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 independently folded [Omichinski, J. G., Clore, G. M., Sakaguchi, K., Appella, E., and Gronenborn, A. M. (1991) FEBS Lett. 292, 25-30; Summers, 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, Hare, D. R., and Arthur, L. O. (1992) Protein Sci. I, 563-574]. As in the interacting finger model, Phe(16) in the NH2-terminal finger and Trp(37) in, the COOH-terminal finger were found to be spatially close, the fluorescence properties of the aromatic residues at positions 16 and 37 in the wild-type and two conservatively substituted (12-53)NCp7 peptides were investigated and compared with those of three negative control derivatives where the finger motifs were not in close contact. Direct distance measurements by Tyr-Trp fluorescence resonance energy transfer of the former derivatives yielded a 7-12 Angstrom interchromophore distance range which is clearly inconsistent with the 12.5-18 Angstrom range measured for the negative controls and thus a random orientation of the zinc finger motifs. In keeping with the proximity of the two finger motifs in the former derivatives, a dramatic decrease in both the native Trp(37) and the conservatively substituted Trp(16) fluorescence quantum yields, correlated to the appearance of an additional subnanosecond lifetime was observed. Moreover, as expected, the amplitude of Trp local motion in these derivatives was significantly lower than that in the negative controls. Finally, refinement of the distance range measured. by fluorescence energy transfer, using the NMR-derived angles of the model with interacting fingers, yielded a mean interchromophore distance of 7.3 +/- 0.7 Angstrom, in good agreement with the NMR-deduced distances of this model. Taken together, our data confirmed the spatial proximity of the two finger motifs, a feature that must be of critical importance for the recognition of the target nucleic acids.