Crystal structures of full length DENV4 NS2B-NS3 reveal the dynamic interaction between NS2B and NS3 upon binding to protease inhibitors

Abstract

Flavivirus is a genus of emerging and re-emerging arboviruses which include many significant human pathogens. Non-structural protein 3 (NS3), a multifunctional protein with N-terminal protease and C-terminal helicase, is essential in viral replication. The NS3 protease together with NS2B cofactor is an attractive antiviral target. A construct with an artificial glycine linker connecting the NS2B cofactor and NS3 protease has been used for structural, biochemical and drug-screening studies. The effect of this linker on dynamics and enzymatic activity of the protease was studied by several biochemical and NMR methods but the findings remained inconclusive. Here, we designed constructs of NS2B cofactor joined to full length DENV4 NS3 in three different manners, namely bNS2B47NS3 (bivalent), eNS2B47NS3(enzymatically cleavable) and gNS2B47NS3 (glycine-rich G4SG4 linker). We report the first crystal structures of linked and unlinked full-length NS2B-NS3 enzyme in its free state and also in complex with Bovine Pancreatic Trypsin Inhibitor (BPTI). These structures demonstrate that the NS2B-NS3 protease mainly adopts a closed conformation. BPTI binding is not essential to but favors the closed conformation by interacting with both NS2B and NS3. The artificial linker between NS2B and NS3 tends to induce the open conformation and interfere with the protease activity. This negative impact on the enzyme structure and function is restricted to the protease domain as the ATPase activities of these constructs are not affected.