Solution structure and backbone dynamics of an engineered arginine-rich subdomain 2 of the hepatitis C virus NS3 RNA helicase

The NS3 protein of the hepatitis C virus (HCV) is a 631 amino acid residue bifunctional enzyme with a serine protease localized to the N-terminal 181 residues and an RNA helicase located in the C-terminal 450 residues. The HC V NS3 RNA helicase consists of three well-defined subdomains which all cont ribute to its helicase activity. The second subdomain of the HCV helicase i s flexibly linked to the remainder of the NS3 protein and could undergo rig id-body movements during the unwinding of double-stranded RNA. It also cont ains several motifs that are implicated in RNA binding and in coupling NTP hydrolysis to nucleic acid unwinding and translocation. As part of our effo rts to use NMR techniques to assist in deciphering the enzyme's structure-f unction relationships and developing specific small molecule inhibitors, we have determined the solution structure of an engineered subdomain 2 of the NS3 RNA helicase of HCV, d(2 Delta)-HCVh, and studied the backbone dynamic s of this protein by N-15-relaxation experiments using a model-free approac h. The NMR studies on this 142-residue construct reveal that overall subdom ain 2 of the HCV helicase is globular and well structured in solution even in the absence of the remaining parts of the NS3 protein. Its solution stru cture is very similar to the corresponding parts in the X-ray structures of the HCV NS3 helicase domain and intact bifunctional HCV NS3 protein. Slow hydrogen-deuterium exchange rates map to a well-structured, stable hydropho bic core region away from the subdomain interfaces. In contrast, the region s facing the subdomain interfaces in the HCV NS3 helicase domain are less w ell structured in d(2 Delta)-HCVh, show fast hydrogen-deuterium exchange ra tes, and the analysis of the dynamic properties of d(2 Delta)-HCVh reveals that these regions of the protein show distinct dynamical features. In part icular, residues in motif V, which may be involved in transducing allosteri c effects of nucleotide binding and hydrolysis on RNA binding, exhibit slow conformational exchange on the milli- to microsecond time-scale. The intri nsic conformational flexibility of this loop region may facilitate conforma tional changes required for helicase function. (C) 2001 Academic Press.