ColinWikholm BIOL368 Week 8

Preparation for Week 9 Journal Club

Unknown Vocabulary

1. chemokine receptors
   • "receptors are G protein-coupled receptors containing 7 transmembrane domains that are found predominantly on the surface of leukocytes." (http://encyclopedia.thefreedictionary.com/chemokine+receptors)
2. haemagglutinin
   • "an antibody that causes the clumping of red blood cells" (http://www.thefreedictionary.com/haemagglutinin)
3. prolate
   • "Having the shape of a spheroid generated by rotating an ellipse about its longer axis." (http://www.thefreedictionary.com/prolate)
4. ternary
   • "consisting of three or groups of three" (http://www.thefreedictionary.com/ternary)
5. complementarity-determining region
   • " are regions within antibodies (also known as immunoglobulins) or T cell receptors where these proteins complement an antigen's shape. Thus, CDRs determine the protein's avidity (roughly, bonding strength) and specificity for specific antigens." (http://encyclopedia.thefreedictionary.com/Complementarity-determining+regions)
6. metastable
   • "Of, relating to, or being an unstable but relatively long-lived state of a chemical or physical system, as of a supersaturated solution or an excited atom." (http://www.thefreedictionary.com/metastable)
7. ectodomain
   • "An ectodomain is the domain of a membrane protein that extends into the extracellular space (the space outside a cell)." (http://encyclopedia.thefreedictionary.com/ectodomain"
8. cryostat
   • " is a device used to maintain low cryogenic temperatures of samples or devices mounted within the cryostat." (http://encyclopedia.thefreedictionary.com/cryostat)
9. prophylactic
   • "Acting to defend against or prevent something, especially disease; protective" (http://www.thefreedictionary.com/prophylactic)
10. monoclonal antibody
    • "Any of the highly specific antibodies produced in large quantity by the clones of a single hybridoma cell." (http://www.thefreedictionary.com/monoclonal+antibody)

Outline

What is the main result (message) presented in this paper?

This study crystallized gp120 and determined the structure of the the gp120-CD4-NAb complex. It also shows the mechanisms of HIV host evasion.

• This is important for understanding HIV-1 structure, and thus the functioning of HIV-1 related to infection of host cells and escape from the immune system. This study proposed the mode of entry consistent with modern perspectives. That is, HIV-1 entry involves primary binding of gp120 to CD4. This causes a conformational change in gp120 that exposes it for chemokine secondary binding or binding by neutralizing antibodies (they are thus called CD4 induced (CD4i) antibodies). If the antibody binds, it prevents secondary binding. If not, chemokine receptors bind to

What is the importance or significance of this work?

Because this study was the first study to elucidate structure of the gp120-CD4-NAb, as well as to crystallize gp120, it confers considerable knowledge to HIV-1’s structure that is relevant to host infection. What is more, it reveals details relevant to the exposure of the gp120 V3 region and its pertinence to targeting by the immune system. As a major epitope, this suggests that V3 mutability may affect host evasion. What is more, it confers specificity for secondary binding to chemokine receptors.

What were the limitations in previous studies that led them to perform this work?

The primary limitations of previous studies was the lack of understanding of HIV-1 glycoprotein structures. In addition, the mechanisms of host cell invasion and immune evasion was previously not understood with respect to target epitopes, mutational effects on protein structures, and subsequent effects on HIV-1 mechanisms and interactions.

What were the methods used in the study?

Protein complex isolation and crystallization

• CD4 domains were derived in hamster ovarian cells.
• the 17b antibody was derived from immortalized B-cells created in response to Epstein-Barr viruses.
• The 17b NAb was fused to a core of gp120 from Drosophila, and mediated by a metallothionein promoter
• The samples were crystallized, suspended in oil, and flash frozen.
• A Fuji BAS2000 scanner was used for diffraction analysis at Brookhaven National Laboratory

Structure determination

• Rotational modelling and refinement was used to identify constituents of the ternary complex crystals.
• After the alpha carbon backbone was determined, secondary modelling and refinement was used to predict the residual N terminus, V1/V2 loop, and V4 regions.

Structural analysis

• Differences in CD4 structure in the ternary complex were compared against the free state (procedure references, not described).
• Interatomic contacts were performed, as well as visual comparison against the SCOP database.

Briefly state the result shown in each of the figures and tables.

• Figure 1. - Shows structural ribbon diagram of gp120-CD4-NAb complex, including the gp120, CD4, Fab 17b heavy and light chains, and side chain of Phe 43.
• Figure 2. - a-c shows Fig. 1 of gp120-CD4-NAb complex rotated by 90o around vertical AXIS. The following components are included in this figure:
  • (a) Ribbon diagram containing alpha helices, beta sheets, and disordered V4 loop;
  • (b) Topology diagram of a
  • (c) gp120-CD4-NAb residues 90–396 and 410–492 are shown as stereo plot of alpha carbon trace, which every 10th and 20th carbon marked uniquely, and disulphide bonds shown as ball and sticks;
  • (d) Structure-sequence alignment of HIV-1 B, O, HIV-1, and SIV, including solvent accessibility.
• Figure 3. - shows ribbon diagram of CD4-gp120 interactions, including:
  • (a) Ribbon diagram of gp120-Cd4 binding, including residue Phe 43
  • (b) Electron density map of Phe 43 cavity
  • (c) gp120-CD4 electrostatic surfaces showing electrostatic potential
  • (d) Surface of contact in gp120-Cd4 binding
  • (e) gp120-CD4 high mutagenic areas
  • (f) gp120 side- and main-chain structures
  • (g) Map of gp120 variability
  • (h) Cavity of Phe 43
  • (i) Interface of gp120-CD4 interaction
  • (j) gp120-CD4 contact points showing Phe 43 and Arg 59
• Figure 4. - Shows structure of neutralizing action of antibobody 17b as a complex with gp120:
  • (a) alpha carbon worm diagram of 17b NAb-gp120 complex
  • (b) 17b NAb-gp120 surface of contact, as well as V3 loop
  • (c) part (b) rotated 90o around horizontal axis
  • (d) electric potential diagram of solvent-exposable surface
  • (e) shows alpha carbon worm diagram of colored gp120 glycoprotein
• Figure 5. - Diagram of core gp120 and its mechanisms governing initiation of HIV-1 membrane fusion with host cell.
• Table 1. - Shows structure of gp120-CD4-NAb complex according to model solutions. Also shows model fit (R-values) and model reliability (Rfree) for each of the structure models.

How do the results of this study compare to the results of previous studies?

• This study was the first to crystallize gp120 (albeit not a perfect product), something novel compared to previous studies. What is more, this study elucidated the gp120-CD4-NAb complex dynamics through distinct structural determination and analysis. This had not been performed previously, either. As a note, this study did not explain limitations of previous studies. The aforementioned new components were implied as novel by the paper, particularly when the paper stated that the structural determination of this paper has no precedent.

Important links

Bioinfomatics Lab: Fall 2016

Class Page: BIOL 368-01: Bioinfomatics Laboratory, Fall 2016

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