scholarly journals NMR structure of a vestigial nuclease provides insight into the evolution of functional transitions in viral dsDNA packaging motors

2020 ◽  
Author(s):  
Bryon P. Mahler ◽  
Pawel J. Bujalowski ◽  
Huzhang Mao ◽  
Erik A. Dill ◽  
Paul J. Jardine ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Molecular Motors ◽  
Solution Structure ◽  
Nuclease Activity ◽  
Structural Basis ◽  
Binding Assays ◽  
Dna Viruses ◽  
Double Stranded Dna ◽  
Fluorescence Binding ◽  
Insight Into

SummaryDouble-stranded DNA viruses use ATP-powered molecular motors to package their genomes. To do so, these motors must efficiently transition between initiation, translocation, and termination modes. Here, we report structural and biophysical analyses of the C-terminal domain of the bacteriophage phi29 ATPase (CTD) that suggest a structural basis for these functional transitions. Sedimentation experiments show that the inter-domain linker in the full-length protein promotes dimerization and thus may play a role in assembly of the functional motor. The NMR solution structure of the CTD indicates it is a vestigial nuclease domain that likely evolved from conserved nuclease domains in phage terminases. Despite the loss of nuclease activity, fluorescence binding assays confirm the CTD retains its DNA binding capabilities and fitting the CTD into cryoEM density of the phi29 motor shows that the CTD directly binds DNA. However, the interacting residues differ from those identified by NMR titration in solution, suggesting that packaging motors undergo conformational changes to transition between initiation, translocation, and termination.

scholarly journals NMR structure of a vestigial nuclease provides insight into the evolution of functional transitions in viral dsDNA packaging motors

2020 ◽  
Vol 48 (20) ◽  
pp. 11737-11749 ◽  
Author(s):  
Bryon P Mahler ◽  
Paul J Bujalowski ◽  
Huzhang Mao ◽  
Erik A Dill ◽  
Paul J Jardine ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Molecular Motors ◽  
Solution Structure ◽  
Nuclease Activity ◽  
Structural Basis ◽  
Binding Assays ◽  
Dna Viruses ◽  
Double Stranded Dna ◽  
Fluorescence Binding ◽  
Insight Into

Abstract Double-stranded DNA viruses use ATP-powered molecular motors to package their genomic DNA. To ensure efficient genome encapsidation, these motors regulate functional transitions between initiation, translocation, and termination modes. Here, we report structural and biophysical analyses of the C-terminal domain of the bacteriophage phi29 ATPase (CTD) that suggest a structural basis for these functional transitions. Sedimentation experiments show that the inter-domain linker in the full-length protein promotes oligomerization and thus may play a role in assembly of the functional motor. The NMR solution structure of the CTD indicates it is a vestigial nuclease domain that likely evolved from conserved nuclease domains in phage terminases. Despite the loss of nuclease activity, fluorescence binding assays confirm the CTD retains its DNA binding capabilities and fitting the CTD into cryoEM density of the phi29 motor shows that the CTD directly binds DNA. However, the interacting residues differ from those identified by NMR titration in solution, suggesting that packaging motors undergo conformational changes to transition between initiation, translocation, and termination. Taken together, these results provide insight into the evolution of functional transitions in viral dsDNA packaging motors.

scholarly journals Principles for enhancing virus capsid capacity and stability from a thermophilic virus capsid structure

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Nicholas P. Stone ◽  
Gabriel Demo ◽  
Emily Agnello ◽  
Brian A. Kelch
Keyword(s):  
Major Capsid Protein ◽  
Extreme Environment ◽  
Structural Basis ◽  
Genome Packaging ◽  
Virus Capsid ◽  
Dna Viruses ◽  
Double Stranded Dna ◽  
High Internal Pressure ◽  
Extracellular Environment ◽  
Catch Bonds

Abstract The capsids of double-stranded DNA viruses protect the viral genome from the harsh extracellular environment, while maintaining stability against the high internal pressure of packaged DNA. To elucidate how capsids maintain stability in an extreme environment, we use cryoelectron microscopy to determine the capsid structure of thermostable phage P74-26 to 2.8-Å resolution. We find P74-26 capsids exhibit an overall architecture very similar to those of other tailed bacteriophages, allowing us to directly compare structures to derive the structural basis for enhanced stability. Our structure reveals lasso-like interactions that appear to function like catch bonds. This architecture allows the capsid to expand during genome packaging, yet maintain structural stability. The P74-26 capsid has T = 7 geometry despite being twice as large as mesophilic homologs. Capsid capacity is increased with a larger, flatter major capsid protein. Given these results, we predict decreased icosahedral complexity (i.e. T ≤ 7) leads to a more stable capsid assembly.

The genome of Oryctes rhinoceros nudivirus provides novel insight into the evolution of nuclear arthropod-specific large circular double-stranded DNA viruses

Virus Genes ◽  
2011 ◽  
Vol 42 (3) ◽  
pp. 444-456 ◽  
Author(s):  
Yongjie Wang ◽  
Olaf R. P. Bininda-Emonds ◽  
Monique M. van Oers ◽  
Just M. Vlak ◽  
Johannes A. Jehle
Keyword(s):  
Dna Viruses ◽  
Oryctes Rhinoceros ◽  
Double Stranded Dna ◽  
Insight Into

scholarly journals Insight into DNA and Protein Transport in Double-Stranded DNA Viruses: The Structure of Bacteriophage N4

2008 ◽  
Vol 378 (3) ◽  
pp. 726-736 ◽  
Author(s):  
Kyung H. Choi ◽  
Jennifer McPartland ◽  
Irene Kaganman ◽  
Valorie D. Bowman ◽  
Lucia B. Rothman-Denes ◽  
...  
Keyword(s):  
Protein Transport ◽  
Dna Viruses ◽  
Double Stranded Dna ◽  
Bacteriophage N4 ◽  
Insight Into

scholarly journals Cryo-EM structure and in vitro DNA packaging of a thermophilic virus with supersized T=7 capsids

2019 ◽  
Vol 116 (9) ◽  
pp. 3556-3561 ◽  
Author(s):  
Oliver W. Bayfield ◽  
Evgeny Klimuk ◽  
Dennis C. Winkler ◽  
Emma L. Hesketh ◽  
Maria Chechik ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Integrity ◽  
Thermus Thermophilus ◽  
Dna Packaging ◽  
Dna Viruses ◽  
Portal Protein ◽  
Single Vertex ◽  
Double Stranded Dna ◽  
Β Sheets

Double-stranded DNA viruses, including bacteriophages and herpesviruses, package their genomes into preformed capsids, using ATP-driven motors. Seeking to advance structural and mechanistic understanding, we established in vitro packaging for a thermostable bacteriophage, P23-45 of Thermus thermophilus. Both the unexpanded procapsid and the expanded mature capsid can package DNA in the presence of packaging ATPase over the 20 °C to 70 °C temperature range, with optimum activity at 50 °C to 65 °C. Cryo-EM reconstructions for the mature and immature capsids at 3.7-Å and 4.4-Å resolution, respectively, reveal conformational changes during capsid expansion. Capsomer interactions in the expanded capsid are reinforced by formation of intersubunit β-sheets with N-terminal segments of auxiliary protein trimers. Unexpectedly, the capsid has T=7 quasi-symmetry, despite the P23-45 genome being twice as large as those of known T=7 phages, in which the DNA is compacted to near-crystalline density. Our data explain this anomaly, showing how the canonical HK97 fold has adapted to double the volume of the capsid, while maintaining its structural integrity. Reconstructions of the procapsid and the expanded capsid defined the structure of the single vertex containing the portal protein. Together with a 1.95-Å resolution crystal structure of the portal protein and DNA packaging assays, these reconstructions indicate that capsid expansion affects the conformation of the portal protein, while still allowing DNA to be packaged. These observations suggest a mechanism by which structural events inside the capsid can be communicated to the outside.

scholarly journals Structural basis for adenylation and thioester bond formation in the ubiquitin E1

2019 ◽  
Vol 116 (31) ◽  
pp. 15475-15484 ◽  
Author(s):  
Zachary S. Hann ◽  
Cheng Ji ◽  
Shaun K. Olsen ◽  
Xuequan Lu ◽  
Michaelyn C. Lux ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Biochemical Analysis ◽  
Bond Formation ◽  
Structural Basis ◽  
Closed Conformation ◽  
Open Conformation ◽  
Thioester Bond ◽  
Catalytic Cysteine ◽  
Conjugating Enzymes ◽  
Insight Into

The ubiquitin (Ub) and Ub-like (Ubl) protein-conjugation cascade is initiated by E1 enzymes that catalyze Ub/Ubl activation through C-terminal adenylation, thioester bond formation with an E1 catalytic cysteine, and thioester bond transfer to Ub/Ubl E2 conjugating enzymes. Each of these reactions is accompanied by conformational changes of the E1 domain that contains the catalytic cysteine (Cys domain). Open conformations of the Cys domain are associated with adenylation and thioester transfer to E2s, while a closed conformation is associated with pyrophosphate release and thioester bond formation. Several structures are available for Ub E1s, but none has been reported in the open state before pyrophosphate release or in the closed state. Here, we describe the structures ofSchizosaccharomyces pombeUb E1 in these two states, captured using semisynthetic Ub probes. In the first, with a Ub-adenylate mimetic (Ub-AMSN) bound, the E1 is in an open conformation before release of pyrophosphate. In the second, with a Ub-vinylsulfonamide (Ub-AVSN) bound covalently to the catalytic cysteine, the E1 is in a closed conformation required for thioester bond formation. These structures provide further insight into Ub E1 adenylation and thioester bond formation. Conformational changes that accompany Cys-domain rotation are conserved for SUMO and Ub E1s, but changes in Ub E1 involve additional surfaces as mutational and biochemical analysis of residues within these surfaces alter Ub E1 activities.

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 Principles for enhancing virus capsid capacity and stability from a thermophilic virus capsid structure

2018 ◽  
Author(s):  
Nicholas P. Stone ◽  
Gabriel Demo ◽  
Emily Agnello ◽  
Brian A. Kelch
Keyword(s):  
Major Capsid Protein ◽  
Extreme Environment ◽  
Structural Basis ◽  
Genome Packaging ◽  
Virus Capsid ◽  
Dna Viruses ◽  
Double Stranded Dna ◽  
High Internal Pressure ◽  
Extracellular Environment ◽  
Catch Bonds

SUMMARYThe capsids of double-stranded DNA viruses protect the viral genome from the harsh extracellular environment, while maintaining stability against the high internal pressure of packaged DNA. To elucidate how capsids maintain stability in an extreme environment, we used cryoelectron microscopy to determine the capsid structure of the thermostable phage P74-26 to 2.8-Å resolution. We find the P74-26 capsid exhibits an overall architecture that is very similar to those of other tailed bacteriophages, allowing us to directly compare structures to derive the structural basis for enhanced stability. Our structure reveals ‘lasso’-like interactions that appear to function like catch bonds. This architecture allows the capsid to expand during genome packaging, yet maintain structural stability. The P74-26 capsid has T=7 geometry despite being twice as large as mesophilic homologs. Capsid capacity is increased through a novel mechanism with a larger, flatter major capsid protein. Our results suggest that decreased icosahedral complexity (i.e. lower T number) leads to a more stable capsid assembly.

scholarly journals Active liquid crystals powered by force-sensing DNA-motor clusters

2021 ◽  
Vol 118 (30) ◽  
pp. e2102873118
Author(s):  
Alexandra M. Tayar ◽  
Michael F. Hagan ◽  
Zvonimir Dogic
Keyword(s):  
Molecular Motors ◽  
Chemical Structure ◽  
Nonequilibrium Dynamics ◽  
Force Sensing ◽  
Critical Threshold ◽  
Nematic Order ◽  
Double Stranded Dna ◽  
Active Liquid ◽  
Energy Consuming ◽  
Insight Into

Cytoskeletal active nematics exhibit striking nonequilibrium dynamics that are powered by energy-consuming molecular motors. To gain insight into the structure and mechanics of these materials, we design programmable clusters in which kinesin motors are linked by a double-stranded DNA linker. The efficiency by which DNA-based clusters power active nematics depends on both the stepping dynamics of the kinesin motors and the chemical structure of the polymeric linker. Fluorescence anisotropy measurements reveal that the motor clusters, like filamentous microtubules, exhibit local nematic order. The properties of the DNA linker enable the design of force-sensing clusters. When the load across the linker exceeds a critical threshold, the clusters fall apart, ceasing to generate active stresses and slowing the system dynamics. Fluorescence readout reveals the fraction of bound clusters that generate interfilament sliding. In turn, this yields the average load experienced by the kinesin motors as they step along the microtubules. DNA-motor clusters provide a foundation for understanding the molecular mechanism by which nanoscale molecular motors collectively generate mesoscopic active stresses, which in turn power macroscale nonequilibrium dynamics of active nematics.

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