Changes in dynamic and static structures of the HIV‐1 p24 capsid protein N‐domain caused by amino‐acid substitution are associated with its viral viability

Abstract

HIV‐1 capsid is comprised of over a hundred p24 protein molecules, arranged as either pentamers or hexamers. Three p24 mutants with amino acid substitutions in capsid N‐terminal domain protein were examined: G60W (α3‐4 loop), M68T (helix 4), and P90T (α4‐5 loop), which exhibited no viability for biological activity. One common structural feature of the three p24 N‐domain mutants, examined by NMR, was the long‐range effect of more β‐structures at the β2‐strand in the N‐terminal region compared with the wild‐type. In addition, the presence of fewer helical structures was observed in M68T and P90T, beyond the broad area from helix 1 to the C‐terminal part of helix 4. This suggests that both N‐terminal beta structures and helices play important roles in the formation of p24 hexamers and pentamers. Next, compared with P90T, we examined cis‐conformation or trans‐conformation of wild‐type adopted by isomerization at G89–P90. Since P90T mutant adopts only a trans‐conformation, comparison of chemical shifts and signal intensities between each spectra revealed that the major peaks (about 85%) in the spectrum of wild‐type correspond to trans‐conformation. Furthermore, it was indicated that the region in cis‐conformation (minor; 15%) was more stabilized than that observed in trans‐conformation, based on the analyses of heteronuclear Overhauser effect as well as the order‐parameter. Therefore, it was concluded that the cis‐conformation is more favorable than the trans‐conformation for the interaction between the p24 N‐terminal domain and cyclophilin‐A. This is because HIV‐1 with a P90T protein, which adopts only a trans‐conformation, is associated with non‐viability of biological activity.