Histidine Triad-like Motif of the Rotavirus NSP2 Octamer Mediates both RTPase and NTPase Activities

Rodrigo Vasquez-Del Carpio, Fernando D. Gonzalez-Nilo, Gonzalo Riadi, Zenobia F. Taraporewala, John T. Patton

Resultado de la investigación: Article

36 Citas (Scopus)

Resumen

Rotavirus NSP2 is an abundant non-structural RNA-binding protein essential for forming the viral factories that support replication of the double-stranded RNA genome. NSP2 exists as stable doughnut-shaped octamers within the infected cell, representing the tail-to-tail interaction of two tetramers. Extending diagonally across the surface of each octamer are four highly basic grooves that function as binding sites for single-stranded RNA. Between the N and C-terminal domains of each monomer is a deep electropositive cleft containing a catalytic site that hydrolyzes the γ-β phosphoanhydride bond of any NTP. The catalytic site has similarity to those of the histidine triad (HIT) family of nucleotide-binding proteins. Due to the close proximity of the grooves and clefts, we investigated the possibility that the RNA-binding activity of the groove promoted the insertion of the 5′-triphosphate moiety of the RNA into the cleft, and the subsequent hydrolysis of its γ-β phosphoanhydride bond. Our results show that NSP2 hydrolyzes the γP from RNAs and NTPs through Mg2+-dependent activities that proceed with similar reaction velocities, that require the catalytic His225 residue, and that produce a phosphorylated intermediate. Competition assays indicate that although both substrates enter the active site, RNA is the preferred substrate due to its higher affinity for the octamer. The RNA triphosphatase (RTPase) activity of NSP2 may account for the absence of the 5′-terminal γP on the (-) strands of the double-stranded RNA genome segments. This is the first report of a HIT-like protein with a multifunctional catalytic site, capable of accommodating both NTPs and RNAs during γP hydrolysis.

Idioma originalEnglish
Páginas (desde-hasta)539-554
Número de páginas16
PublicaciónJournal of Molecular Biology
Volumen362
N.º3
DOI
EstadoPublished - 22 sep 2006

Huella dactilar

Nucleoside-Triphosphatase
Rotavirus
Histidine
RNA
Catalytic Domain
Double-Stranded RNA
Hydrolysis
Genome
RNA-Binding Proteins
RNA triphosphatase
Carrier Proteins
Nucleotides
Binding Sites

ASJC Scopus subject areas

  • Molecular Biology

Citar esto

Vasquez-Del Carpio, Rodrigo ; Gonzalez-Nilo, Fernando D. ; Riadi, Gonzalo ; Taraporewala, Zenobia F. ; Patton, John T. / Histidine Triad-like Motif of the Rotavirus NSP2 Octamer Mediates both RTPase and NTPase Activities. En: Journal of Molecular Biology. 2006 ; Vol. 362, N.º 3. pp. 539-554.
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title = "Histidine Triad-like Motif of the Rotavirus NSP2 Octamer Mediates both RTPase and NTPase Activities",
abstract = "Rotavirus NSP2 is an abundant non-structural RNA-binding protein essential for forming the viral factories that support replication of the double-stranded RNA genome. NSP2 exists as stable doughnut-shaped octamers within the infected cell, representing the tail-to-tail interaction of two tetramers. Extending diagonally across the surface of each octamer are four highly basic grooves that function as binding sites for single-stranded RNA. Between the N and C-terminal domains of each monomer is a deep electropositive cleft containing a catalytic site that hydrolyzes the γ-β phosphoanhydride bond of any NTP. The catalytic site has similarity to those of the histidine triad (HIT) family of nucleotide-binding proteins. Due to the close proximity of the grooves and clefts, we investigated the possibility that the RNA-binding activity of the groove promoted the insertion of the 5′-triphosphate moiety of the RNA into the cleft, and the subsequent hydrolysis of its γ-β phosphoanhydride bond. Our results show that NSP2 hydrolyzes the γP from RNAs and NTPs through Mg2+-dependent activities that proceed with similar reaction velocities, that require the catalytic His225 residue, and that produce a phosphorylated intermediate. Competition assays indicate that although both substrates enter the active site, RNA is the preferred substrate due to its higher affinity for the octamer. The RNA triphosphatase (RTPase) activity of NSP2 may account for the absence of the 5′-terminal γP on the (-) strands of the double-stranded RNA genome segments. This is the first report of a HIT-like protein with a multifunctional catalytic site, capable of accommodating both NTPs and RNAs during γP hydrolysis.",
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Histidine Triad-like Motif of the Rotavirus NSP2 Octamer Mediates both RTPase and NTPase Activities. / Vasquez-Del Carpio, Rodrigo; Gonzalez-Nilo, Fernando D.; Riadi, Gonzalo; Taraporewala, Zenobia F.; Patton, John T.

En: Journal of Molecular Biology, Vol. 362, N.º 3, 22.09.2006, p. 539-554.

Resultado de la investigación: Article

TY - JOUR

T1 - Histidine Triad-like Motif of the Rotavirus NSP2 Octamer Mediates both RTPase and NTPase Activities

AU - Vasquez-Del Carpio, Rodrigo

AU - Gonzalez-Nilo, Fernando D.

AU - Riadi, Gonzalo

AU - Taraporewala, Zenobia F.

AU - Patton, John T.

PY - 2006/9/22

Y1 - 2006/9/22

N2 - Rotavirus NSP2 is an abundant non-structural RNA-binding protein essential for forming the viral factories that support replication of the double-stranded RNA genome. NSP2 exists as stable doughnut-shaped octamers within the infected cell, representing the tail-to-tail interaction of two tetramers. Extending diagonally across the surface of each octamer are four highly basic grooves that function as binding sites for single-stranded RNA. Between the N and C-terminal domains of each monomer is a deep electropositive cleft containing a catalytic site that hydrolyzes the γ-β phosphoanhydride bond of any NTP. The catalytic site has similarity to those of the histidine triad (HIT) family of nucleotide-binding proteins. Due to the close proximity of the grooves and clefts, we investigated the possibility that the RNA-binding activity of the groove promoted the insertion of the 5′-triphosphate moiety of the RNA into the cleft, and the subsequent hydrolysis of its γ-β phosphoanhydride bond. Our results show that NSP2 hydrolyzes the γP from RNAs and NTPs through Mg2+-dependent activities that proceed with similar reaction velocities, that require the catalytic His225 residue, and that produce a phosphorylated intermediate. Competition assays indicate that although both substrates enter the active site, RNA is the preferred substrate due to its higher affinity for the octamer. The RNA triphosphatase (RTPase) activity of NSP2 may account for the absence of the 5′-terminal γP on the (-) strands of the double-stranded RNA genome segments. This is the first report of a HIT-like protein with a multifunctional catalytic site, capable of accommodating both NTPs and RNAs during γP hydrolysis.

AB - Rotavirus NSP2 is an abundant non-structural RNA-binding protein essential for forming the viral factories that support replication of the double-stranded RNA genome. NSP2 exists as stable doughnut-shaped octamers within the infected cell, representing the tail-to-tail interaction of two tetramers. Extending diagonally across the surface of each octamer are four highly basic grooves that function as binding sites for single-stranded RNA. Between the N and C-terminal domains of each monomer is a deep electropositive cleft containing a catalytic site that hydrolyzes the γ-β phosphoanhydride bond of any NTP. The catalytic site has similarity to those of the histidine triad (HIT) family of nucleotide-binding proteins. Due to the close proximity of the grooves and clefts, we investigated the possibility that the RNA-binding activity of the groove promoted the insertion of the 5′-triphosphate moiety of the RNA into the cleft, and the subsequent hydrolysis of its γ-β phosphoanhydride bond. Our results show that NSP2 hydrolyzes the γP from RNAs and NTPs through Mg2+-dependent activities that proceed with similar reaction velocities, that require the catalytic His225 residue, and that produce a phosphorylated intermediate. Competition assays indicate that although both substrates enter the active site, RNA is the preferred substrate due to its higher affinity for the octamer. The RNA triphosphatase (RTPase) activity of NSP2 may account for the absence of the 5′-terminal γP on the (-) strands of the double-stranded RNA genome segments. This is the first report of a HIT-like protein with a multifunctional catalytic site, capable of accommodating both NTPs and RNAs during γP hydrolysis.

KW - NSP2

KW - NTPase activity

KW - rotavirus replication

KW - RTPase activity

KW - viroplasm formation

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U2 - 10.1016/j.jmb.2006.07.050

DO - 10.1016/j.jmb.2006.07.050

M3 - Article

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AN - SCOPUS:33748269289

VL - 362

SP - 539

EP - 554

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 3

ER -