The principal neutralizing determinant (PND) of human immunodeficiency virus (HIV) is located inside the third variable loop (designated the V3 loop) of the envelope glycoprotein gp120. The V3 loop is typically 35 amino-acids long, and the 1st and the 35th residues in the loop are invariant cystines involved in a disulfide-bridge. Although PNDs from different HIV isolates contain a conserved GPG-sequence, the amino acids flanking the conserved sequence show hypervariability among HIV isolates; the GPG and the two flanking regions are collectively referred to as the GPG-crest or the PND. The amino acid sequence variability in the GPG-crest gives rise to different antigenic specificities for different PNDs from different HIV isolates. By combining two-dimensional nuclear magnetic resonance (2D NMR) and molecular modeling techniques, we have developed a method to study (1) the global tertiary fold of the V3 loops of HIV and (2) the local structure of the PND at the tip of the V3 loop. In this article, we report the results of our structural studies on the V3 loop of a Thailand HIV isolate. The sequential assignment is made by combining DQF-COSY, TOCSY, and NOESY/ROESY experiments. Various intra- and inter-residue inter-proton distances are estimated by full-matrix analyses of the NOESY data at 100 and 400 ms of mixing times and of the ROESY data at 60 and 200 ms of mixing times. 100 inter-residue distances are used as structural constraints in a simulated annealing procedure to derive energetically stable structures. Two functional motifs in the V3 loop, i.e., the glycosylation site and the GPG-crest, form defined structures: a turn is located at the glycosylation site, and the GPG-crest forms a protruding domain with a type-II GPGQ turn. The other regions of the V3 loop are rather flexible--especially the C-terminal DIRKAYC-stretch. These flexible regions of the V3 loop lead to conformational flexure of the entire V3 loop without altering the local structures of the glycosylation site or the GPG-crest. However, the ROESY experiments revealed no slow exchange among different V3 loop conformations, and therefore the flexible conformations are in fast exchange within the NMR time scale. The extent of this conformational flexibility is also discussed.
Amino Acid Sequence, HIV, HIV Envelope Protein gp120, Humans, Magnetic Resonance Spectroscopy, Models, Molecular, Molecular Sequence Data, Peptide Fragments, Protein Conformation, Protein Folding, Protein Structure, Secondary, Thailand