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

Area of Science:

• Biophysics
• Structural Biology
• Chemical Physics

Background:

• The conserved Gly-Pro-Gly-Arg (GPGR) motif in the HIV-1 gp120 V3 loop is crucial for antibody binding.
• Previous studies suggested potential hydrogen bonding within the GPGR motif in antibody-bound V3 loop peptides.
• Antibody-dependent conformational flexibility of the V3 loop is implicated in HIV-1 neutralization.

Purpose of the Study:

• To investigate the presence of specific hydrogen bonds between arginine side chains and glycine backbone carbonyls in the GPGR motif of the HIV-1 gp120 V3 loop peptide RP135.
• To determine if the antibody 0.5beta induces or stabilizes such hydrogen bonds in the RP135/0.5beta complex.
• To compare structural findings with previous crystal structure data and solid-state NMR measurements.

Main Methods:

• Solid-state Nuclear Magnetic Resonance (NMR) spectroscopy using rotational echo double resonance (REDOR) on frozen solutions.
• Preparation of 15N-labeled arginine and 13C-labeled glycine carbonyls in the RP135 peptide.
• 13C-detected 13C-15N REDOR measurements on RP135/0.5beta peptide/antibody complexes.
• Validation of REDOR technique using a synthetic helical peptide MB(i+4)EK.
• Molecular modeling and dynamics calculations integrating solid-state and liquid-state NMR restraints.

Main Results:

• REDOR measurements demonstrated the absence of hydrogen bonding between arginine side chains and glycine backbone carbonyls in the GPGR motif of the RP135/0.5beta complex.
• This finding contrasts with observations in a different V3 loop peptide/antibody complex (RP142/59.1).
• The results support previously suggested antibody-dependent conformational variations in the GPGR motif.
• REDOR measurements on MB(i+4)EK confirmed the method's sensitivity for detecting relevant 13C-15N distances.
• Molecular modeling indicated compatibility between solid-state and liquid-state NMR data, reducing ambiguity.

Conclusions:

• The specific hydrogen bond proposed to exist in other V3 loop-antibody complexes is not present in the RP135/0.5beta complex.
• Antibody binding induces distinct conformational states in the V3 loop's GPGR region.
• Solid-state NMR REDOR is a valuable tool for probing inter-atomic distances and hydrogen bonding in biomolecular complexes.
• Integrated structural and dynamics modeling provides a more comprehensive understanding of peptide-antibody interactions.