Rational design and functional expression of a constitutively active single-chain NS4A-NS3 proteinase.
BACKGROUND
The proteinase domain of the hepatitis C virus NS3 protein is involved in the maturation of the viral polyprotein. A central hydrophobic domain of the NS4A protein is required as a cofactor for its proteolytic activity. The three-dimensional structure of the proteinase domain alone and complexed with an NS4A-derived peptide has been solved recently and revealed that the N terminus of the proteinase is in near proximity to the C terminus of the cofactor. To study the molecular basis of the enzyme activation by its cofactor and to overcome the difficulties of structural and functional investigation associated with a two-species complex, we rationally designed a link to bridge the two molecules in order to have a single polypeptide construct.


RESULTS
The engineered construct led to the production of a stable, monomeric protein with proteolytic activity that is independent from the addition of a synthetic peptide representing the cofactor domain of the NS4A protein. The protein is active on both protein and synthetic peptide substrates. Spectroscopic and kinetic analysis of the recombinant NS4A-NS3 single-chain proteinase demonstrated features superimposable with the isolated NS3 proteinase domain complexed with the NS4A cofactor.


CONCLUSIONS
We designed a very tight connection between the NS3 and NS4A polypeptide chains with the rationale that this would allow a more stable structure to be formed. The engineered single-chain enzyme was indistinguishable from the NS3 proteinase complexed with its NS4A cofactor in all enzymatic and physico-chemical properties investigated.
Pasquo
A
A
Istituto di Ricerche di Biologia Molecolare (IRBM), P. Angeletti, Pomezia (Rome), Italy.
Nardi
M C
MC
Dimasi
N
N
Tomei
L
L
Steinkühler
C
C
Delmastro
P
P
Tramontano
A
A
De Francesco
R
R
D001483:Base Sequence; D004789:Enzyme Activation; D016174:Hepacivirus / Q000737:chemistry*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus