HIV gp120 Envelope Glycoprotein: Structure & Interactions with CD4 and Antibodies

1. Kwong, P.D., Wyatt, R., Robinson, J., Sweet, R.W., Sodroski, J., Hendrickson, W.A. (1998) Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393:648-659. Nicki Harmon, Samantha Hurndon, & Zeb Russo Bioinformatics Lab 10/19/2011

2. Outline • HIV causes the destruction of CD4 lymphocytes • GP120 is a primary binding region for HIV • Crystal structure at 2.5 Å of a HIV gp120 core with associated proteins • Secondary Structure in GP120 • CD4 is bound to gp120 in a depression formed by the interface of the inner and outer domains • Antibody interactions with gp120 • Overview of gp120 activity during HIV fusion • Protein Production, Crystalization, & Data Collection

3. HIV causes destruction of CD4 lymphocytes • Entry of HIV virus into host cells is mediated by viral envelope glycoproteins • These glycoproteins are arranged in oligomeric, most likely trimeric spikes along the surface of the virion • The surface of the spike is primarily gp120 • gp120 contains five variable regions (V1-V5) • both conserved and variable gp120 regions are heavily glycosylated • this glycosylation probably modulates the immunogenicity and antigenicity of gp120 • gp120 is the main target for antibodies • gp120 will bind to glycoprotein on CD4 and acts as main receptor • gp120 binds to the most amino-terminal of the four immunoglobulin like domains of CD4

4. Outline • HIV causes the destruction of CD4 lymphocytes • GP120 is a primary binding region for HIV • Crystal structure at 2.5 Å of a HIV gp120 core with associated proteins • Secondary Structure in GP120 • CD4 is bound to gp120 in a depression formed by the interface of the inner and outer domains • Antibody interactions with gp120 • Overview of gp120 activity during HIV fusion • Protein Production, Crystalization, & Data Collection

5. GP120 is a primary binding region for HIV • mutagenesis has found critical regions in both gp120 and CD4 for binding • CD4 binding induces a conformation change in gp120 which exposes/forms a chemokine receptor • This chemokine receptor for CCR5 and CXCR4 serve as obligate secondary receptors for HIV entry into the cell • There are other more conserved regions of gp120 that seem to be involved in chemokine-receptor binding • CD4i (CD4 induced) antibodies block the binding of the gp120-CD4 complex to the chemokine receptor • HIV and related retroviruses belong to a class of enveloped fusogenic viruses, all which require post-translational cleavage for activation. • since gp120 is so important in receptor binding and in interactions with antibodies, info about it is important

6. Outline • HIV causes the destruction of CD4 lymphocytes • GP120 is a primary binding region for HIV • Crystal structure at 2.5 Å of a HIV gp120 core with associated proteins • Secondary Structure in GP120 • CD4 is bound to gp120 in a depression formed by the interface of the inner and outer domains • Antibody interactions with gp120 • Overview of gp120 activity during HIV fusion • Protein Production, Crystalization, & Data Collection

7. Crystal structure at 2.5 Å of a HIV gp120 core with associated proteins • Gp120 in red, CD4 in yellow, CD4i antibody 17b in dark and light blue • Due to the fact that gp120 is extensively glycosylated and shows great conformational heterogeneity, radical modification of the protein surface was devised to image it.

8. Outline • HIV causes the destruction of CD4 lymphocytes • GP120 is a primary binding region for HIV • Crystal structure at 2.5 Å of a HIV gp120 core with associated proteins • Secondary Structure in GP120 • CD4 is bound to gp120 in a depression formed by the interface of the inner and outer domains • Antibody interactions with gp120 • Overview of gp120 activity during HIV fusion • Protein Production, Crystalization, & Data Collection

9. Secondary Structure in GP120 • core is made up of 25 β-sheets, 5 α-helices, and 10 defined loop segments • the polypeptide chain is folded into two main domains along with some digressions from this body • Inner domain contains a two-helix, two-sheet bundle with a small five sheet β-sandwich at its termini-proximal end and a projection from the distal side where the V1/V2 stem originates. • Outer domain is a stacked double barrel that lies alongside inner domain so that the both barrel axes are roughly parallel to each other. • There is a ‘minidomain’ which is comprised of four antiparallel β-sheets that create a ‘bridging sheet’ that is in contact with both the inner and outer domains • structure based alignment shows conservation despite the variability in HIV strains

10. Great similarities between HIV-1,HIV-2, and SIV • α-Carbon trace shows the conservation of disulfide bridges • Sequence alignment shows similarity between HIV strains and SIV as well a s solvent accessability

11. Outline • HIV causes the destruction of CD4 lymphocytes • GP120 is a primary binding region for HIV • Crystal structure at 2.5 Å of a HIV gp120 core with associated proteins • Secondary Structure in GP120 • CD4 is bound to gp120 in a depression formed by the interface of the inner and outer domains • Antibody interactions with gp120 • Overview of gp120 activity during HIV fusion • Protein Production, Crystalization, & Data Collection

12. CD4 is bound to gp120 in a depression formed by the interface of the inner and outer domains • Electron density in the Phe43 binding site • Electrostatic potential across the surfaces of gp120 and CD4

13. More binding of CD4 and gp120 • 3d shows contact surfaces • 3e shows mutational hotspots on both CD4 and gp120 • 3f elucidates side vs main-chain contributions to gp120 surface • 3g demonstrates gp120 sequence variability • 3h shows the Phe43 cavity

14. A closer look at the Phe43 cavity

15. Outline • HIV causes the destruction of CD4 lymphocytes • GP120 is a primary binding region for HIV • Crystal structure at 2.5 Å of a HIV gp120 core with associated proteins • Secondary Structure in GP120 • CD4 is bound to gp120 in a depression formed by the interface of the inner and outer domains • Antibody interactions with gp120 • Overview of gp120 activity during HIV fusion • Protein Production, Crystalization, & Data Collection

16. Antibody interactions with gp120 • Interactions between gp120 and CD4i antibody, highlighting the V3 region

17. Outline • HIV causes the destruction of CD4 lymphocytes • GP120 is a primary binding region for HIV • Crystal structure at 2.5 Å of a HIV gp120 core with associated proteins • Secondary Structure in GP120 • CD4 is bound to gp120 in a depression formed by the interface of the inner and outer domains • Antibody interactions with gp120 • Overview of gp120 activity during HIV fusion • Protein Production, Crystalization, & Data Collection

18. Overview of gp120 activity during HIV fusion to lymphocytes

19. Outline • HIV causes the destruction of CD4 lymphocytes • GP120 is a primary binding region for HIV • Crystal structure at 2.5 Å of a HIV gp120 core with associated proteins • Secondary Structure in GP120 • CD4 is bound to gp120 in a depression formed by the interface of the inner and outer domains • Antibody interactions with gp120 • Overview of gp120 activity during HIV fusion • Protein Production, Crystallization, & Data Collection

20. Protein Production, Crystalization, & Data Collection • Two domain CD4 was created in Chinese hamster ovarian cells • 17b antibody was isolated from a HIV-1 infected individual • Core gp120 taken from Drosophilia • After crystallization, took many models of each protein and compared them to crystal imagery

21. Thank You!