Bacteriophage Lambda gpNu1 and <i>Escherichia coli</i> IHF Proteins Cooperatively Bind and Bend Viral DNA:  Implications for the Assembly of a Genome-Packaging Motor

Ortega, Marcos E.; Catalano, Carlos Enrique

doi:10.1021/bi052284b

Cited by 38 publications

(69 citation statements)

References 60 publications

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“…The initiation complex as discussed here (Fig. 4C) is similar to what has been proposed for phage λ except that in T4-like phages two rings of small terminases bind to the genomic DNA, whereas in phage λ a dimer of small terminase binds to the initiation sequences (23,48).…”

Section: Discussionsupporting

confidence: 71%

Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages

Sun

Gao

Kondabagil

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

123

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Tailed DNA bacteriophages assemble empty procapsids that are subsequently filled with the viral genome by means of a DNA packaging machine situated at a special fivefold vertex. The packaging machine consists of a "small terminase" and a "large terminase" component. One of the functions of the small terminase is to initiate packaging of the viral genome, whereas the large terminase is responsible for the ATP-powered translocation of DNA. The small terminase subunit has three domains, an N-terminal DNA-binding domain, a central oligomerization domain, and a C-terminal domain for interacting with the large terminase. Here we report structures of the central domain in two different oligomerization states for a small terminase from the T4 family of phages. In addition, we report biochemical studies that establish the function for each of the small terminase domains. On the basis of the structural and biochemical information, we propose a model for DNA packaging initiation.T4-like phages | X-ray crystal structure M ost tailed bacteriophages and some eukaryotic viruses employ a molecular motor to package their genomic DNA into preformed empty capsids (procapsids or proheads) (1-3). A large amount of negatively charged DNA is packaged into the confined space within the capsid to near crystalline density, creating a pressure of about 60 atm inside the head (4). To counteract the increasing pressure during DNA packaging, the motor needs to generate forces of up to 60 pN (4-6), which is about 20 times the force generated by myosin motors. The energy for genome packaging is provided by ATP hydrolysis.Generally, DNA packaging machines consist of three components (3, 7). The first component is a dodecameric portal protein located at the special fivefold vertex of the capsid through which the DNA is threaded into the head. Crystal structures of portal proteins from phages φ29 (8), SPP1 (9), and P22 (10) show that they form cone-shaped structures with a central cylindrical channel about 36 Å wide. Cryoelectron microscopy (cryo-EM) studies of phages φ29 (8), SPP1 (11), T4 (12), P22 (13,14), and ϵ15 (15) show that the wider end of the cone-shaped portal is inside the capsid, and the narrower end protrudes out of the capsid. The portal provides a site of attachment for the DNA packaging motor to the procapsid and for the tail to the filled capsid. To what extent the portal participates in DNA packaging is not clear, but it might act as a valve to stop the DNA from escaping the head during successive strokes of the packaging motor (16) and when completely packaged (17). It was also proposed that the portal might be involved in sensing when the head is fully packaged (18,19).The second component of the DNA packaging machine is the large "terminase" motor protein which has both an ATPase activity to provide energy for packaging and a nuclease activity for packaging initiation and termination (20). In most DNA phages, the newly replicated genome is a branched concatemer without any accessible free ends (3). It needs to be cleaved in ...

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Section: Discussionsupporting

confidence: 71%

Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages

Sun

Gao

Kondabagil

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

123

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“…We did not use DNA-interaction data available for the bacteriophage λ small terminase (13,25) because of significant differences in DBD folds and oligomeric states (dimeric species reported for λ are not observed in the SF6, Sf6, and phBC6A51 small terminases) and because of DNA bending in λ involving an additional host factor (26). Interestingly, when modeling the DNA, guided by HTH-DNA complexes of closest structural homologues (27), the DNA orientation is roughly parallel to the oligomer axis.…”

Section: Discussionmentioning

confidence: 99%

Structural basis for DNA recognition and loading into a viral packaging motor

Buttner

Chechik

Ortiz-Lombardı́a

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

106

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Genome packaging into preformed viral procapsids is driven by powerful molecular motors. The small terminase protein is essential for the initial recognition of viral DNA and regulates the motor's ATPase and nuclease activities during DNA translocation. The crystal structure of a full-length small terminase protein from the Siphoviridae bacteriophage SF6, comprising the N-terminal DNA binding, the oligomerization core, and the C-terminal β-barrel domains, reveals a nine-subunit circular assembly in which the DNA-binding domains are arranged around the oligomerization core in a highly flexible manner. Mass spectrometry analysis and four further crystal structures show that, although the full-length protein exclusively forms nine-subunit assemblies, protein constructs missing the C-terminal β-barrel form both nine-subunit and ten-subunit assemblies, indicating the importance of the C terminus for defining the oligomeric state. The mechanism by which a ring-shaped small terminase oligomer binds viral DNA has not previously been elucidated. Here, we probed binding in vitro by using EPR and surface plasmon resonance experiments, which indicated that interaction with DNA is mediated exclusively by the DNA-binding domains and suggested a nucleosome-like model in which DNA binds around the outside of the protein oligomer.bacteriophage SPP1 | DNA packaging | virus assembly | X-ray crystallography T he virus genome in tailed dsDNA bacteriophages and in the evolutionarily related herpes viruses is packaged into a preformed empty procapsid (1-3). A powerful ATP-fueled molecular machine drives the DNA with a speed of up to 1;800 bp∕s through the portal protein embedded in a unique vertex of the icosahedral procapsid (2-4). The molecular motor usually consists of the small and large terminase proteins. The small terminase plays a dual role in virus particle assembly: It (i) recognizes viral DNA during the initiation of packaging and (ii) modulates the ATPase and nuclease activities of the large terminase during DNA translocation (5, 6). After filling a procapsid, the rest of the DNA is then docked to the portal entrance of another procapsid where the process of DNA translocation is repeated (7).X-ray structures have been determined for portal proteins from bacteriophages φ29 (8), SPP1 (9), and P22 (10) and also for large terminases from bacteriophages T4 (6), RB49 (6), and SPP1 (11). Three-dimensional information on small terminases is limited to the cryo-EM structure of phage P22 small terminase (12), the NMR structure of the DNA-binding domain of phage λ gpNu1 (13), and the crystal structure of phage Sf6 small terminase (14). In the absence of accurate three-dimensional data for all three motor components of one particular phage, mapping of functional information to the structure and modeling molecular interactions between individual components is challenging. We have addressed this issue by extending the structural information on Bacillus subtilis bacteriophages SPP1 and SF6, two very closely related viruses of the Siphovi...

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“…The SPM also predicts the dependence of K obs of IHF-H′ DNA binding (or any other process) on the concentration of a nonelectrolyte like glycine betaine: (5) In equation 5, δ GB is a correction term (|δ GB | ≪ 1) for GB nonideality and concentration scale conversion. For glycine betaine, the composite term (K p − 1) ΔB H 2 O in Eq 5 has been dissected into contributions from different types of biopolymer surface.…”

Section: Thermodynamic Analysis Of Coulombic and Hofmeister-osmotic Cmentioning

confidence: 99%

Formation of a Wrapped DNA–Protein Interface: Experimental Characterization and Analysis of the Large Contributions of Ions and Water to the Thermodynamics of Binding IHF to H′ DNA

Meulen

Saecker

Record

2008

Journal of Molecular Biology

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To characterize driving forces and driven processes in formation of a large-interface, wrapped protein-DNA complex analogous to the nucleosome, we have investigated the thermodynamics of binding the 34 bp H′ DNA sequence to the E. coli DNA-remodeling protein Integration Host Factor (IHF). Isothermal titration calorimetry (ITC) and fluorescence resonance energy transfer (FRET) are applied to determine effects of salt concentration (KCl, KF, KGlutamate (KGlu) A novel analysis of the large effects of anion identity on K obs , SK obs and on ΔH°o bs dissects coulombic, Hofmeister and osmotic contributions to these quantities. This analysis attributes anionspecific differences in K obs , SK obs and ΔH°o bs to (i) displacement of a large number of waters of hydration (estimated to be 1.0 (± 0.2) × 10 3 ) from the 5340 Å 2 of IHF and H′ DNA surface buried in complex formation, and (ii) significant local exclusion of F − and Glu − from this hydration water, relative to the situation with Cl − , which we propose is randomly distributed. To quantify net water release from anionic surface (22% of the surface buried in complexation, mostly from DNA phosphates), we determined the stabilizing effect of glycine betaine (GB) on K obs : dlnK obs /d[GB] = 2.7 ± 0.4 at constant KCl activity, indicating the net release of 150 H 2 O from anionic surface.

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Bacteriophage Lambda gpNu1 and Escherichia coli IHF Proteins Cooperatively Bind and Bend Viral DNA: Implications for the Assembly of a Genome-Packaging Motor

Cited by 38 publications

References 60 publications

Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages

Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages

Structural basis for DNA recognition and loading into a viral packaging motor

Formation of a Wrapped DNA–Protein Interface: Experimental Characterization and Analysis of the Large Contributions of Ions and Water to the Thermodynamics of Binding IHF to H′ DNA

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