Probing hydrogen bonds in the antibody-bound HIV-1 gp120 V3 loop by solid state NMR REDOR measurements

We describe solid state NMR measurements on frozen solutions of the complex of the 24-residue HIV-1 gp120 V3 loop peptide RP135 with the Fab fragment of the anti-gp120 antibody 0.5 beta, using rotational echo double resonance (REDOR). In order to probe possible hydrogen bonding between arginine side chains and glycine backbone carbonyls in the region of the conserved Gly-P ro-Gly-Arg (GPGR) motif of the V3 loop, RP135 samples were prepared with N- 15 labels at the eta nitrogen positions of arginine side chains and C-13 la bels at glycine carbonyl positions and C-13-detected C-13-N-15 REDOR measur ements were performed on peptide/antibody complexes of these labeled sample s. Such hydrogen bonding was previously observed in a crystal structure of the V3 loop peptide/antibody complex RP142/59.1 [Ghiara et al. (1994) Scien ce, 264, 82-85], but is shown by the REDOR measurements to be absent in the RP135/0.5 beta complex. These results confirm the antibody-dependent confo rmational differences in the GPGR motif suggested by previously reported so lid state NMR measurements of phi and Psi backbone dihedral angles in the R P135/0.5 beta complex. In addition, we describe REDOR measurements on the h elical synthetic peptide MB(i+4)EK in frozen solution that establish our ab ility to detect C-13-N-15 dipole-dipole couplings in the distance range app ropriate to these hydrogen bonding studies. We also report the results of m olecular modeling calculations on the central portion RP135, using a combin ation of the solid state NMR restraints of Weliky et al. [Nat. Struct. Biol ., 6, 141-145, 1999] and the liquid state NMR restraints of Tugarinov et al . (Nat. Struct. Biol., 6, 331-335, 1999]. The dynamics calculations demonst rate the mutual compatibility of the two sets of experimental structural re straints and reduce ambiguities in the solid state NMR restraints that resu lt from symmetry and signal-to-noise considerations.