KP Ramirez Week 7

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Top 10 Definitions

1. Ectodomain: Part of a membrane protein that extends into the extracellular space. Ectodomains are usually the part of a protein that initiate contact with surface which leads to signal transduction. In SARS-CoV the ectodomain of the spike protein is responsible for attachment to and entry into cells during infection.
2. Glycocalyx: Extracellular polymeric material produced by some bacteria epithelia and other cells. Initially applied to a polysaccharide matrix excreted by epithelial cells forming a coating on the surface of epithelial tissue.
3. Monoclonal: Cells that are defined as a group produced from a single ancestral cell by repeated cellular replication. Thus from a singe "clone" the process of replication can occur in vivo, or may be stimulated in vitro for laboratory manipulations.
4. Fusogenic: Facilitating fusion of the viral envelope with the cellular plasma membrane.
5. Epitope: Associated with the antigenic molecule to which the T-cell receptor respond. A site on the large molecule against which an antibody will be produced and will bind.
6. Chemokine Receptor: Cytokine receptors found on the surface of certain cells, which interact with a type of ctyokine called a chemokine. There are 19 distinct chemokine described in mammals.
7. Oligomer: An oligomer consists of less than fine monomer units, in contrast to a polymer that at least in principle, consists of an unlimited number of polymers.
8. Isomorphism: A kind of mapping between objects which show a relationship between two properties of operations. If there exists an isomorphism between two structures, we call the two structures Isomorphic.
9. Proteolysis: Directed degradation of proteins by cellular enzymes called proteases or by intramolecular digestion.
10. Crystallography: The experimental science of determining the arrangement of atoms in solids.

Outline

Introduction

• Describes the effect HIV-1 has on the human body including destroying CD-4+ lymphocytes. This is best explored when understanding the structure of this virus.
• The viral envelope contains glycoproteins and oligomeric spikes, trimeric spikes which are anchored by gp41, a transmembrane envelope glycoprotein.
• There are five variable regions. Region 1-4 form loops exposed to the surface which are bonded at their bases by disulfide bonds.
• Basic Entry and binding can be studied through conserved regions of gp120, a glycoprotein target for neutralizing antibodies elicited during infection, which form discontinuous structures that are essential for interacting with gp41 ectodomain and the viral receptors.
• Human Immunodeficiency viruses enter host cells gp120 envelope glycoprotein binding to the Cd4 glycoprotein.
• CD4 structure is similar to CDR2 region in terms of gp120 binding. Once it binds it causes conformational changes in gp120 which expose or forms binding sites for CCR5 and CXCR4, secondary receptors for viral entry.
• CD4 causes conformational changes in envelope glycoproteins causing shedding of gp120 from complex.
  1. To understand the role of the gp120 glycoprotein in receptor binding and interactions with neutralizing antibodies, this could lead to future understanding of HIV-1 resistance.

Structure determination

• The process used was crystallization by modifying the protein surface exploring gp120 from strain HXBc2 of HIV-1.
• The structure of gp120 resulted in being distinctive to other structures that were explored in the past. This included nno similarities in the inner domains.

Figure 1 Presents the overal structure of gp120, Fab17b in blue, and the N-termial of CD4 in yellow

Figure 2 Presents the structure core of gp120, indicating spacial proximity between strands and H bonding.

Interfacial cavities

• Analysis of the solvent accessible surface of the ternary complex revealed a number of topological surface cavities.
• The gp120-CD4 interface were unusually large, the second was from a pocket in gp120 at the interface between the inner and outer domain.
• The cavity served as a water buffer between gp120 and CD4, it was postulated that this may have helped the virus escape from antibodies against the CD4 binding site.
• Given the frequency of gp120 sequence divergence the residue that line this cavity provide little direct contact to CD4 they do affect gp120-CD4 interactions, thus mutations at Thr257 and Trp427 can substantially reduce binding, changes in cavity lining residue can also affect the binding of antibodies directed against the CD4 binding site.
• Despite the unusual cavity laden interface between gp120 and CD4 this structure reflects the true character of interaction. The missing gp120 loops and termini could not conceivably have a role in filling these cavities.

Antibody Interface

• Explored 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 and all four strands mad substantial contact with 17b, which suggested that the integrity of the sheets were necessary for 17b binding. It had a hydrophobic center surrounded by basic periphery. The basic gp120 complements the acidic 17b surface but only one salt bridge was observed.
• This presented that 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.

Oligomer and gp41 interaction gp120 exist as a trimeric complex with gp41 on the virion surface, a large electroneutral surface on the inner domain.

• Mutagenic and antibody binding analyses indicate that 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 interative regions will extent away from core gp120 toward the viral membrane that is conserved.

Conformational Change in core gp120

• Abundant evidence suggests that CD4 binding induces a conformational change in gp120.
• However if the conformation of gp120 were preserved in absense of CD4 the Phe43 cavity would be a pocket and present a dilemma. The residues would instead by inexplicably hydrophobic inside of a pocket, and would connect to the bridging sheet.
• It was also observed that there was no detectable binding of Fab17b to core gp120 unless CD4 is present.
• The Phe43 cavity serves as the linchpin and without it the structure might collapse.

Viral evasion of immune responses Most envelope protein surface is hidden from humoral immune responses by glycosylation and oligomeric occlusion.

• Most neutralizing antibodies access only two surfaces: the one that overlaps the CD4 binding site and the one that over laps the Chemokine receptor site.
• Conformation without CD4 may expose underlying side chain variability.
• Understanding the specific mechanism of immune evasion may be a prerequisite to the design of an effective vaccine.

Mechanistic implications for virus entry

• Virus entry implies the HIV surface proteins fuse the viral membrane with the target cell membrane.
• gp120 is a crucial participant in the control and initiation of fusion, it functions in positioning and locating a cell capable of productive viral infection.
• This is a simplistic system which comprises only two membranes: the viral oligomer and the two host receptors.
• Crystallography is used to provide us with how to understand the intermediate state where gp120 is bound to CD4, and the fusion active state of the gp41.
• The process is initiated by binding of HIV-1 to the cellular receptor CD4, this orients the viral spike . This follows with the N and C termini of the gore gp120 toward the viral membrane while 17b epitope/chemokine receptor binding site of gp120 surface faces the target cell membrane.
• CD4 induces conformational changes in gp120 which results in the creation of a metastable oligomer. CD4 binding results in movement of the V2 loop. This stabilizes the bridging sheet. It also changes teh V3 region. The CD4 site not only orients the gp120 surface implicated in chemokine receptors binding to face the target cell it also forms and exposes the site itself.
• The next step in HIV-1 entry is the interaction of gp120-CD4 complex with the chemokine receptor. It is encased in the host membrane and binding will move the gp120 bridging sheet close to the target membrane. The chemokine receptor binding triggers additional conformational changes in teh HIV-1 envelope.
• Further understanding will require snapshots of other intermediates.

Methods Protein production, crystallization and data collection the two-domain cd4 was produced in hamster ovarian cells, the monoclonal antibody 17b and core gp120 from drosophila schneider 2 lines under the control of an inducible metallothionein promoter. protein purification and ternary complex crystallization was run. Best crystals were small needles of cross section of 30-40 micrometers, were crosslinked by vapor diffustion glutaraldehyde treatment and transferred to immiscible oil. Oscillation data was collected. Data processing and reductio were achieved using DENZO and SCALEPACK

Table 1'

Structure determination and refinement In order to locate the position of the Fab 17b in the ternary complex crystals, rotational searches with 52 different Fab models were made with the program MERLOT. The Fab fragments were aligned by superposition of their variable domains to compare rotational solutions. Four models showed more than 10% discrimination between the highest and second highest solutions, no consistent rotational solutions were found. Discrimination between correct and incorrect solutions was achieved by using confirmatory searches with variable portion of the Fab. this was only successful with molecule B of 1hil. this was Rigid-body refinement of the 1hil solution. Translation searches used in rigid body refined Fab as a partial structure to help descriminate the correct solution. 2 distinctive solutions were found the 25 rotational solution of 3Cd4 and the 61st rotational solution of 1cdh these were virtually identical. The Calpha backbone could be modelled, defining the secondary structure and with computer aided sequence alignment to help to position the amino acid sequence leaving only regions around the n terminus, the v1/v2 loop and the V4 loop uncertain.

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