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

1. Dual conformations for the HIV-1 gp120 V3 loop in complexes with different neutralizing Fabs (1999) Structure 7: 131-142 Alex George J’aime Moehlman Amanda Wavrin Bobek Seddighzadeh Stanfield RL, Cabezas E, Satterthwait AC, Stura EA, Profy AT and Wilson IA Journal Club Presentation BIOL 398-01/S10: Bioinformatics Laboratory March 9, 2010

2. Outline • Background on gp120 protein • Significance of the V3 Loop • Background on antibodies • Introduction to the paper • Results • Discussion • conformational structures

3. gp120 Is a Viral Surface Glycoprotein • gp120 is a glycoprotein cleaved from gp160 precursor and remains in non-covalent association with gp40 • gp120 is anchored to the viral membrane by gp40 where it plays a vital role in the specificity of the virus • Binds to CD4 cells and at least one other coreceptor on the target cell’s surface • CCR5T-Cells; CXCR4 macrophages

4. Binding of gp120 to the CD4 Cell Receptor and Coreceptor http://www.nature.com/nrd/journal/v2/n7/images/nrd1134-f3.gif

5. Characteristics of the V3 Loop of HIV-1 gp120 • V3 domain of gp120- is disulfide-linked loop • Approximately 40 amino acids • High degree of sequence diversity amongst viral isolates • Required for viral entry into the cell • Membrane fusion • Interacts with cell surface chemokine receptors on T cells and macrophages

6. V3 loop Is an Immunogenic Site • Accessibility to V3 loop depends on: • Viral isolate type • Interaction with CD4 • Causes a conformational change • As a result of gp120-CD4 interaction, the V3 loop becomes susceptible to neutralizing antibodies and proteases

7. Conformational variations in gp120 and the V3 loop can modulate gp120 function • La Rosa et al. Study • The amino acid composition of the loop are highly conserved in some positions and variable in others • E.i. the MN sequence are conserved in 80% of 244 isolates • This suggests that these amino acid positions have a key structural and functional role to virus infectivity

8. Antibodies Can Help Reveal Tertiary Conformation of V3 Loop • Investigation of the neutralizing antibodies and their complexes with V3 loop reveal: • Tertiary conformation of the V3 loop • Which can lead to an understanding in co-receptor usage • An understanding of why some antibodies are viral-specific and others recognize many different strains • Antibodies neutralize the virus by preventing viral-cell membrane fusion

9. Antibody Fab Fragments 50.1, 59.1 and 58.2 • Antibody Fab fragments: • Fab 50.1: specific for MN viral strain • Fab 59.1: strongly neutralize IIIB and weakly neutralize MN • Fab 58.2: highly potent and broadly neutralizing antibody • Further analysis revealed a double bend around GRAFY contrary to the study • Found they could replace the ALA residue in the double bend with Aib residue to provide a more rigid scaffold for vaccine or drug design

10. Introduction to the Stanfield et al. Paper • La Rosa study determined conserved amino acid sequences in the V3 loop are significant to structure • Examine antibodies in complex with the V3 loop to determine the tertiary conformation • Attempts to identify why some antibodies are viral specific while others recognize many different viral strains

11. The abbreviated amino acid sequences for the V3 peptide RP70, His Loop, Ser Loop and Aib142 Detailed view of the His and Ser Loop hydrazone bond a) b)

12. Results from X-Ray Crystallography of Fab 58.2 in complex with Aib142, His loop and the Ser loop X-ray structure for the Fab-V3 complexes agreed with the La Rosa et al. predictions

13. 3-Dimensional views of the Fab 58.2 complex with: Aib142 Histidine Loop Serine Loop Red sequence= highly conserved region Cyan= Light Chain Purple= Heavy Chain

14. Comparing the 3D structures of the H1 loops in Fab Fragments 58.2 (red), N10 (yellow) and AN02 (blue)

15. Predicted structures from the determination of electron densities of Aib142 (a,b), the His loop (c) and the Ser loop (d)

16. Surface Picture of Fab 58.2 indicating acidic (red), basic (blue) and neutral (white) regions Indicates the acidic binding pocket for ArgP322 of Aib 142 b) Stereoview of the combining site

17. Summary of the number of Van der Waals interactions between Fab 58.2 and its bound peptides, Aib142, the Histidine loop and the Serine loop

18. Summary of the number of Hydrogen bonds and salt bridge interactions between Fab 58.2 and its bound peptides, Aib142, the Histidine loop and the Serine loop

19. a) The conformations of the V3 loop bound to Fabs 50.1 (yellow), 59.1 (purple) and 58.2 bound to Aib142 (blue) and His loop (green) Summary the turn angles displayed in Figure 6 Figure 6

20. Biological Implications • The V3 loop is involved in virus infectivity. • There is a high conservation at the tip of the gp120 V3 loop, surrounded by regions of high diversity. • This suggests that structural conservation is related to biological function. • Studies have suggested that one or more amino acid residue alterations within the V3 loop leads to changes in viral tropism.

21. Further Research • Unanswered questions provide a direction for further studies, with intent of revealing the biological role of the V3 loop. • This could open up the possibility of drug design or vaccine development.

22. References • Stanfield RL, Cabezas E, Satterthwait AC, Stura EA, Profy AT and Wilson IA. (1999) Dual conformations for the HIV-1 gp120 V3 loop in complexes with different neutralizing Fabs. Structure 7: 131-142