis or as serious as retinal degeneration and the treatment regimen vary accordingly. Within these contingencies, our aim with this chapter is to shed light on molecular bases of SARS-CoV-2 infection, systemic invasiveness following ocular transmission, manifestation and permanent effects on ocular tissues.

scholarly journals The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

2016 ◽  
Author(s):  
Brendan J. Hilbert ◽  
Janelle A. Hayes ◽  
Nicholas P. Stone ◽  
Rui-Gang Xu ◽  
Brian A. Kelch
Keyword(s):  
Dna Binding ◽  
Active Site ◽  
Dna Cleavage ◽  
Atpase Domain ◽  
Dna Translocation ◽  
Genome Packaging ◽  
Virus Maturation ◽  
Nuclease Domain ◽  
Dna Packaging Motor ◽  
Large Terminase

AbstractMany viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the ATPase that powers DNA translocation and an endonuclease that cleaves the concatemeric genome both at initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage is still mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nucleolysis. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of nucleolysis suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid shell during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the procapsid upon completion of packaging unlocks the nuclease domains to cleave DNA.

scholarly journals A dual allosteric pathway drives human mitochondrial Lon

2021 ◽  
Author(s):  
Genis Valentin Gese ◽  
Saba Shahzad ◽  
Carlos Pardo-Hernandez ◽  
Anna Wramstedt ◽  
Maria Falkenberg ◽  
...  
Keyword(s):  
Active Sites ◽  
Matrix Protein ◽  
Atp Hydrolysis ◽  
Allosteric Regulation ◽  
Mitochondrial Transcription Factor ◽  
Allosteric Mechanism ◽  
Mitochondrial Transcription Factor A ◽  
Substrate Processing ◽  
Molecular Bases ◽  
Allosteric Pathway

The hexameric, barrel-forming, AAA+ protease Lon is critical for maintaining mitochondrial matrix protein homeostasis. Efficient substrate processing by Lon requires the coordinated action of six protomers. Despite Lon's importance for human health, the molecular bases for Lon's substrate recognition and processing remain unclear. Here, we use a combination of biochemistry and electron cryomicroscopy (cryo-EM) to unveil the structural and functional basis for full-length human mitochondrial Lon's degradation of mitochondrial transcription factor A (TFAM). We show how opposing protomers in the Lon hexamer barrel interact through their N-terminal domains to give what resembles three feet above the barrel and help to form a triangular pore located just above the entry pore to the barrel. The interactions between opposing protomers constitute a primary allosteric regulation of Lon activity. A secondary allosteric regulation consists of an inter-subunit signaling element in the ATPase domains. By considering the ATP or ADP load in each protomer, we show how this dual allosteric mechanism in Lon achieves coordinated ATP hydrolysis and substrate processing. This mechanism enforces sequential anti-clockwise ATP hydrolysis resulting in a coordinated hand-over-hand translocation of the substrate towards the protease active sites.

scholarly journals Single-Stranded DNA Translocation of E. coli UvrD Monomer Is Tightly Coupled to ATP Hydrolysis

2012 ◽  
Vol 418 (1-2) ◽  
pp. 32-46 ◽  
Author(s):  
Eric J. Tomko ◽  
Christopher J. Fischer ◽  
Timothy M. Lohman
Keyword(s):  
Atp Hydrolysis ◽  
Dna Translocation ◽  
Single Stranded Dna ◽  
E Coli ◽  
Tightly Coupled
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