Biochemical characterization of recombinant hepatitis C virus nonstructural protein 4B: evidence for ATP/GTP hydrolysis and adenylate kinase activity.
While nonstructural protein 4B (NS4B) from hepatitis C virus (HCV) is absolutely required for viral propagation, a full understanding of the enzymatic properties of this protein is lacking. Previous studies suggest that NS4B is located at the endoplasmic reticulum and that the protein structure consists of four central transmembrane domains with the N- and C-termini located in the cytoplasm of the host cell. To characterize the enzymatic activity of NS4B, the full-length protein with a C-terminal His tag was expressed in Sf9 insect cells and stabilized with nonionic detergents during purification. Chemical cross-linking experiments using GTP-gamma-azidoanilide and ATP-gamma-azidoanilide and equilibrium binding analyses with GTPgammaS and ATPgammaS show that both GTP and ATP are bound by NS4B, with ATP displaying a higher affinity. Analyses of enzymatic reactions catalyzed by NS4B indicate that the terminal phosphate groups of ATP, GTP, and GDP are removed to produce ADP, GDP, and GMP, respectively. The k(cat) for hydrolysis of GTP by purified NS4B compared favorably with the k(cat) for hydrolysis of GTP by Ras-p21 in the absence of GTPase activating proteins (GAPs). In addition to the hydrolysis of NTP and NDP substrates, adenylate kinase activity was detected in purified preparations of NS4B with the reverse reaction 2ADP --> ATP + ADP, yielding a larger k(cat) compared to that of the forward reaction ATP + AMP --> 2ADP. These studies suggest that HCV NS4B possesses both adenylate kinase activity and nucleotide hydrolase activity. Mutation of amino acids in the Walker A and B motifs of NS4B resulted in decreased affinity for both GTPgammaS and ATPgammaS as well as decreased ATP hydrolysis and AK activity.
Thompson
Aaron A
AA
Department of Biochemical Pharmacology, La Jolla Laboratories, Pfizer Global Research and Development Inc., San Diego, California 92121, USA.
Zou
Aihua
A
Yan
Jiangli
J
Duggal
Rohit
R
Hao
Weidong
W
Molina
David
D
Cronin
Ciarán N
CN
Wells
Peter A
PA
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D000263:Adenylate Kinase / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D004789:Enzyme Activation / Q000502:physiology; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D016174:Hepacivirus / Q000201:enzymology*; D006867:Hydrolases / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*
C093542:NS4 protein, hepatitis C virus; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000263:Adenylate Kinase; D006867:Hydrolases