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. Crystallization Crystallize protein to cross-section of 30-40um
Measure diffraction patterns with Bragg’s Law to determine space between electrons/atoms (2d model)
Originally crystals diffracted more than 2A, but were able to reduce the limit to 2.5A

11. Multiple Isomorphous Replacement
Electrons oscillate during diffraction, must phase them to find electron distribution (location) by inclusion of evenly spaced heavy metals/atom compounds
Tried over 20 different heavy-atom solutions
Isomorphism highest between K3IrCl6, and K2OsCl6 and Native form.

12. Molecular Replacement
Used Fourier Analysis (mathematical method) to determine 3d pattern of these heavy-atoms (Electron Density Model)
K3IrCl6 modelled as 9 partially occupied sites (2sites of occupancy)
Poor data quality, small isomorphous differences
K2OsCl6 (4 sites of occupancy), highest site at same as 2nd highest for K3IrCl6

13. Density Modification to Improve Electron Density Model
Correlations in region internal to domain 1 of CD4 between experimental electron density and calculated model
Linkage of unmodeled density (PRISM program)
Recipricol-space averaging of the PRISM modeled density
Real-space model subtraction (XPLOR)
Solvent flattening
Histrogram matching
Negative-Density Truncation

14. Structure Solution Data
R-value is difference in experimental structure vs hypothetical structure
Table 1

15. 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

16. 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

17. 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

18. 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

19. 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

20. 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

21. 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

22. 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

23. 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

24. 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

25. 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.

26. 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.

27. 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.

28. Figure 4

29. 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.

30. Conformational change in core gp120
Evidence for CD4-induced conformational change:
1. Structural dilemma with Phe43 cavity
2. Characteristics of 17b binding to core gp120
3. Comparison with theory
Evolutionary algorithms of known sequence variants of gp120 gives secondary structure predictions with high reliability
4. Phe43 cavity is the nexus of the CD4 interface -- lying b/w the inner and outer domain and bridging sheet-- without Phe3 structure might collapse!
Although abundant evidence to suggest that CD4 binding induces a conformational change in gp120, this evidence derives from intact gp120 with V loops in place or from oligomeric gp120-gp41 complex
No evidence yet to explain the nature of the conformational change which occurs within core gp120 itself -- studying the ternary complex structure could tell us what these changes are
Evidence for CD4-induced conformational change:
If the new conformation of gp120 activated by binding of CD4 were preserved in the absence of CD4 = structural dilemma with Phe43 cavity
Why? Cavity lining residues have few structural restrictions yet residues highly conserved and hydrophobic if exposed in a pocket. This pocket structure is therefore intimately connected to the bridging sheet (another structural dilemma in absence of CD4)
Structures seen in presence of CD4 would be sensitive would be sensitive to orientational shifts b/w the inner and outer domains
Characteristics of 17b binding to core gp120:
Do not observe detectable binding of Fab 17b to core gp120 UNLESS CD4 is present
Because no direct CD4-17b contacts in structure, effect of CD4 must be to stabilize bridging-sheet minidoman to which 17b binds (suggesting conformational change induced by CD4 binding)
Binding of CD4 to gp120 not limited to an unmasking of Ab epitope.
Comparison with theory
Evolutionary algorithms of known sequence variants of gp120 gives secondary structure predictions with high reliability
Compared with structure predicted by authors, theory is accurate except at three places where it is wrong: at Phe43 cavity or locations in contact with CD4
Phe43 cavity is the nexus of the CD4 interface -- lying b/w the inner and outer domain and bridging sheet = without which structure might collapse
How does CD4 binding lead to this state? Exceptional CD4 binding thermodynamics suggest answer is a large conformational change in gp120 occurs upon CD4 binding
CD4 inserts Phe43 to stabilize the cavity and the interaction b/w itself and gp120

31. Figure 5: Diagram of gp120 initiation of fusion.
State 1: single monomer of gp V1/V2 loops = partially block the CD4 binding site
Following CD4 binding -- conformational change depicted as an inner/outer domain shift and formation on Phe43 cavity
Chemokine receptor binds to the bridging sheet and the V3 loop causing orientational shift of core gp120
Triggers further changes which leads to fusion of viral and target membranes

32. Viral Evasion of Immune Response
Analysis of antigenic structure of gp120 shows that most of the envelope protein surface is hidden from humoral immune response by glycosylation and oligomeric blockage
Most neutralizing antibodies access only two surfaces: CD4-bidning site and chemokine-receptor binding site
Conformational changes in core gp120
CD4-binding site recognition prevented by: recessed nature of the binding pocket and topographical surface mismatch
Chemokine receptor region recognition prevented by: conformational change; steric blockage; surface mismatch 
Viral Evasion of Immune Response:
How?
Analysis of antigenic structure of gp120 shows that most of the envelope protein surface is hidden from humoral immune response by glycosylation and oligomeric blockage
Most neutralizing antibodies access only two surfaces: one that overlaps CD4-bidnign site (shielded by V1/V2 loop) and another that overlaps the chemokine-receptor binding site (Shielded by V2/V3 loops)
Conformational changes in core gp120
CD4-bidngin site recognition by neutralizing Ab prevented by…
Recessed nature of the binding pocket
Topographical surface mismatch
Chemokine receptor region recognition by neutralizing Ab prevented by…
Conformational change may hide conserved epitope
Steric blockage may take place b/w CD4 and target membrane
Surface mismatch may camouflage chemokine-receptor binding site on V3 loop

33. Mechanistic implications for virus entry
Gp120 crucial for fusion of HIV to cell surface
Given data presented here, suggest this may be a simple process comprising viral oligomer and two host receptors.
Gp120 functions in: POSITIONING
Gp120 function in: TIMING
Entry process initiated by binding of HIV-1 to cellular receptor CD4 -- orients viral spike
CD4 binding induces conformational change in gp120
Mechanistic implications for virus entry:
During virus entry, HIV entry proteins fuse the viral membrane with target cell membrane. Gp120 crucial for fusion of HIV to cell surface
Given data presented here, suggest this may be a simple process: Comprises two membrane: viral oligomer and two host receptors. Two snapshots: an intermediate stare in which gp120 bound to CD4 and a final "fusion-active" state of gp41 ectodomain.
Gp120 functions in: POSITIONING
Locating a cell capable of productive viral infection
Anchoring virus to cell surface
Orienting viral spike next to target membrane
Gp120 function in: TIMING
Holding gp41 in specific conformation and triggering release of fusion peptides of trimeric gp41
Entry process initiated by binding of HIV-1 to cellular receptor CD4
Proposed structure suggests: this binding orients viral spike: orients the N and C termini of gp120 towards viral membrane and the 17b chemokine receptor binding site on gp120 surface faces target cell.
CD4 binding induces conformational change in gp120
Structure described here describes state in atomic detail
CD4 binding results in movement of V2 loop, which partially blocks V3 loop and CD4 interacting epitopes
Also creates/stabilizes bridging sheet
CD4 binding changes V3 region -- altering exposure of V3 epitopes
Uncovered V3 and CD4i epitopes create chemokine-receptor binding site
THUS: CD4 binding not only orients the gp12 surface implicated in chemokine receptor binding to face target cell but also forms and exposes site itself
Next step: Interaction of gp120-CD4 complex with chemokine receptor
Gp120 contacts dominate interaction with chemokine receptor
Because most of chemokine receptor encased in host membrane, binding will move gp120 closer to target membrane
Chemokine receptor binding believed to trigger additional conformational changes in envelope glycoprotein trimer leading to exposure of gp120 ectodomain
Perhaps chemokine receptors triggers gp41 exposure by taking gp120 protomers away from the trimer axis
Structure of gp120/CD4/17b antibody ternary complex described here revels many ,molecular aspects of HIV-1 entry including atomic structure of gp120, explicit interaction with CD4 and conserved binding for the chemokine receptor
Still unknown: Details of apo state of core gp120, oligomeric structure, interactions with chemokine receptors, etc. Further understanding will require snapshots of intermediates

34. Mechanistic implications for virus entry
CD4 binding results in movement of V2 loop, which partially blocks V3 loop and CD4 interacting epitopes
Also creates/stabilizes bridging sheet
CD4 binding changes V3 region -- altering exposure of V3 epitopes
Next step: Interaction of gp120-CD4 complex with chemokine receptor
Binding will move gp120 closer to target membrane
MAIN POINT: Structure of gp120/CD4/17b antibody ternary complex described here revels many ,molecular aspects of HIV-1 entry

35. 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.