Structure of an HIV gp120 envelope glycoprotein in complex with the CD4

receptor and a neutralizing human antibody.

Kevin Paiz-RamirezJanelle N. RuizRyan Willhite

Angela GaribaldiBiology 398.01

Professor Kam D. Dahlquist, Ph.D.Department of Biology

Loyola Marymount UniversityMarch 2nd, 2009

• 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

Outline

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

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.

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

Figure 1Red- 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, 648-659(18 June 1998)doi:10.1038/31405

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

Figure 2

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, 648-659(18 June 1998)doi:10.1038/31405

A- Ribbon diagram

B- Topology diagram

C- Helices shown as corkscrews and labeled (1–5)

D- structure-based sequence alignment

Methods of Determination

• Crystallization by modifying protein surface

• Deglycosylation of gp120 variants

• Molecular replacement

• Isomorphous replacement

• Density modification

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

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.

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

Density Modification to Improve Electron Density Model

1. Correlations in region internal to domain 1 of CD4 between experimental electron density and calculated model

2. Linkage of unmodeled density (PRISM program)

3. Recipricol-space averaging of the PRISM modeled density

4. Real-space model subtraction (XPLOR)5. Solvent flattening6. Histrogram matching7. Negative-Density Truncation

Structure Solution Data

Table 1

•R-value is difference in experimental structure vs hypothetical structure

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

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

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

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

•Left side CD4 surface shown in yellow, gp120 imprint in red

Figure 3d

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

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

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, purpleFigure 3g

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

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

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

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.

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.

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.

Figure 4

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.

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!

Figure 5: Diagram of gp120 initiation of fusion.

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 

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

• 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

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.