SPATIAL PROXIMITY OF THE HIV-1 NUCLEOCAPSID PROTEIN ZINC FINGERS INVESTIGATED BY TIME-RESOLVED FLUORESCENCE AND FLUORESCENCE RESONANCE ENERGY-TRANSFER

The three-dimensional structure of peptides encompassing the two zinc- saturated finger motifs of the nucleocapsid protein NCp7 of HIV-1 has been reported by several groups. Whereas the folded structures of the finger motifs were in good agreement, discrepancies existed concerning their spatial relationship since the fingers were found either close to each other [Morellet, N., Jullian, N., De Rocquigny, H., Maigret, B ., Darlix, J. L., and Rogues, B. P. (1992) Embo J. 11, 3059-3065] or i ndependently folded [Omichinski, J. G., Clore, G. M., Sakaguchi, K., A ppella, E., and Gronenborn, A. M. (1991) FEBS Lett. 292, 25-30; Summer s, M. F., Henderson, L. E., Chance, M. R., Bess, J. W., Jr., South, T. L., Blake, P. R., Sagi, I., Perez-Alvarado, G., Sowder, R. C., III, H are, D. R., and Arthur, L. O. (1992) Protein Sci. I, 563-574]. As in t he interacting finger model, Phe(16) in the NH2-terminal finger and Tr p(37) in, the COOH-terminal finger were found to be spatially close, t he fluorescence properties of the aromatic residues at positions 16 an d 37 in the wild-type and two conservatively substituted (12-53)NCp7 p eptides were investigated and compared with those of three negative co ntrol derivatives where the finger motifs were not in close contact. D irect distance measurements by Tyr-Trp fluorescence resonance energy t ransfer of the former derivatives yielded a 7-12 Angstrom interchromop hore distance range which is clearly inconsistent with the 12.5-18 Ang strom range measured for the negative controls and thus a random orien tation of the zinc finger motifs. In keeping with the proximity of the two finger motifs in the former derivatives, a dramatic decrease in b oth the native Trp(37) and the conservatively substituted Trp(16) fluo rescence quantum yields, correlated to the appearance of an additional subnanosecond lifetime was observed. Moreover, as expected, the ampli tude of Trp local motion in these derivatives was significantly lower than that in the negative controls. Finally, refinement of the distanc e range measured. by fluorescence energy transfer, using the NMR-deriv ed angles of the model with interacting fingers, yielded a mean interc hromophore distance of 7.3 +/- 0.7 Angstrom, in good agreement with th e NMR-deduced distances of this model. Taken together, our data confir med the spatial proximity of the two finger motifs, a feature that mus t be of critical importance for the recognition of the target nucleic acids.