A model of a gp120 V3 peptide in complex with an HIV-neutralizing antibody based on NMR and mutant cycle-derived constraints.

The 0.5beta monoclonal antibody is a very potent strain-specific HIV-neutralizing antibody raised against gp120, the envelope glycoprotein of HIV-1. This antibody recognizes the V3 loop of gp120, which is a major neutralizing determinant of the virus. The antibody-peptide interactions, involving aromatic and negatively charged residues of the antibody 0.5beta, were studied by NMR and double-mutant cycles. A deuterated V3 peptide and a Fab containing deuterated aromatic amino acids were used to assign these interactions to specific V3 residues and to the amino acid type and specific chain of the antibody by NOE difference spectroscopy. Electrostatic interactions between negatively charged residues of the antibody Fv and peptide residues were studied by mutagenesis of both antibody and peptide residues and double-mutant cycles. Several interactions could be assigned unambiguously: F96(L) of the antibody interacts with Pro13 of the peptide, H52(H) interacts with Ile7, Ile9 and Gln10 and D56(H) interacts with Arg11. The interactions of the light-chain tyrosines with Pro13 and Gly14 could be assigned to either Y30a(L) and Y32(L), respectively, or Y32(L) and Y49(L), respectively. Three heavy-chain tyrosines interact with Ile7, Ile20 and Phe17. Several combinations of assignments involving Y32(H), Y53(H), Y96(H) and Y100a(H) may satisfy the NMR and mutagenesis constraints, and therefore at this stage the interactions of the heavy-chain tyrosines were not taken into account. The unambiguous assignments [F96(L), H52(H) and D56(H)] and the two possible assignments of the light-chain tyrosines were used to dock the peptide into the antibody-combining site. The peptide converges to a unique position within the binding site, with the RGPG loop pointing into the center of the groove formed by the antibody complementary determining regions while retaining the beta-hairpin conformation and the type-VI RGPG turn [Tugarinov, V., Zvi, A., Levy, R. & Anglister, J. (1999) Nat. Struct. Biol. 6, 331-335].

Thermodynamic analysis of the interaction between the 0.5beta Fv fragment and the RP135 peptide antigen derived from the V3 loop of HIV-1 gp120.

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.

Contribution of arginine residues in the RP135 peptide derived from the V3 loop of gp120 to its interaction with the Fv fragment of the 0.5beta HIV-1 neutralizing antibody.

The construction, expression, and purification of an active Fv fragment of the 0.5beta monoclonal human immunodeficiency virus type 1 (HIV-1) neutralizing antibody is reported. The interaction between the Fv fragment and the RP135 peptide derived from the V3 loop of gp120 from HIV-1IIIB was studied by varying the salt concentration and by mutating arginine residues in the peptide. The mutations R4A, R8A and R11A (which correspond to residues 311, 315, and 318 in gp120 of HIV-1IIIB) reduce the binding free energy by 0.22 (+/- 0. 20), 4.32 (+/- 0.16), and 1.58 (+/- 0.17) kcal mol-1, respectively. The salt-dependent components of their contributions to binding are 0.02 (+/- 0.22), -0.55 (+/- 0.18), and -0.97 (+/- 0.19) kcal mol-1, respectively. The magnitudes of the mutational effects and the extent of shielding by 1 M NaCl suggest that Arg-8 is involved in a buried salt bridge in the peptide-Fv fragment complex, whereas Arg-11 is involved in a more solvent-exposed electrostatic interaction.

On the choice of reference mutant states in the application of the double-mutant cycle method.

Pairwise interactions in proteins can be detected and, in certain circumstances, their strength measured by applying the method of double-mutant cycles. Such cycles comprise wild type protein, two single mutants and the corresponding double mutant. The analysis of double-mutant cycles is most straightforward when the mutations are to alanine since interactions are mostly removed without new interactions being formed. Here, 'not-to-alanine' double-mutant cycles are analysed. It is shown that a 'not-to-alanine' double-mutant cycle can be decomposed into four double-mutant cycles with mutations only to alanine. The coupling energy corresponding to the 'not-to-alanine' double-mutant cycle is expressed as a function of the coupling energies of these four cycles.