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 398.01 Professor Kam D. Dahlquist, Ph.D. Department of Biology Loyola Marymount University March 2nd, 2009

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

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

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

Structure Solution Data Table 1

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 Figure 3d Left side CD4 surface shown in yellow, gp120 imprint in red

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, purple Figure 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.

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.