AninditaVarshneya BIOL368 Week 8

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Journal Club Preparation

Vocabulary

  1. oligomeric
  2. chemokine receptor
  3. fusogenic
  4. ectodomain
  5. isomorphous
  6. ellipsoid
  7. staves
  8. B factors
  9. interfacial cavity
  10. electroneutral surface

Outline

  • What is the main result (message) presented in this paper?
    • HIV interacts with the CD4 glycoprotein and a chemokine receptor using several structural components that ultimately allow the virus to fuse with the cellular membrane and elicit a neutralizing response against the immune system. Some of these important structures include:
      • A protein complex including a two-domain fragment of human CD4 and an antigen-binding fragment that prevents ligand attachment to the chemokine receptor
      • A "cavity-laden" CD4-gp120 interface
      • A conserved binding site for the chemokine receptor
      • Conformational change upon binding with CD4
      • And other mechanisms for evading the immune response created by the human cells
    • HIV defends itself from humoral immune response by keeping most of its gp120 (viral entry gene) proteins buried.
    • The two major antibody neutralizing sites are the CD4 binding site and the chemokine-receptor binding site which are hidden by regions V1/V2 loop and V2 and V3 loops respectively
    • Conformational changes made by the protein further hide the protein from immune response
    • gp120 is incredibly crucial for HIV viral entry, and is therefore an incredibly important protein for the progression of HIV
      • locates cells, anchors the virus to the cell, orients the viral spike, and holds the protein responsible for fusion to the membrane until the accurate time
      • not explanatory of the entire mechanism, but does explain various aspects of the overarching framework
    • Mechanism for viral entry
      • CD4 binds to HIV-1
      • Orients the viral spike through conformational changes
      • Changes the location of the V3 region which contains proteolytic enzymes
      • Uncovers the chemokine binding receptor
      • Chemokine receptor is bound to and causes more conformational change which exposes gp41 ectodomain
    • As of publication, authors did not know how gp120 interacts with the chemokine receptor or how exactly its structure changed in apo state and oligomeric structure, or the conformational change required for the movement of the gp41 ectodomain
      • gp41 is especially important because it is essential to the fusion process
  • What is the importance or significance of this work?
    • This work is incredibly important as it provides a basis off of which future researches could analyze mediation points that would prevent or reduce viral entry of HIV into immune cells, thereby decreasing the overall progression of the HIV virus.
  • What were the limitations in previous studies that led them to perform this work?
    • Limitations from previous studies were not exactly identified by the authors of the paper. Considering the novelty of the paper, however, I can guess that previous researchers lacked the technology to accurately determine the exact protein structures of the most important proteins affecting viral entry. Because, for the most part, structure defines function, the structure of the proteins in conjunction with other modes of analysis provided clarity regarding the mechanism of viral entry into cells.
  • What were the methods used in the study?
    • Protein production, crystallization, and data collection
      • Two-domain CD4 were created using Chinese hamster ovarian cells
      • Biochemical manipulations: deglycosylations, papsin digestion
      • Protein purification and crystallization
    • Structure determination and refinement
      • Fab models created with MERLOT
      • Patterson correlations
      • chi-squared tests from SCALEPACK
      • Molecular replacement phases and phasing parameters using MLPHASE
      • PRISM modelled density
    • Structure Analysis
      • root-mean-square residue deviation
      • Structure alignments made with visual comparison using SCOP database
      • Automatic searches using PrISM
  • Briefly state the result shown in each of the figures and tables.
    • Figure 1
      • This figure indicated the overall structure of the gp120 protein and the two domains of CD4
      • The positions of the gp120 termini indicate that the viral membrane is at the top of the diagram which means that the target membrane is at the bottom.
      • Drawn with RIBBONS
    • Figure 2
      • Several views of the core of the gp120 protein
      • a) ribbon diagram with alpha helices and beta sheets are color coded
      • b) topology diagram meant to provide more information regarding the diagrams in (a) and (c), spacial proximity indicates hydrogen bonding (strong)
      • c) steroplot of an alpha-carbon trace, disulphide connections are specially depicted
      • d) structure based sequence alignment of HIV-1 B, C, O, HIV-2, and SIV, variability and consistencies between sequences are marked, residues directly interacting with CD4 are marked, direct contact is more restrictive than loss of solvent-accessible surface
    • Figure 3
      • a) ribbon binding between gp120 and CD4
      • b) electron density in Phe43 cavity
      • c) electrostatic surfaces of CD4 and gp120, found at solvent-accessible surfaces,
      • d) CD4-gp120 contact surface, fits together like puzzle pieces within a CD4 imprint
      • e) CD4-gp120 contact areas prone to mutations are highlighted
      • f) side-chains and main-chains in the gp120 protein
      • g) variability in sequences on the gp120 protein surface
      • h) phe43 cavity matches the orientation of the gp120 protein
      • i) interactions between the CD4-gp120 contact surface
      • j) residues on gp120 that are involved with direct contact with Phe43 and Arg59
    • Figure 4
      • neutralizing antibody interface
      • a) c-alpha diagram of two genes in the HIV virus
      • b) contact surface between gp120 and 17b and the V3 loop
      • c) a rotated version of b to show the 17b epitope
      • d) the 17b region is the most electropositive region in the molecule
    • Figure 5
      • A summary diagram depicting the initiation of gp120 fusion, gp120 V1 and V2 bind to CD4 causing a conformational change causing both the inner and outer domains of the gp120 protein to move to allow for an opening near the Phe43 residue, chemokine receptor attaches to the bridging sheet and V3 loop which causes another shift of the gp120 protein ultimately allowing the viral membrane to fuse with the cellular membrane
    • Table 1
      • r-values indicate the accuracy and quality of a model from x-ray crystallography
      • Rfree correlated with accuracy of model fit
      • Important because it allowed for accurate electron density maps which were vital to mapping out the structure of the gene as they indicated which types of bonds existed between particular residues
  • How do the results of this study compare to the results of previous studies.
    • This study used newer technology to put together findings that built off of the work of previous researchers. Though the researchers did not clearly or explicitly indicate using previous researchers work, it is implied through their references that their findings are merely a congregation of previous research with more definite protein structure to clarify the mechanism of viral entry completed by HIV

PowerPoint

HIV Journal Club

Acknowledgements

Colin Wikholm, Isai Lopez, Jordan T. Detamore and I worked together to understand our paper and create the PowerPoint. We also met outside of class on Monday afternoon to complete our PowerPoint and practice our presentation. While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source.

References


Other Links

User Page: Anindita Varshneya

Bioinfomatics Lab: Fall 2016

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

Weekly Assignments Individual Journal Assignments Shared Journal Assignments

SURP 2015

Links: Electronic Lab Notebook