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 truncated V3 peptides.
  • 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.
• The Aib₁₄₂ peptide was created using chemical synthesis while the Ser and His peptide loops were generated using solid phase synthesis.
• The complexes were crystallized using the sitting drop, vapor diffusion method at 22.5 degrees Celsius
• The structures determined through the use of the X-PLOR computer program, PC refinement, and an in-house Harada translation function. Various programs were used to generate the data and renderings given in the Tables and Figures.

Explanations of Figures and Tables

• 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 three 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 formation of the final Fab 58.2-Peptide complexes.
• Table 1: Table 1 describes the X-ray diffraction data and statistics for each of the three Fab 58.2-peptide complexes. Unfortunately, due to my limited knowledge of the in-depth details of X-ray crystallography, unfamiliarity with the conventions of data presentation in the field, and the lack of further explanation of this data in the paper I am unable to interpret most of the information given in this table.
• 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.
• Figure 3: Figure 3 shows the structural conformation of the H1 loop of the Fab 58.2 antibody in comparison to the structure of the H1 loops of two other Fab antibodies, N10 (yellow) and AN02 (blue), with similar H1 loop length. The H1 loop structure of AN02 represents the expected canonical structure for the length of the loop. Fab 58.2 (red) shows a similar structure to the other two H1 loops from residues 20-29 and 35-37, but differs distinctly from the other two H1 loops between residues 31-34 forming pronounced inner(31-32) and outer(33-34) kinks.
• Figure 4: Figure 4 shows the electron densities of some of the various structures examined in the experiment.(a) Displays the electron density of the GPGR residues in the Aib₁₄₂ peptide complex and indicates a type 1 β turn. (b) Displays the electron density of the RAibFY residues in the Aib₁₄₂ peptide complex. (c)and(d) Display the electron density of the entire peptide sequence of the His-loop and Ser-loop peptides excluding the J-Z hydrazone linkage due to insufficient density.
• Figure 5: Figure 5 shows the Fab 58.2-Aib₁₄₂ complex with the Fab 58.2 shown using the space-filling model with regions of polarity shown as red and blue, red representing negative charges and blue representing positive charges. The Aib₁₄₂ is bound to a highly negative pocket of the Fab 58.2 structure. The stereoview of all the Fab 58.2 residues that interact with the peptide at the binding site is also shown.
• Table 2: Table 2 gives a list of each of the Van der Waals interactions between the specific residues of Fab 58.2 and the specific residues of each of the three peptides in complex. The table shows the Fab 58.2 residues that interact with the common residues amongst all three of the peptides as well as the unique interactions between the Fab 58.2 residues and certain peptide specific residues. The common residue interactions are listed under the "In all complexes" column and the unique interactions are listed for each specific residue under each of the individual peptide columns. It is seen that there are 3 instances of interactions that are completely unique to a specific peptide and two instances in which there are no peptide specific interactions.
• Table 3: Table 3 shows the various hydrogen bonds and salt bridges taking place between the Fab 58.2 residues and the various residues of the three peptides in complex. Again due to my lacking knowledge of crystallography data conventions and lack of explanation in the paper it is difficult to render further interpretation of the data.
• Figure 6: Figure 6 shows the comparison of the structural data of the Fab 50.1-peptide and 59.1-peptide complexes obtained by the lab in previous experiments and the structural data of the Fab 58.2-Aib₁₄₂ and Fab 58.2-His complexes studied in this experiment. The comparison shows that the main chain residues of all of the peptides share a similar structural configuration, but differ when examining the conformational structure developed by the GPGR loop residues. The peptides of Fab 50.1 and 59.1 show a type 2 β-turn at the GPGR residues while both of the Fab 58.2 peptide conformations show a type 1 β-turn at the GPGR residues.
• Table 4: Table 4 lists the φ and ψ angle values for conserved residues among the peptides used to form the Fab 50.1-peptide and 59.1-peptide complexes from the previous experiments and the Fab 58.2-Aib₁₄₂ complex. When examining the angles of the GPGR residues between the three peptides, the angles between the 50.1 and 59.1 residues are considerably similar, but are markedly different from the angles of the same residues in the Aib₁₄₂ peptide. The difference in these angles represents the conformational differences between the GPGR residues of the previous experiment's peptides and the current experiment's peptides.

Discussion

• The GPGR region of the V3 peptide is highly conserved throughout viral variants and is biologically relevant to the binding of antibodies to gp120.
• The GPGR region of the V3 peptide has been seen to adopt multiple different structural conformations based on it's environment and binding partner. These variations may relate to variations in biological function or binding potential of the V3 loop.
• This study's findings disagree with previous studies done by the same lab in the different conformational structures taken by the GPGR β-turn, however this disagreement actually furthers their theory that different types of antibodies will cause different conformational shifts in the V3 structure.
• In agreement with the epitope mapping of the Jellis study, this paper concludes that the residues Gly^P319, Pro^P320 and Arg^P322 are especially important for anti-body binding to gp120.
• Although not conclusively proven, this paper asserts that the effects of different V3 loop conformations could lead to changes in biological functions and interactions of gp120.
• Future experiments should include the crystallization of a complete V3 peptide sequence rather than truncated versions of the peptide which has been used so far. Intact gp120 complexes would provide for more definite conclusions to be made.

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.

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