Bobak Seddighzadeh Week 7

Journal Club: Dual conformations for the HIV-1 gp120 V3 loop in complexes with different neutralizing Fabs

Definitions

1. Syncytium: A tissue characterized by cytoplasmic continuity, or a large mass of cytoplasm not separated into individual cells and containing many nuclei.
2. Fab: a fragment of immunoglobulin prepared by papain treatment. Fab fragments consist of one light chain linked through a disulphide bond to a portion of the heavy chain and contain one antigen binding Site.
3. Antigen: Any substance that’s recognized as foreign by the immune system and triggers an immune response, stimulating the production of an antibody that specifically reacts with it.
4. Tropic: A turning towards or having an affinity for.
5. Paucity: smallness of number or scarcity.
6. Ternary complex: describes the tripartite combination of such as for an enzyme-cofactor-substrate for a multi-substrate enzyme.
7. Epitope: That part of an antigenic molecule to which the t-cell receptor responds, a site on a large molecule against which an antibody will be produced and to which it will bind.
8. Isomorphous: Similarity in form, as in organisms of different ancestry.
9. Chemokine receptor: cytokine receptors found on the surface of certain cells, which interact with a type of cytokine called a chemokine.
10. Domain: A part of a molecule or structure with common physio-chemical features or properties.

Works Cited:

• Biology-online.org dictionary

What is the main result presented in this paper?

The main results of the paper presented the structure for Fab 58.2 in complex with one linear Aib-containing peptide and two cyclic constrained peptides. Fab 58.2 is a highly potent and broadly neutralizing antibody that can neutralize T-tropic and M-tropic viral strains. Furthermore, they surmised from Fab 50.1 and 59.1 that the Ala residue can be replaced with an Aib residue and introduced with hydrazone bonds to constrain the peptide to provide a more rigid scaffold for vaccine and drug design.

What is the importance or significance of this work?

The importance of their work is that they are studying they investigating the neutralizing antibodies and their complexes with the V3 loop. This can provide inside into the tertiary conformation of the V3 loop, which can lead to an understanding in the co-receptor usage and the progression of the disease. It can also provide insight as to why some antibodies are viral-specific and others many recognize many different strains. Thus, we can have a larger template of information to work with designing drugs and vaccines. They are using molecular structure of the V3 loop and antibodies to design better antibodies, which can save many lives if successful.

What were the limitations in previous studies that led them to perform this work?

In previous studies they have found that the V3 loop becomes more sensitive to neutralizing antibodies and proteases when gp120 is bound to CD4. It has been noted that sequence changes to the V3 region can affect which cells are infected. For example, T-tropic V3 sequences are more basic than M tropic sequences. However, no distinct patterns between the differences in residues in the V3 region and phenotypic changes have been determined. It is likely that likely that conformational variation in the gp120 and the V3 loop can modulate gp120 function. In previous studies, crystal structures of pg120, CD4 and the antibody Fab fragments have been determined, but they removed the V3 loop. Therefore, the lack of structural information on the V3 loop led them to this study.

What were the methods used in the study? The methods of the study

A) Preparation of Aib142 peptide:

1. Chemical synthesis prepared the Aib142 peptide
2. The N terminus was acetylated and the Cys side chain was protected with Acm group
3. The peptide was cleaved from the resin
4. The peptide was purified using a reverse phase column on a HPLC system.
5. The cyclic peptides were synthesized by solid phase synthesis and purified to single peaks by HPLC and identities confirmed by mass spectrometry.

B) Crystallizations:

• All crystallizations were carried out using sitting drop, vapor diffusion method at 22.5 degrees Celsius.

C) Determining complex structures:

• The Fab58.2/Ser loop complex structure was determined using coordinates of Fab 59.1 minus its peptide.
• Structures were determined using X-PLOR rotation function and PC refinement

Briefly state the result shown in each of the figures and tables

Figure 1:

• Fab 58.2 , 50.1, 59.1 attack peptide RP70
• Aib142 is a linear peptide crystallized with 58.2 and 59.1
• The his and Ser loop are constrained with hydrazone linkage between J and Z residues

Table 1

• Summarizes the data collection and refinement statistics of the crystal structures for Fab 58.2.
• The crystals of the two Fab peptides were isomorphous differing by only one amino acid

Figure 2:

• Reveals the x-ray structures of Fab 58.2-Aib 142 complex, Fab 58.2-His loop complex, and Fab 58.2-Ser loop complex.
• Distinguished between light chain, heavy chain, and conserved sequences

Table 2:

• Displays the Van der Waals contacts between Fab 58.2 and bound peptides
• A total of 124, 103, and 77 van der Waals contacts are made between Fab and Aib142, *His loop, and Ser loop complexes

Figure 3:

• Compares the conformation of H1 loops between Fabs 58.2, N10, AN02.
• Fabs 58.2 and N10 differ around several residues at their tips.
• There’s an increase in flexibility as H1 loop increases in length

Table 3

• Shows the hydrogen bonds and salt bridge interactions between the Fab 58.2 peptide complexes
• In each complex charge-charge interactions are found between Argp322 of the peptide and AspL94 and GluH95 of the Fab
• A total of 11,7, and 7 H bonds and salt bridge interactions were made between the peptides and Fab

Figure 4

• Residues GraibFy is a double bend
• 11 of the 24 residues are visble for the 58.2-Aib142 complex
• Electron density is present for most of the cyclic peptides, with a minor break in density around the hydraxone linker in the His loop. The ser loop complex was left out because of its weak electron density.
• How do the results of this study compare to the results of previous studies (See Discussion).

Table 4

• Shows the mainchain dihedral angles for the peptides bound to Fab peptides.
• A similar mainchain torsion angle is shared between resides before and after the GPGR sequence.

Figure 5

• Reveals the Fab 58.2 binding pocket with Aib142
• Figure 5 essentially proposes a mechanism for antibody binding based on amino acid interactions.
• Argp322 on Fab 58.2 is bound in a deep pocket forming a charge-charge interaction with AspL94 and GluH95 of Aib142 peptide

Figure 6

• Reveals the dual conformations of the V3 loop
• All four peptides share the same mainchain torsion angles
• HIGPG residues in Fabs 50.1 and 59.1 can be superimposed with low rmsds, but Fab 58.2 exudes a different turn.

How do the results of this study compare to the results of previous studies

Studies in the past have shown that changes in the amino acid residues of the V3 loop leads to changes in viral tropism however no correlation has been shown as to how it affects the conformation. However this study takes this initiative to towards understanding the conformational flexibility of the loop. They use the crystal structure s of Fab fragments in complex with V3 loop peptides to do so.