Kwong et. al. (1998) Nature Paper

My partners are Zeb Russo and Samantha Hurndon and we are working on the Kwong paper for Journal Club.

Vocabulary Terms

Oligometric- a molecule consisting of two, three, or four monomers; less than five. TheFreeDictionary
Glycosylated- the process of adding sugar units to protein molecules. Biology Online
Antigenicity- the capacity of a molecule or an antigen to induce an immune response that is to be recognized by and interact with an immunologically specific anitobdy or T-cell receptor. Biology Online
Trimeric- a reaction product composed of three identical molecules. TheFreeDictionary
Chemokine- any of various cytokines produced in acute and chronic inflammation that mobilize and activate white blood cells. TheFreeDictionary
Isomorphous- having a similar structure or appearance but being of different ancestry. TheFreeDictionary
Ellipsoid- The refractile outer portion of the inner member of a rod or cone cell, it is located between the myoid and the outer member of the cell and contains mitochondria. TheFreeDictionary
Murine- of, or relating to, a member of the rodent family. Biology Online
Cryogenic- systems at very low temperatures. Biology Online
Microcalorimeter- a calorimeter for measuring very small amounts of heat, in which the heat source and a small heating coil are placed in identical vessels and the amount of current through the coil is varied until the temperatures of the vessels are identical, as indicated by thermocouples. TheFreeDictionary

Outline

Introduction

The envelope protein gp120 is crucial for HIV to enter human cells.
Gp120's interaction with CD4 glycoprotein and chemokine receptor causes a fusion between viral and cellular membrane.
This study shows the X-ray crystal structure of gp120 at 2.5 A resolution.
The X-ray displays gp120 with a two-domain fragment of human CD4 and an anitgen-binding fragment that blocks chemokine-receptor binding.
The overall result of the crystal structure is that gp120 undergoes a conformational change when it binds to CD4.
HIV can be compared to the related SIV because they both take over their hosts through the destruction of CD4.
Both of these viruses contain specific regions (V1-V5).
First 4 regions (V1-V4) form loops that contain disulfide bonds.
It has been determined that gp 120 binds to the most amino-terminal of the domains of CD4.
Through mutagenesis, the regions on CD4 that are critical for gp120 binding have been discovered.
The V3 loop is the main factor in chemokine receptor binding.
HIV comes from a family of retroviruses that includes diseases such as influenza.
These viruses all require post-translational cleavage for activation.
The exterior coat proteins of these viruses are distinct allowing them to penetrate membranes in different ways.
Since gp120 is crucial for HIV to invade human cells, this study focuses on it's structure in order to understand how it functions.

Structural Determination

In order to understand the relation between structure and function, gp120 glycoprotein was radically modified at the surface to see what would result.
Cuts were made at the termini and variable loops in different combinations with various gp120 with various gp120 strains.
Over 20 different combinations of gp120 variants were obtained and crystallized.
The gp120 crystallized from HxBC2 of HIV-1 was manipulated to have deletions of the 52 and 19 residues; itlost over 90% of it's carbohydrates but retained 80% of the non-variable loop protein.
In this manipulated model, Gly-Ala-Gly was substituted in for 67 V1/V2 loop residues and 32 V3 loop residues; there was also removal of all sugar groups between the N-terminus residues.
Gp120 was shortened but was done so in a way to retain it's ability to interact with CD4 and antibodies.
The ternary structure was determined through techniques such as molecular replacement, isomorphous replacement, and density modification.
The final model that was composed had a total of 7,877 atoms.
Gp120: 90-396 and 410-492 (except the loop substitutions)
CD4: 1-181 residues
Antibody: Light chain 1-213 residues and Heavy Chain 1-229 residues
There were also 602 water molecules, 11 N-acteylglucosamine, and 4 fucose residues.
Figure 1: this figure shows the truncated version of the gp120 without it's N and C terminal ends, the 2 N-terminal domains of CD4, and the light and heavy chain of the Fab 17b antibody.

Structure of gp120

The core gp120 contains 25beta strands, 5alpha helices, and 10 loops.
Figure 2: this figure shows the polypeptide chain of gp120 folded into 2 domains.
The inner domain has 2 helices, 2-stranded bundle with a 5 stranded beta where the V1/V2 stem is located.
There is no similarities between the inner domain and atomic structure.
The outer domain has a stacked double barrel and is parallel.
Figure 4: this figure shows the carbon-trace patterns on gp120 and where the V3 loop is located from different angles.
HIV-2 is 35% identical to the HXBc2 strain, 77% and 51% for HIV-1 C and O traces.

CD4-gp120 Interaction

Cd4 is bound in a depression at the interface of the domains and bridging sheet of gp120.
Surface areas are smaller due to large cavities in the interface.
The CD4-gp120 surface has a complementary electrostatic potential at the contact sites.
Figure 3: this figure shows electron density in the CD4-gp120 interaction; it also displays cavities on the interfacial surfaces.
The atomic contacts made on the interface as Van der Waal and Hydrogen bonds.
These interactions are compatible with previous data studying mutations of these surfaces.
Phe43 and Arg59 on CD4 have contact on multiple sites on gp120's residues (Asp368, Glu370, Trp427).
Phe43 accounts for 23% of the contact.
Lys29 on CD4 is the only residue to make an ionic hydrogen bond.
Most interfacial interactions occur on main chain atoms.
Phe43 has highly conserved hydrophobic residues that have a functional significance.

Interfacial Cavities

There are 2 unusually large cavities at the interface.
The larger cavity is hydrophilic on both surfaces.
Residues on gp120 have a variable section surrounding highly conserved areas that play an important role in binding to CD4.
Phe43 is spherical and is located at the interactions of domains with the bridging sheets.

Antibody Interface

17b has an acidic surface.
It's location suggests that it is a necessity for antigen binding.
The CD4i epitopes are disguised from the immune system due to the V2 and V3 loops that are nearby.

Chemokine-Receptor Site

The Chemokine Receptor CCR5 overlaps the 17b epitope.
The basic and polar gp120 residues are important for it's interaction with this receptor.
The electrostatic interactions between the bridging sheet (basic) and the chemokine receptor (acidic) are possibly what drive the conformational change that allows the virus to enter the cell.

Oligomer and gp41 Interactions

gp120 is a trimer complex with gp41.
The full length N and C terminal ends (not the shortened versions that were previously used) are crucial for interaction with gp41.

Conformational Change in Core gp120

The conformational change happens when the gp120 is bound to CD4, from loops and the gp120-gp41 complex.
17b does not bind to gp120 unless CD4 is present, although the two are never in contact.
Phe43 act as a stabilizing structure, in it's absence structure is endanger of collapsing.

Viral Evasion of Immune Responses

Most of the envelope protein's surfaces are hidden from immune responses due to glycosylation.
There are two free surfaces; the first overlaps CD4 binding site which is shielded by the V1-v2 loops and the second overlaps the chemokine-receptor sites which is shielded by the V2-V3 loops.

Mechanistic Implications for Virus Entry

During viral entry, the HIV surface proteins fuse both membranes together.
Figure 5: this figure shows how the gp120 fuses with the chemokine receptors.
gp120 controls and initiates this membrane fusion.
It also locates cells that are vulnerable to infection and initiates this same binding and fusion processes.
The alignments of the N and C terminal ends of gp120 that face the viral membrane and the 17b/chemokine-receptor binding sites on gp 120 that face the target cell membrane, are consistent with the proposed structure in this experiment.