PDB-7jsg: Adeno-Associated Virus 2 Rep68 HD-Heptamer-ssDNA with ATPgS

Basic information

• Entry: Database: PDB / ID: 7jsg
• Title: Adeno-Associated Virus 2 Rep68 HD-Heptamer-ssDNA with ATPgS

Components

• DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
• Protein Rep68

• Keywords: VIRAL PROTEIN/DNA / AAV / Protein-DNA / AAA+ / SF3 / HUH / VIRAL PROTEIN / VIRAL PROTEIN-DNA complex

Function / homology

Function:

symbiont-mediated arrest of host cell cycle during G2/M transition / permeabilization of host organelle membrane involved in viral entry into host cell / symbiont entry into host cell via permeabilization of inner membrane / viral DNA genome replication / symbiont-mediated perturbation of host cell cycle G1/S transition checkpoint / endonuclease activity / DNA replication / DNA helicase / host cell nucleus / ATP hydrolysis activity / DNA binding / ATP binding / metal ion binding

Domain/homology:

Rep protein catalytic-like / : / Rep protein catalytic domain like / Parvovirus (PV) NS1 nuclease (NS1-Nuc) domain profile. / Parvovirus non-structural protein 1, helicase domain / Parvovirus non-structural protein NS1 / Helicase, superfamily 3, DNA virus / Superfamily 3 helicase of DNA viruses domain profile. / P-loop containing nucleoside triphosphate hydrolase

Component:

DNA / DNA (> 10) / Protein Rep68

• Biological species: Adeno-associated virus - 2; synthetic construct (others)
• Method: ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.2 Å
• Authors: Escalante, C.R.

Funding support

United States, 1items

Organization	Grant number	Country
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)	GM124204	United States

• Citation: Journal: Nucleic Acids Res / Year: 2020; Title: The Cryo-EM structure of AAV2 Rep68 in complex with ssDNA reveals a malleable AAA+ machine that can switch between oligomeric states.; Authors: Vishaka Santosh / Faik N Musayev / Rahul Jaiswal / Francisco Zárate-Pérez / Bram Vandewinkel / Caroline Dierckx / Molly Endicott / Kamyar Sharifi / Kelly Dryden / Els Henckaerts / Carlos R Escalante / ; Abstract: The adeno-associated virus (AAV) non-structural Rep proteins catalyze all the DNA transactions required for virus viability including, DNA replication, transcription regulation, genome packaging, and during the latent phase, site-specific integration. Rep proteins contain two multifunctional domains: an Origin Binding Domain (OBD) and a SF3 helicase domain (HD). Studies have shown that Rep proteins have a dynamic oligomeric behavior where the nature of the DNA substrate molecule modulates its oligomeric state. In the presence of ssDNA, Rep68 forms a large double-octameric ring complex. To understand the mechanisms underlying AAV Rep function, we investigated the cryo-EM and X-ray structures of Rep68-ssDNA complexes. Surprisingly, Rep68 generates hybrid ring structures where the OBD forms octameric rings while the HD forms heptamers. Moreover, the binding to ATPγS promotes a large conformational change in the entire AAA+ domain that leads the HD to form both heptamer and hexamers. The HD oligomerization is driven by an interdomain linker region that acts as a latch to 'catch' the neighboring HD subunit and is flexible enough to permit the formation of different stoichiometric ring structures. Overall, our studies show the structural basis of AAV Rep's structural flexibility required to fulfill its multifunctional role during the AAV life cycle.

History

Deposition	Aug 14, 2020	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Dec 9, 2020	Provider: repository / Type: Initial release
Revision 1.1	Dec 16, 2020	Group: Database references / Category: citation / citation_author Item: _citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2	Dec 23, 2020	Group: Database references / Category: citation Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3	Mar 6, 2024	Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2 Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

Assembly

Deposited unit

A: Protein Rep68

B: Protein Rep68

C: Protein Rep68

D: Protein Rep68

E: Protein Rep68

F: Protein Rep68

G: Protein Rep68

N: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')

Summary:

• 431 kDa, 8 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	431,487	8
Polymers	431,487	8
Non-polymers	0	0
Water	0	0

1

Summary:

• Idetical with deposited unit
• defined by author

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555		1

Components

#1: Protein

A B C D E F G
Protein Rep68

Details:

Mass: 60908.703 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Adeno-associated virus - 2 / Gene: Rep68
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P03132, DNA helicase

#2: DNA chain

N DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')

Details:

Mass: 5126.320 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

Experimental details

Experiment

• Experiment: Method: ELECTRON MICROSCOPY
• EM experiment: Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

Sample preparation

• Component: Name: Rep68 Helicase in complex with ssDNA (poly-dT) / Type: COMPLEX / Details: Heptameric complex model of helicase domain / Entity ID: all / Source: MULTIPLE SOURCES
• Molecular weight: Value: 0.4245 MDa / Experimental value: YES

Source (natural)

ID	Entity assembly-ID	Organism	Ncbi tax-ID
1	1	Adeno-associated virus - 2	10804
2	1	synthetic construct (others)	32630

• Source (recombinant): Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
• Buffer solution: pH: 7.9

Buffer component