4R05

Crystal structure of the refolded DENV3 methyltransferase

Summary for 4R05

• Entry DOI: 10.2210/pdb4r05/pdb
• Descriptor: Nonstructural protein NS5 (2 entities in total)
• Functional Keywords: methyltransferase, flavivirus, transferase
• Biological source: Dengue virus 3
• Total number of polymer chains: 2
• Total formula weight: 60139.17

Authors

Brecher, M.B.,Li, Z.,Zhang, J.,Chen, H.,Lin, Q.,Liu, B.,Li, H.M. (deposition date: 2014-07-29, release date: 2014-11-12, Last modification date: 2023-09-20)

Primary citation

Brecher, M.B.,Li, Z.,Zhang, J.,Chen, H.,Lin, Q.,Liu, B.,Li, H.
Refolding of a fully functional flavivirus methyltransferase revealed that S-adenosyl methionine but not S-adenosyl homocysteine is copurified with flavivirus methyltransferase.
Protein Sci., 24:117-128, 2015

PubMed 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.

PubMed: 25352331
DOI: 10.1002/pro.2594

Experimental method

X-RAY DIFFRACTION (2.1 Å)