Characterization of the nucleotide binding properties of SV40 T antigen using fluorescent 3'(2')-O-(2,4,6-trinitrophenyl)adenine nucleotide analogues.
ATP binding to the large tumor (T) antigen encoded by the simian virus 40 (SV40) genome plays an essential role in the replication of viral DNA [Fanning, E., and Knippers, R. (1992) Annu. Rev. Biochem. 61, 55-85]. To better explore the functions of T antigen during the replication process, we have studied the interactions of T antigen with fluorescent 3'(2')-O-(2,4,6-trinitrophenyl) (TNP) adenine nucleotide analogues. Binding of TNP-ATP and TNP-ADP was accompanied by an 8-fold fluorescence enhancement and a concomitant blue shift (11 nm) of the maximal emission wavelength; the intrinsic protein tryptophan fluorescence was quenched maximally by 50%. Both signals were utilized to characterize the nucleotide binding activity of T antigen. TNP-ATP and TNP-ADP bound to the ATP binding site with dissociation constants of 0.35 microM and 2.6 microM. TNP substitution enhanced the affinity of ADP for T antigen by approximately 11-fold. The binding stoichiometry was 1 mol of TNP nucleotide per mole of monomer T antigen. The binding of TNP-ATP was more temperature dependent than that of TNP-ADP. The enthalpy change contributed nearly half of the energy for TNP-ATP binding, whereas binding of TNP-ADP was primarily entropy driven. Both TNP-ATP and TNP-ADP were strong inhibitors of the T antigen ATPase activity, confirming the high affinities of the TNP nucleotides for the ATP binding site. Like the parent nucleotides, they also induced T antigen hexamer formation. Using the TNP nucleotides as fluorescent probes, we have measured the affinity of various nucleotides and analogues for T antigen. The results indicate that the nucleotide binding specificity of T antigen was similar to that of the prokaryotic helicases Dna B and Rep, suggesting closely related ATP binding sites in the three DNA helicases.
Huang
S G
SG
Institute of Physical Biochemistry, University of Munich, Germany. Sghuang@pbm.med.uni-muenchen.de
Weisshart
K
K
Fanning
E
E
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005456:Fluorescent Dyes; D008274:Magnesium / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism* / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism; D011108:Polymers / Q000378:metabolism; D011485:Protein Binding; D013539:Simian virus 40 / Q000201:enzymology / Q000276:immunology*; D012965:Sodium Chloride / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature
C009587:2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate; D000952:Antigens, Polyomavirus Transforming; D005456:Fluorescent Dyes; D009711:Nucleotides; D010710:Phosphates; D011108:Polymers; D012965:Sodium Chloride; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000244:Adenosine Diphosphate; C036745:2',3'-(O-(2,4,6-trinitrocyclohexadienylidine))adenosine 5'-diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D008274:Magnesium; C018279:sodium phosphate