Abstract:
HIV-1 Nef modulates disease progression through interactions with over 30 host proteins. Individual chains fold into
membrane-interacting N-terminal and C-terminal core (Nefcore) domains respectively. Nef exists as small oligomers near
membranes and associates into higher oligomers such as tetramers or hexadecamers in the cytoplasm. Earlier structures of
the Nefcore in apo and complexed forms with the Fyn-kinase SH3 domain revealed dimeric association details and the role of
the conserved PXXP recognition motif (residues 72–78) of Nef in SH3-domain interactions. The crystal structure of the
tetrameric Nef reported here corresponds to the elusive cytoplasmic stage. Comparative analyses show that subunits of
Nefcore dimers (open conformation) swing out with a relative displacement of ,22 A°
and rotation of ,174u to form the
‘closed’ tetrameric structure. The changes to the association are around Asp125, a conserved residue important for viral
replication and the important XR motif (residues 107–108). The tetramer associates through C4 symmetry instead of the 222
symmetry expected when two dimers associate together. This novel dimer-tetramer transition agrees with earlier solution
studies including small angle X-ray scattering, analytical ultracentrifugation, dynamic laser light scattering and our
glutaraldehyde cross-linking experiments. Comparisons with the Nefcore—Fyn-SH3 domain complexes reveal that the PXXP
motif that interacts with the SH3-domain in the dimeric form is sterically occluded in the tetramer. However the 151–180
loop that is distal to the PXXP motif and contains several protein interaction motifs remains accessible. The results suggest
how changes to the oligomeric state of Nef can help it distinguish between protein partners.
