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Journal of Virology, January 2009, p. 1060-1070, Vol. 83, No. 2
0022-538X/09/$08.00+0     doi:10.1128/JVI.01325-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Mechanism of NS2B-Mediated Activation of NS3pro in Dengue Virus: Molecular Dynamics Simulations and Bioassays{triangledown}

Zhili Zuo,1 Oi Wah Liew,1 Gang Chen,1 Pek Ching Jenny Chong,1 Siew Hui Lee,1 Kaixian Chen,2 Hualiang Jiang,2 Chum Mok Puah,1* and Weiliang Zhu2*

Centre for Biomedical & Life Sciences, Singapore Polytechnic, 500 Dover Road, Singapore 139651,1 Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, People's Republic of China2

Received 25 June 2008/ Accepted 20 October 2008

The NS2B cofactor is critical for proteolytic activation of the flavivirus NS3 protease. To elucidate the mechanism involved in NS2B-mediated activation of NS3 protease, molecular dynamic simulation, principal component analysis, molecular docking, mutagenesis, and bioassay studies were carried out on both the dengue virus NS3pro and NS2B-NS3pro systems. The results revealed that the NS2B-NS3pro complex is more rigid than NS3pro alone due to its robust hydrogen bond and hydrophobic interaction networks within the complex. These potent networks lead to remodeling of the secondary and tertiary structures of the protease that facilitates cleavage sequence recognition and binding of substrates. The cofactor is also essential for proper domain motion that contributes to substrate binding. Hence, the NS2B cofactor plays a dual role in enzyme activation by facilitating the refolding of the NS3pro domain as well as being directly involved in substrate binding/interactions. Kinetic analyses indicated for the first time that Glu92 and Asp50 in NS2B and Gln27, Gln35, and Arg54 in NS3pro may provide secondary interaction points for substrate binding. These new insights on the mechanistic contributions of the NS2B cofactor to NS3 activation may be utilized to refine current computer-based search strategies to raise the quality of candidate molecules identified as potent inhibitors against flaviviruses.

* Corresponding author. Mailing address for C. M. Puah: Centre for Biomedical & Life Sciences, Singapore Polytechnic, 500 Dover Road, Singapore 139651. Phone: (65) 6772 1896. Fax: (65) 6870 8004. E-mail: puah{at}sp.edu.sg. Mailing address for W. Zhu: Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, People's Republic of China. Phone: 86-21-50805020. Fax: 86-21-50807088. E-mail: wlzhu{at}mail.shcnc.ac.cn

{triangledown} Published ahead of print on 29 October 2008.

Journal of Virology, January 2009, p. 1060-1070, Vol. 83, No. 2
0022-538X/09/$08.00+0     doi:10.1128/JVI.01325-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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