A crystal structure of the dengue virus non-structural protein 5 (NS5) polymerase delineates interdomain amino acid residues that enhance its thermostability and de novo initiation activities

Siew Pheng Lim, Jolene Hong Kiew Koh, Cheah Chen Seh, Chong Wai Liew, Andrew D. Davidson, Leng Shiew Chua, Ramya Chandrasekaran, Tobias C. Cornvik, Pei Yong Shi, Julien Lescar

Research output: Contribution to journalArticle

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Abstract

The dengue virus (DENV) non-structural protein 5 (NS5) comprises an N-terminal methyltransferase and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. Both enzymatic activities form attractive targets for antiviral development. Available crystal structures of NS5 fragments indicate that residues 263-271 (using the DENV serotype 3 numbering) located between the two globular domains of NS5 could be flexible. We observed that the addition of linker residues to the N-terminal end of the DENV RdRp core domain stabilizes DENV1-4 proteins and improves their de novo polymerase initiation activitiesby enhancing the turnoverofthe RNA and NTP substrates. Mutation studies of linker residues also indicate their importance for viral replication.We report the structure at 2.6-Å resolution of an RdRp fragment from DENV3 spanning residues 265-900 that has enhanced catalytic properties compared with the RdRp fragment (residues 272-900) reported previously. This new orthorhombic crystal form (space group P21212) comprises two polymerases molecules arranged as a dimer around a non-crystallographic dyad. The enzyme adopts a closed "preinitiation" conformation similartothe one that was captured previouslyin space group C2221 with one molecule per asymmetric unit. The structure reveals that residues 269-271 interact with the RdRp domain and suggests that residues 263-268 of the NS5 protein from DENV3 are the major contributors to the flexibility between its methyltransferase and RdRp domains. Together, these results should inform the screening and development of antiviral inhibitors directed against the DENV RdRp.

Original languageEnglish (US)
Pages (from-to)31105-31114
Number of pages10
JournalJournal of Biological Chemistry
Volume288
Issue number43
DOIs
StatePublished - Oct 25 2013
Externally publishedYes

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Viral Structural Proteins
RNA Replicase
Dengue Virus
Viruses
Crystal structure
Amino Acids
Proteins
Methyltransferases
Antiviral Agents
Molecules
Dimers
Conformations
Screening
RNA
Mutation
Crystals
Substrates
Enzymes

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

A crystal structure of the dengue virus non-structural protein 5 (NS5) polymerase delineates interdomain amino acid residues that enhance its thermostability and de novo initiation activities. / Lim, Siew Pheng; Koh, Jolene Hong Kiew; Seh, Cheah Chen; Liew, Chong Wai; Davidson, Andrew D.; Chua, Leng Shiew; Chandrasekaran, Ramya; Cornvik, Tobias C.; Shi, Pei Yong; Lescar, Julien.

In: Journal of Biological Chemistry, Vol. 288, No. 43, 25.10.2013, p. 31105-31114.

Research output: Contribution to journalArticle

Lim, Siew Pheng ; Koh, Jolene Hong Kiew ; Seh, Cheah Chen ; Liew, Chong Wai ; Davidson, Andrew D. ; Chua, Leng Shiew ; Chandrasekaran, Ramya ; Cornvik, Tobias C. ; Shi, Pei Yong ; Lescar, Julien. / A crystal structure of the dengue virus non-structural protein 5 (NS5) polymerase delineates interdomain amino acid residues that enhance its thermostability and de novo initiation activities. In: Journal of Biological Chemistry. 2013 ; Vol. 288, No. 43. pp. 31105-31114.
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abstract = "The dengue virus (DENV) non-structural protein 5 (NS5) comprises an N-terminal methyltransferase and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. Both enzymatic activities form attractive targets for antiviral development. Available crystal structures of NS5 fragments indicate that residues 263-271 (using the DENV serotype 3 numbering) located between the two globular domains of NS5 could be flexible. We observed that the addition of linker residues to the N-terminal end of the DENV RdRp core domain stabilizes DENV1-4 proteins and improves their de novo polymerase initiation activitiesby enhancing the turnoverofthe RNA and NTP substrates. Mutation studies of linker residues also indicate their importance for viral replication.We report the structure at 2.6-{\AA} resolution of an RdRp fragment from DENV3 spanning residues 265-900 that has enhanced catalytic properties compared with the RdRp fragment (residues 272-900) reported previously. This new orthorhombic crystal form (space group P21212) comprises two polymerases molecules arranged as a dimer around a non-crystallographic dyad. The enzyme adopts a closed {"}preinitiation{"} conformation similartothe one that was captured previouslyin space group C2221 with one molecule per asymmetric unit. The structure reveals that residues 269-271 interact with the RdRp domain and suggests that residues 263-268 of the NS5 protein from DENV3 are the major contributors to the flexibility between its methyltransferase and RdRp domains. Together, these results should inform the screening and development of antiviral inhibitors directed against the DENV RdRp.",
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T1 - A crystal structure of the dengue virus non-structural protein 5 (NS5) polymerase delineates interdomain amino acid residues that enhance its thermostability and de novo initiation activities

AU - Lim, Siew Pheng

AU - Koh, Jolene Hong Kiew

AU - Seh, Cheah Chen

AU - Liew, Chong Wai

AU - Davidson, Andrew D.

AU - Chua, Leng Shiew

AU - Chandrasekaran, Ramya

AU - Cornvik, Tobias C.

AU - Shi, Pei Yong

AU - Lescar, Julien

PY - 2013/10/25

Y1 - 2013/10/25

N2 - The dengue virus (DENV) non-structural protein 5 (NS5) comprises an N-terminal methyltransferase and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. Both enzymatic activities form attractive targets for antiviral development. Available crystal structures of NS5 fragments indicate that residues 263-271 (using the DENV serotype 3 numbering) located between the two globular domains of NS5 could be flexible. We observed that the addition of linker residues to the N-terminal end of the DENV RdRp core domain stabilizes DENV1-4 proteins and improves their de novo polymerase initiation activitiesby enhancing the turnoverofthe RNA and NTP substrates. Mutation studies of linker residues also indicate their importance for viral replication.We report the structure at 2.6-Å resolution of an RdRp fragment from DENV3 spanning residues 265-900 that has enhanced catalytic properties compared with the RdRp fragment (residues 272-900) reported previously. This new orthorhombic crystal form (space group P21212) comprises two polymerases molecules arranged as a dimer around a non-crystallographic dyad. The enzyme adopts a closed "preinitiation" conformation similartothe one that was captured previouslyin space group C2221 with one molecule per asymmetric unit. The structure reveals that residues 269-271 interact with the RdRp domain and suggests that residues 263-268 of the NS5 protein from DENV3 are the major contributors to the flexibility between its methyltransferase and RdRp domains. Together, these results should inform the screening and development of antiviral inhibitors directed against the DENV RdRp.

AB - The dengue virus (DENV) non-structural protein 5 (NS5) comprises an N-terminal methyltransferase and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. Both enzymatic activities form attractive targets for antiviral development. Available crystal structures of NS5 fragments indicate that residues 263-271 (using the DENV serotype 3 numbering) located between the two globular domains of NS5 could be flexible. We observed that the addition of linker residues to the N-terminal end of the DENV RdRp core domain stabilizes DENV1-4 proteins and improves their de novo polymerase initiation activitiesby enhancing the turnoverofthe RNA and NTP substrates. Mutation studies of linker residues also indicate their importance for viral replication.We report the structure at 2.6-Å resolution of an RdRp fragment from DENV3 spanning residues 265-900 that has enhanced catalytic properties compared with the RdRp fragment (residues 272-900) reported previously. This new orthorhombic crystal form (space group P21212) comprises two polymerases molecules arranged as a dimer around a non-crystallographic dyad. The enzyme adopts a closed "preinitiation" conformation similartothe one that was captured previouslyin space group C2221 with one molecule per asymmetric unit. The structure reveals that residues 269-271 interact with the RdRp domain and suggests that residues 263-268 of the NS5 protein from DENV3 are the major contributors to the flexibility between its methyltransferase and RdRp domains. Together, these results should inform the screening and development of antiviral inhibitors directed against the DENV RdRp.

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