1. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody.
Kevin Paiz-Ramirez
Janelle N. Ruiz
Ryan Willhite
Angela Garibaldi
Biology
Professor Kam D. Dahlquist, Ph.D.
Department of Biology
Loyola Marymount University
March 2nd, 2009

2. Outline Structure Determination Purpose of Study Methods
Electron Density in Phe43 Cavity
Interfacial cavities
Antibody Interface
Chemokine Receptor site
Oligomer & gp41 interactoin
Conformational changes
Viral evasion & Immune response
Mechanistic implication for virus entry
References

3. Exploring HIV-1 Structure
Entry of HIV involves a sequential interaction of
the envelope glycoprotein (gp120)
CD4 glycoprotein
chemokine receptor (primary receptor)
CD4i (antibodies that block gp120-CD4 complexes to the chemokine receptor)
CCR5 and CXCR4 for HIV-1 (secondary receptors)
CD4 binding induces conformational changes in the gp120
Entry of HIV is mediated by envelope glycoproteins
5 variable regions
Variable and non variable regions are glycosylated
V3 loop determines specificity

4. Exploring HIV-1 Structure
Gp41 (transmembrane coat proteins) variants found in all enveloped viruses
N-terminal fusion peptides which participate in membrane fusion
Enveloped viruses tend to be characteristic in entry
Direct membrane penetration (HIV)
HIV causes destruction of CD4 T cells which ultimately leads to AIDS.

5. Purpose of Study Gp120 glycoprotein has important role in
receptor binding
interactions with neutralizing antibodies
Information about the gp120 structure is important for understanding HIV infection
Assist in designing therapeutic strategies.
Overall purpose is to observe the mechanism of HIV entry and intervene

6. Figure 1 Red- gp120 Orange/yellow- N terminal 2 domains of CD4
Light blue- Fab17b
Purple/blue- Heavy chain
From the following article:Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibodyPeter D. Kwong, Richard Wyatt, James Robinson, Raymond W. Sweet, Joseph Sodroski and Wayne A. HendricksonNature 393, (18 June 1998)doi: /31405

7. Determining The Structure
Devised a crystallization strategy that modified the protein surface
Obtained crystals of
a ternary complex composed of a truncated form of gp120
the N-terminal two domains (DID2) of CD4
Fab from the human neutralizing monoclonal antibody 17
The ternary structure was solved by
combinations of molecular replacement
isomorphous replacement
density modification techniques

8. Figure 2 A- Ribbon diagram B- Topology diagram
C- Helices shown as corkscrews and labeled (1–5)
D- structure-based sequence alignment
Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibodyPeter D. Kwong, Richard Wyatt, James Robinson, Raymond W. Sweet, Joseph Sodroski and Wayne A. HendricksonNature 393, (18 June 1998)doi: /31405

9. Methods of Determination
Crystallization by modifying protein surface
Deglycosylation of gp120 variants
Molecular replacement
Isomorphous replacement
Density modification

10. Structure Solution Data
Table 1

11. Ribbon Diagram of CD4-gp120
gp120 in red
CD4 in yellow
Residue Phe 43 of CD4 reaches into heart of gp120
Gp120 has recessed binding pocket

12. Electron Density in Phe43 Cavity
Gp120 model in red
CD4 model carbon atoms in yellow
Nitrogen atoms in blue
Oxygen atoms in red
Phe43 of CD4 reaches up to contact cavity
Upper middle region has central unidentified density
Hydrophobic residues line back of cavity, around c.u.d

13. Electrostatic Surfaces of CD4 and gp120
Electrostatic potential at
solvent-accessible surface
Dark blue is most positive
Deep red is most negative
Right side gp120 recessed binding pocket marked by Yellow Cα worm of CD4
Left side shows CD4 surface with red Cα worm of gp120

14. CD4-gp120 Contact Surface Right side gp120 surface in red
Surface is within 3.5A surface to atom distance of CD4 (shown in yellow)
Serves as imprint of CD4 on gp120 surface
Figure 3d
Left side CD4 surface shown in yellow, gp120 imprint in red

15. CD4-gp120 Mutational Hot-spots
On the right, gp120 surface in red
Gp120 residues that affect CD4 binding are highlighted (high effect cyan, moderate effect green)
Left, residues important in gp120 binding on CD4 surface (cyan-high, green-moderate)
White is surface of H20 filled cavity at CD4-gp120 interface
Figure 3e

16. Side-Chain/Main-Chain Contribution to gp120 Surface
Main-chain atoms portion of surface, green
Side-chain atoms portion of surface, white
Cα of glycine atoms portion of surface, red
Higher surface concentration of main-chain atoms in regions corresponding to CD4 imprint
Figure 3f

17. Sequence Variability Mapped to gp120 Surface
Scale of white (conserved) to red (highly variable)
Carbohydrate residues are:
N-acetylglucosamine is blue
Fucose is blue
Asn-proximal N-acetylglucosamines, purple
Figure 3g

18. Phe43 Cavity Surface of Phe43 cavity in blue, buried in gp120.
Cα worm representation of gp120 (red)
Green shows secondary structure predictions that were incorrect
Figure 3h

19. Cd4-gp120 Interface
Shows 6 segments of gp120 (single lines) interacting with CD4 (double lines)
Arrows show main-chain direction
Side Chain of Phe43 also shown
Figure 3i

20. Gp120 Contacts around Phe43 and Arg59 of CD4
Shows residues on gp120 involved in direct contact with Phe43 or Arg59
Dashed Lines are electrostatic interactions
Bold lines are side chains of Phe43, Arg59, and parts of gp120 that interact with them
Hydrophobic interactions between Phe43 (CD4) and Trp 427, Glu 370, Gly 473 and Ile 371 (gp120) and between Arg59 (CD4) and Val430 (gp120)
Figure 3j

21. Interfacial Cavities
Analysis of the surface of the ternary complex revealed topological surface cavities.
The gp120-CD4 interface were unusually large. The cavity served as a water buffer between gp120 and CD4.
The residue that line this cavity provide little direct contact to CD4. This can reduce binding. This can affect the binding of antibodies against the CD4 binding site.
Despite the unusual cavity laden interface between gp120 and CD4 the missing gp120 loops and termini could not have a role in filling these cavities.

22. Antibody Interface
Concerning the 17b antibody, a broadly neutralizing human monoclonal isolated from HIV infected individuals that bind to CD4 induced gp120 epitrope, that overlaps into the chemokine receptor binding site.
The 17b contact surface is very acidic.
Tested against four stranded bridging sheet. Contact suggested that the sheets were necessary for 17b binding.
The interaction between 17b and gp120 involves a hydrophobic central region flanked with periphery by charged regions
There is no direct CD4-17b contact.

23. Chemokine-Receptor site
The chemokine receptor, CCR5 overlaps with 17b, both are induced upon CD4 binding and both involve highly conserved residues.
The hydrophobic and acidic surface of 17b heavy chain may mimic the tyrosine rich acidic N-Terminal region of CCR5.

24. Figure 4

25. Oligomer and gp41 interaction
gp120 exist as a trimeric complex with gp41 on the virion surface.
The N and C termini of full length gp120 are the most important regions for interaction with the gp41 glycoprotein.
It was expected that gp41 interactive regions will extent away from core gp120 toward the viral membrane that is conserved.

26. References
Kwong Peter, Wyatt Richard, Robinson James, Sweets Raymond, Sodroski Joseph, and Wayne Hendrickson. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Proc Natl Acad Sci USA 1998.