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 Å and rotation of ∼174° 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.
Highlights
HIV-1 Nef is an important accessory protein that is attributed with multiple distinct functions such as immune evasion [1,2], virion infectivity [3,4] and support for viral replication [5,6] and survival
We report the crystallization of the full-length HIV-1 Nef after experimentation with naturally occurring Nef variants from patient samples and were successful with one such sample
Crystallization and structure description We attempted crystallization of the full-length HIV-1 Nef (RP14) with several cloned samples derived from patients and were successful with the sample with sequence as deposited in the GenBank with accession number: GQ184340
Summary
HIV-1 Nef is an important accessory protein that is attributed with multiple distinct functions such as immune evasion [1,2], virion infectivity [3,4] and support for viral replication [5,6] and survival. These functions are regulated through the interactions of Nef with more than 30 different partner proteins in the membrane anchored and cytoplasmic states respectively [7,8], It is puzzling as to how this small ,27 kDa protein can control multiple important functions. A population of the myristoylated form, as supported by affinity experiments, exists in the cytoplasm where the N-terminal myristoylated segment is suggestedly sequestered in the hydrophobic pockets between helices aA and aB of the C-terminal domain [12,13]
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