Refolding of a fully functional flavivirus methyltransferase revealed that S-adenosyl methionine but not S-adenosyl homocysteine is copurified with flavivirus methyltransferase

Protein Sci. 2015 Jan;24(1):117-28. doi: 10.1002/pro.2594. Epub 2014 Dec 11.

Abstract

Methylation of flavivirus RNA is vital for its stability and translation in the infected host cell. This methylation is mediated by the flavivirus methyltransferase (MTase), which methylates the N7 and 2'-O positions of the viral RNA cap by using S-adenosyl-l-methionine (SAM) as a methyl donor. In this report, we demonstrate that SAM, in contrast to the reaction by-product S-adenosyl-l-homocysteine, which was assumed previously, is copurified with the Dengue (DNV) and West Nile virus MTases produced in Escherichia coli (E. coli). This endogenous SAM can be removed by denaturation and refolding of the MTase protein. The refolded MTase of DNV serotype 3 (DNV3) displays methylation activity comparable to native enzyme, and its crystal structure at 2.1 Å is almost identical to that of native MTase. We characterized the binding of Sinefungin (SIN), a previously described SAM-analog inhibitor of MTase function, to the native and refolded DNV3 MTase by isothermal titration calorimetry, and found that SIN binds to refolded MTase with more than 16 times the affinity of SIN binding to the MTase purified natively. Moreover, we show that SAM is also copurified with other flavivirus MTases, indicating that purification by refolding may be a generally applicable tool for studying flavivirus MTase inhibition.

Keywords: S-adenosyl methionine; flavivirus; methyltransferase; refolding.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Crystallography, X-Ray
  • Dengue / virology*
  • Dengue Virus / chemistry
  • Dengue Virus / enzymology*
  • Dengue Virus / metabolism
  • Humans
  • Methyltransferases / chemistry
  • Methyltransferases / isolation & purification
  • Methyltransferases / metabolism*
  • Models, Molecular
  • Protein Refolding
  • S-Adenosylhomocysteine / isolation & purification
  • S-Adenosylhomocysteine / metabolism*
  • S-Adenosylmethionine / isolation & purification
  • S-Adenosylmethionine / metabolism*
  • West Nile Fever / virology*
  • West Nile virus / chemistry
  • West Nile virus / enzymology*
  • West Nile virus / metabolism

Substances

  • S-Adenosylmethionine
  • S-Adenosylhomocysteine
  • Methyltransferases

Associated data

  • PDB/3EVA
  • PDB/3P8Z
  • PDB/4R05