Nicki Harmon, Samantha Hurndon, & Zeb Russo Bioinformatics Lab 10/19/2011

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!