Isai Lopez Individual Journal 8

Vocabulary

1. Glycosylated: "The addition of saccharides to proteins or lipids to form a glycoprotein or glycolipid." http://medical-dictionary.thefreedictionary.com/Glycosylated
2. Immunogenicity: "The ability of a substance to provoke an immune response or the degree to which it provokes a response" http://medical-dictionary.thefreedictionary.com/immunogenicity
3. Mutagenesis: "Formation or development of a mutation." http://medical-dictionary.thefreedictionary.com/mutagenesis
4. Fusogenic: "Facilitating fusion, especially relating to cells." http://www.yourdictionary.com/fusogenic
5. Hemagglutinin: "An antibody that causes agglutination of erythrocytes." http://medical-dictionary.thefreedictionary.com/hemagglutinin
6. Isomorphous: "Having the same form or shape, or being morphologically equal." http://medical-dictionary.thefreedictionary.com/isomorphous
7. Monoclonal: "Derived from a single cell; pertaining to a single clone." http://medical-dictionary.thefreedictionary.com/monoclonal
8. Prolate: "(Mathematics) having a polar diameter of greater length than the equatorial diameter" http://www.thefreedictionary.com/prolate
9. Linchpin: "A central cohesive element" http://www.thefreedictionary.com/linchpin
10. Glycocalyx: "The glycoprotein-polysaccharide covering that surrounds many cells." http://medical-dictionary.thefreedictionary.com/glycocalyx

Outline

• The main result of the paper is the discovery and reporting of the structure of a complex of factors important in investigating how HIV binds to and enters CD4 T cells. The exact combination of factors includes an external binding protein located on the viral envelope (gp120), a fragment of CD4, and a fragment of antibody that blocks chemokine-receptor binding.
• The importance of this discovery, especially relative to its time, is that it allows for a lens through which future scientific work can examine the specifics of how HIV invades CD4 T cells, as well as how the gp120 protein binds to CD4 and exposes itself to antibodies. With this tool guiding researchers, it gives information that will ideally help target the virus more aggressively and effectively.
• Little information about limitations of previous studies is given in the paper, however, the article does identify that the extracellular portion of CD4 had been characterized. Because of the importance of the gp120 protein in the interaction with CD4, the article cites the importance of characterizing gp120 because of the protein’s role in infiltrating the host’s immune cells. Also noted is the fact that binding between gp120 and CD4 causes a conformational change in gp120 that exposes portions which allow HIV antibodies to bind to the virus. As such, the researchers saw it necessary to characterize the structure of the entire complex, including the gp120 protein, the antibody, and the portion of CD4 that binds to the rest of the complex.

Methods

• Protein Production, Crystallization and Data Collection
  • CD4 from hamster ovarian cells, 17b antibody from an HIV-1 infected host B-cell, and gp120 from Drosophila fly
  • Biochemical Procedures including deglycosylation and papain digestion
  • Protein purification as well as ternary complex crystallization
  • Vapor-diffusion glutaraldehyde treatment
  • Flash freezing in nitrogen
  • Data processing done using two programs: DENZO and SCALEPACK
• Structure Determination and Refinement:
  • MERLOT used to create models of Fab fragments
  • Superposition of variable regions for comparison
  • XPLOR used to produce a Patterson correlation
  • SCALEPACK to produce a chi-square
  • Fourier analysis to identify heavy-atom sites
  • MLPHARE for refinement of phasing parameters
  • PRISM used to model the density
  • XPLOR also used to perform model subtraction
  • MAPMAN used to put together density maps
  • Program O used to model alpha-Carbon backbone as well as to align the sequence
  • Secondary structure of the protein predicted using program PHD
• Structure Analysis
  • Visual determination of structural alignment using SCOP
  • PrISM used to search sequences

Figures

• Fig. 1: Model was made using RIBBONS program and shows the general structure of the complex including gp120, Fab 17b, and the two domains of CD4 bound together. The structure reveals the relative positions of the viral envelope as well as the target membranes. As shown, the target envelope is at the bottom of the picture, under 17b, while the top denotes the location of the viral envelope
• Fig. 2a: Ribbon diagram of core gp120 revealing the locations of alpha helices and beta sheets, and the connections of amino acids between them
• Fig. 2b: Topology diagram of the same molecule. The advantage here is that we can more easily visualize the overall direction of the 3D structure as well as get a relative sense of the strengths of hydrogen bonding using proximity of the chains, where closer chains associate more tightly and form stronger H-bonds
• Fig. 2c: This structure shows the alpha-Carbon trace for the core gp120. Here we have marked alpha carbons along the structure of the protein as well as numbers that help denote the location of marked residues. In addition, the 7 disulfide bonds are marked on this diagram.
• Fig 2d: This shows a sequence alignment with 5 different strains. Three from HIV-1, one from HIV-2, and one from SIV. Marked along the alignments are specific parts of gp120 such as alpha helices, beta chains, and variable regions. Also marked are those residues which directly connect with CD4.
• Fig 3a: Ribbon diagram showing the binding of gp120 and CD4
• Fig 3b: Figure shows the electron density map for the cavity where Phe43 of CD4 interacts with gp120
• Fig 3c: Shows the relative values for electrostatic potential along the surfaces of CD4 and gp120. One side of the picture shows the model rotated 180 degrees across a vertical axis
• Fig 3d: Shows the contact surfaces of the gp120 protein with CD4. What this figure gives us is an imprint of the CD4 molecule on the surface of gp120.
• Fig 3e: This figure shows mutational hotspots for both gp120 and CD4. The highlights on gp120 denote residues whose mutations cause a significant effect on CD4 binding.
• Fig 3f: Figure highlights the contributions from side-chain and main-chain to the surface of gp120. Contributions from main-chain is done in green, while side-chain contributions are shown in white. There appears to be a correlation between the main chain atom contribution and the imprint of CD4
• Fig 3g: Variability in the sequence corresponding to different parts of the surface of gp120. The colors range from white, which is low variability, to brick-red, which is the highest variability.
• Fig 3h: Shown here is the Phe43 cavity, with the protein stripped down to wire connectivity to allow for better visualization.
• Fig 3i: Shown here is a schematic representation of the interaction between gp120 and CD4, with structural elements labelled along with important amino acids from the parts of gp120
• Fig 3j: Shown here are certain specific amino acid residues on gp120 which interact with Phe43 and Arg59 on CD4
• Fig 4a: Alpha-Carbon diagram of gp120 and the 17b Fab segment, color coded to show the different domains of gp120
• Fig 4b: Shows the contact surface of gp120, 17b Fab, and the V3 loop. Also parts are color coded to make them easier to visualize.
• Fig 4c: Figure is the same as above, but shows the figure rotated 90 degrees around a horizontal axis to see the epitope of 17b Fab more easily.
• Fig 4d: Map of electrostatic potential of the surface of the complex. The most electropositive parts of the surface are those that correspond to the 17b epitope.
• Fig 4e: Figure showing alpha-Carbon model of gp120, where the color coding is the same as in 4a, rotated 90 degrees about a horizontal axis.
• Fig 5: Summary denoting the process by which gp120 protein fuses with CD4. V1/V2 shift from their original positions after CD4 binding, which forms the Phe43 cavity. At this point, a chemokine receptor binds to the bridging sheet as well as the V3 loop, then causing a second conformational change at gp120, finally allowing for fusion of the membranes.
• Table 1: In this table, a summary of data from the complex structure determination is shown. An r-value is given to show the accuracy of the model produced using X-Ray Crystallography. The importance of this table is in its measurement of accuracy of the data, especially when talking about electron density and the models created, which gave researchers an idea of how close their models were.

Previous Studies

• This study used ideas and models proposed from earlier research studies to create a new model that was difficult to compare to earlier works. Most of the comparisons to other models were for specific sequences, which were by and large consistent with the work they had done. The research done here reflects a characterization that had not yet been done using sequences that had been used by previous researchers.

Acknowledgments

• I worked with Colin Wikholm Jordan Detamore and Anu Varshneya throughout the duration of this journal entry outside of class, as well as communication through text message and Facebook message.
• While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source. *Isai Lopez 00:43, 25 October 2016 (EDT):

References

• Kwong, P. D., Wyatt, R., Robinson, J., Sweet, R. W., Sodroski, J., & Hendrickson, W. A. (1998). Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature, 393(6686), 648-659. DOI: 10.1038/31405

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