SASDBU4: Vaccinia primase D5 protein fragment containing the D5N and helic...

Basic information

• Entry: Database: SASBDB / ID: SASDBU4

Sample

Vaccinia primase D5 protein fragment containing the D5N and helicase domain

• Primase D5 protein fragment containing the D5N and helicase domain (protein), Vaccinia D5 323-785, Vaccinia virus

Function / homology

Function:

helicase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / host cell cytoplasm / hydrolase activity / ATP binding

Domain/homology:

DNA primase/nucleoside triphosphatase, C-terminal / Poxvirus D5 protein-like / Bacteriophage/plasmid primase, P4, C-terminal / D5 N terminal like / Helicase, superfamily 3, DNA virus / Superfamily 3 helicase of DNA viruses domain profile. / P-loop containing nucleoside triphosphate hydrolase

Component:

Uncoating factor OPG117

• Biological species: Vaccinia virus
• Citation: Journal: J Virol / Year: 2016; Title: Domain Organization of Vaccinia Virus Helicase-Primase D5.; Authors: Stephanie Hutin / Wai Li Ling / Adam Round / Gregory Effantin / Stefan Reich / Frédéric Iseni / Nicolas Tarbouriech / Guy Schoehn / Wim Pascal Burmeister / ; Abstract: Poxviridae are viruses with a large linear double-stranded DNA genome coding for up to 250 open reading frames and a fully cytoplasmic replication. The double-stranded DNA genome is covalently circularized at both ends. Similar structures of covalently linked extremities of the linear DNA genome are found in the African swine fever virus (asfarvirus) and in the Phycodnaviridae We are studying the machinery which replicates this peculiar genome structure. From our work with vaccinia virus, we give first insights into the overall structure and function of the essential poxvirus virus helicase-primase D5 and show that the active helicase domain of D5 builds a hexameric ring structure. This hexamer has ATPase and, more generally, nucleoside triphosphatase activities that are indistinguishable from the activities of full-length D5 and that are independent of the nature of the base. In addition, hexameric helicase domains bind tightly to single- and double-stranded DNA. Still, the monomeric D5 helicase construct truncated within the D5N domain leads to a well-defined structure, but it does not have ATPase or DNA-binding activity. This shows that the full D5N domain has to be present for hexamerization. This allowed us to assign a function to the D5N domain which is present not only in D5 but also in other viruses of the nucleocytoplasmic large DNA virus (NCLDV) clade. The primase domain and the helicase domain were structurally analyzed via a combination of small-angle X-ray scattering and, when appropriate, electron microscopy, leading to consistent low-resolution models of the different proteins.; IMPORTANCE: Since the beginning of the 1980s, research on the vaccinia virus replication mechanism has basically stalled due to the absence of structural information. As a result, this important class of pathogens is less well understood than most other viruses. This lack of information concerns in general viruses of the NCLDV clade, which use a superfamily 3 helicase for replication, as do poxviruses. Here we provide for the first time information about the domain structure and DNA-binding activity of D5, the poxvirus helicase-primase. This result not only refines the current model of the poxvirus replication fork but also will lead in the long run to a structural basis for antiviral drug design.

Contact author

• Stephanie Hutin

Models

• Model #534: ; Type: dummy / Software: DAMMIF (r3709) / Radius of dummy atoms: 3.00 A / Chi-square value: 1.096209 / P-value: 0.000004; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: Vaccinia primase D5 protein fragment containing the D5N and helicase domain; Specimen concentration: 1.42-2.20
• Buffer: Name: Tris / Concentration: 20.00 mM / pH: 7 / Composition: 150 mM NaCL, 5% glycerol, 1mM DTT

Entity #342

Name: Vaccinia D5 323-785 / Type: protein
Description: Primase D5 protein fragment containing the D5N and helicase domain
Formula weight: 53.48 / Num. of mol.: 6 / Source: Vaccinia virus / References: UniProt: P21010
Sequence:

GAMGNKLFNI AQRILDTNSV LLTERGDYIV WINNSWKFNS EEPLITKLIL SIRHQLPKEY SSELLCPRKR KTVEANIRDM LVDSVETDTY PDKLPFKNGV LDLVDGMFYS GDDAKKYTCT VSTGFKFDDT KFVEDSPEME ELMNIINDIQ PLTDENKKNR ELYEKTLSSC LCGATKGCLT FFFGETATGK STTKRLLKSA IGDLFVETGQ TILTDVLDKG PNPFIANMHL KRSVFCSELP DFACSGSKKI RSDNIKKLTE PCVIGRPCFS NKINNRNHAT IIIDTNYKPV FDRIDNALMR RIAVVRFRTH FSQPSGREAA ENNDAYDKVK LLDEGLDGKI QNNRYRFAFL YLLVKWYKKY HVPIMKLYPT PEEIPDFAFY LKIGTLLVSS SVKHIPLMTD LSKKGYILYD NVVTLPLTTF QQKISKYFNS RLFGHDIESF INRHKKFANV SDEYLQYIFI EDISSP

Experimental information

• Beam: Instrument name: ESRF BM29 / City: Grenoble / 国: France / Type of source: X-ray synchrotronSynchrotron / Wavelength: 0.93 Å / Dist. spec. to detc.: 2.43 mm
• Detector: Name: Pilatus 1M

Scan

Title: Vaccinia virus primase D5, amino acids 323-785 / Measurement date: Sep 26, 2014 / Storage temperature: 20 °C / Cell temperature: 20 °C / Exposure time: 2 sec. / Number of frames: 2000 / Unit: 1/nm /

	Min	Max
Q	0.1547	4.9693

Distance distribution function P(R)

Sofotware P(R): GNOM 4.6 / Number of points: 311 /

	Min	Max
Q	0.1973	1.665
P(R) point	10	320
R	0	14.45

Result

Type of curve: single_conc /

	Experimental	Porod
MW	345 kDa	-
Volume	-	570 nm3

	P(R)	P(R) error	Guinier	Guinier error
Forward scattering, I0	29	0.04	29.5	0.04
Radius of gyration, Rg	4.7 nm	0.006	4.8 nm	0.03

	Min	Max
D	-	14.5
Guinier point	2	24