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 0.5beta, 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-Pro-Gly-Arg (GPGR) motif of the V3 loop, RP135 samples were prepared with 15N labels at the eta nitrogen positions of arginine side chains and 13C labels at glycine carbonyl positions and 13C-detected 13C-15N REDOR measurements were performed on peptide/antibody complexes of these labeled samples. Such hydrogen bonding was previously observed in a crystal structure of the V3 loop peptide/antibody complex RP142/59.1 [Ghiara et al. (1994) Science, 264, 82-85], but is shown by the REDOR measurements to be absent in the RP135/0.5beta complex. These results confirm the antibody-dependent conformational differences in the GPGR motif suggested by previously reported solid state NMR measurements of phi and psi backbone dihedral angles in the RP135/0.53 complex. In addition, we describe REDOR measurements on the helical synthetic peptide MB(i+4)EK in frozen solution that establish our ability to detect 13C-15N dipole-dipole couplings in the distance range appropriate to these hydrogen bonding studies. We also report the results of molecular modeling calculations on the central portion RP135, using a combination 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 demonstrate the mutual compatibility of the two sets of experimental structural restraints and reduce ambiguities in the solid state NMR restraints that result from symmetry and signal-to-noise considerations.

01 Jun 1998·NeuroscienceQ3 · MEDICINE

The human immunodeficiency virus-1 envelope protein gp120 binds through its V3 sequence to the glycine site of N-methyl-d-aspartate receptors mediating noradrenaline release in the hippocampus

Q3 · MEDICINE

Article

Author: Anna Maria Pittaluga ; Maurizio Raiteri ; R. Pattarini

Recent results show that the HIV-1 protein gp120 can enhance N-methyl-D-aspartate receptor-mediated release of noradrenaline from CNS nerve endings. We now investigate the mechanism of this action, including the structural determinants of the gp120 effect and the nature of its binding sites. The N-methyl-D-aspartate-evoked release of [3H]noradrenaline from rat hippocampal synaptosomes was potentiated similarly by gp120 and gp160; gp41 was ineffective. The regions of gp120 involved appear to be outside the CD4-binding domain of the protein, because gp120 retained its activity after pretreatment with N-carbomethoxycarbonyl-D-prolyl-D-phenylalanine, a compound known to inhibit binding of gp120 to CD4 receptors. Moreover, sequences of gp120 critical for binding to CD4 did not mimic the effect of gp120. Preincubation of synaptosomes with anti-galactocerebroside antibodies did not affect gp120 activity. The protein effect was retained by peptides mimicking its V3 sequence, including the cyclic V3 "universal peptide" and the linear V3 sequence BRU-C-34-A, but not RP-135 (a central portion of BRU-C-34-A). The block of the N-methyl-D-aspartate-induced [3H]noradrenaline release by 7-chlorokynurenate, an antagonist at the N-methyl-D-aspartate receptor glycine site, was competitively reversed by glycine, by V3 and by BRU-C-34-A. When added with N-methyl-D-aspartate, V3 was three to four orders of magnitude more potent than glycine (EC50 values: about 20 pM and 150 nM, respectively) in enhancing [3H]noradrenaline release. Gp120 did not release glycine or serine from synaptosomes, thus excluding indirect actions through these agents. To conclude, gp120 may act following recognition by its V3 sequence of a high-affinity site possibly coincident with the glycine site of N-methyl-D-aspartate receptors present on hippocampal terminals of noradrenergic neurons. Considering the importance of N-methyl-D-aspartate receptor activation and of noradrenaline in cognitive processes, the effects of gp120 and V3 described here may be relevant to the pathology of AIDS dementia.

01 Dec 1997·Journal of Biological ChemistryQ2 · BIOLOGY

Thermodynamic Analysis of the Interaction between the 0.5β Fv Fragment and the RP135 Peptide Antigen Derived from the V3 Loop of HIV-1 gp120

Q2 · BIOLOGY

Article

Author: Amnon Horovitz ; Gabriel A. Faiman

The Fv fragment of the 0.5beta monoclonal antibody has recently been constructed, expressed, and purified. It binds with nanomolar affinity to the immunogenic RP135 peptide that is derived from the principal neutralizing determinant of HIV-1 in the third hypervariable region of gp120. Here, we analyzed the temperature-dependence of binding of the 0.5beta Fv fragment to the RP135 peptide and a series of mutants thereof. Our results show that there is almost complete enthalpy-entropy compensation in the effects of mutations in the peptide on binding to the Fv, indicating that the mutations do not change the binding mechanism. There is good correlation, for residues within the antigenic epitope, between mutational effects on DeltaCp and calculated values of DeltaDeltaCp based on the extent of burial of polar and non-polar surface areas of amino acids. The value of DeltaCp for the binding of the 0.5beta Fv fragment to the wild-type RP135 peptide is found to be -5.0 (+/- 0.9) kcal K-1 mol-1 in the presence of 0.1% Tween-20 but only -0.1 (+/- 0.9) kcal K-1 mol-1 in its absence. This result has important implications for the successful application of the structural parameterization approach to predicting changes in heat capacity that accompany binding reactions carried out in the presence of detergent or protein-stabilizing agents.

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Indication	Highest Phase	Country/Location	Organization	Date
HIV Infections	Phase 1	-	Merck Sharp & Dohme Corp.	-
HIV Infections	Phase 1	-	Repligen Corp.	-

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