Chris Rhodes Week 7

The paper we are preparing is "Dual conformations for the HIV-1 gp120 V3 loop in complexes with different neutralizing Fabs" Stanfield et al. (1999) The full article can be found here Stanfield (1999)

10 Terms

1. Chemokine:
2. Macrophage:
3. Syncytia:
4. Principle Neutralizing Determinant:
5. CXCR4:
6. CCR5:
7. Epitope:
8. Antihapten Antibody:
9. MN viral Sequence:
10. Tropism:

Outline

Introduction

• The V3 domain of gp120 of HIV-1 viruses represent essential sites of CD4-Virus interaction necessary to facilitate viral and CD4 membrane fusion allowing for the initiation of the viral infection of the CD4 cell.
• The V3 peptide loop has multiple effects on viral interactions including tropism and antibody neutralization.
• Previous research has indicated that the amino acid sequences around the stems of the V3 loops of different viral variants were highly variable while the amino acid sequences comprising the tip of the loop (GPGR) were highly conserved among the variants suggesting that the amino acids that comprise the tip are essential for function.
• Though the function of the V3 loop is fairly well understood,the various structural conformations of the V3 amino acids and their effects on viral binding potential and viral progression are still unknown.
• Previous studies have not been able to relate specific changes in the V3 sequence with a change in phenotype, however there could be a relationship between V3 amino acid conformations and gp120 functionality.
• The lab has previously researched crystal structures of Fab 50.1-V3 and Fab 59.1-V3 complexes in an attempt to find the conformations adopted by the V3 peptide upon binding to various antibodies shown to have different styles of viral neutralization.
  • These two previous studies did not prioritize the study of the (GPGR) loop tip as the current study does.
• Understanding how the structural conformation of the V3 peptide affects its function could provide invaluable insight into HIV-1 immunology and potential medical treatments for the disease.

Materials and Methods

• This experiment uses the neutralizing antibody Fab 58.2 crystallized in complex with three different V3 partial peptides detailed in Figure 1.
  • Histidine Loop: JHIGPGRAFGZG
  • Serine Loop: JSIGPGRAFGZG
  • Aib₁₄₂: YNKRKRIHIGPGRAibFYTTKNIIGC
• Aib is used to replace the alanine in the V3 peptide in order to establish a stable and uniform structure for the peptide. If the alanine was not replaced, the structure of peptide would be too variable to perform accurate crystallization.

Explanations of Figures

• FIgure 1: Figure 1 details the protein sequences of each of the peptides used to form complexes with the Fab 58.2 antibody as well as the complete sequence of the V3 peptide used to create the experimental peptides. It also gives a pictorial representation of how the hydrazone linkage of the J and Z residues of the His and Ser-loop peptides forms the stabilized loop used in the final Fab 58.2 complex.
• Figure 2: Figure 2 shows computer generated representations of the crystalized structures of the entire Fab 58.2-Peptide complex for Fab 58.2 bound to (a) the linear Aib₁₄₂ containing peptide, (b) the His-loop peptide, and (c) the Ser-loop peptide. The two loop peptide complexes show very similar Fab 58.2 structures while the linear Aib₁₄₂ peptide complex shows a distinctly larger gap between the upper 2 domains containing the peptide and the lower 2 domains, which when compared to the loop peptide complexes seem much more compressed or tightly packed.

Important Quotes

• We have been investigating several HIV-1 neutralizing antibodies and their complexes with V3 peptides in order to determine the tertiary conformation of the V3 loop and to understand why some antibodies are viral-specific while others recognize many different viral strains.
• Knowledge of the conformation of this loop may help to explain coreceptor usage and the changes that take place in the virus upon conversion from a primary M-tropic isolate to the T-tropic strains associated with disease progression
• Recent studies using intact viral particles rather than soluble monomeric gp120, however, indicate that V3 neutralizing antibodies may also prevent the binding of HIV-1 to CD4+ human cells
• Fab 58.2 is a highly potent and broadly neutralizing antibody that has been reported to neutralize both T-tropic and M-tropic viral strains, as well as several strains that have been passaged only once or twice.
• Pro, Gly and Arg are the most commonly found residues in positions i+1, i+2 and i+3 of a type II β turn
• These epitope mapping results indicate that residues GlyP319, ProP320 and ArgP322 are especially important for antibody binding, in agreement with the crystal structure that shows that ArgP322 forms a charge–charge interaction with the Fab and residues GlyP319 and ProP320 are critical for determining the conformation of the tip of the V3 loop. Residue GlyP319, which of course is a highly conformationally flexible residue, may also be important for allowing the loop to adjust to more than one distinct conformation. GlyP321, the i+2 residue in the type I β turn of the peptide in the 58.2–peptide complexes, is not absolutely required, in agreement with the diversity of residues that are found at the i+2 position of type I turns. This result contrasts with the alanine replacement experiment on peptide binding to Fab 59.1. When GlyP321 is replaced by an Ala residue, the affinity of Fab 59.1 for the peptide is reduced to about 32% of that of the native peptide, in good agreement for the strong preference for a Gly residue at the i+2 position of a type II turn, as found in the 59.1–peptide complex.
• These two peptides both adopt torsion angles for a type II β turn around residues GPGR. Thus, it was surprising to observe that the peptide bound to Fab 58.2 had torsion angles for a type I β turn around these same residues
• We clearly observe two different conformations for the V3 loop while bound to different neutralizing antibodies, and two conformations for the tip of the loop in solution. These V3 loop conformations are biologically relevant, as the antibodies bind to viral gp120 to effect their neutralization activity. These conformations may represent two different and distinct secondary structural motifs that may or may not relate to viral phenotype, or they could merely indicate that the V3 loop is flexible and able to adopt different conformations. The only reported crystals of gp120 to date have been obtained for protein where this loop was truncated at its base; crystals of gp120 with the intact loop have not yet been reported.
• The high degree of conservation of the Gly-Pro-Gly-Arg/Gln motif at the tip of the gp120 V3 loop, surrounded by regions of high sequence diversity, suggests this structural conservation is related to biological function.