Amanda N. Wavrin Week 7

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Article Summary-Dual conformations for the HIV-1 gp120 V3 loop in complexes with different neutralizing Fabs

Dual conformations for the HIV-1 gp120 V3 loop in complexes with different neutralizing Fabs

Terms and Definitions

  1. Fab fragments-Fab fragments consist of one light chain linked through a disulphide bond to a portion of the heavy chain and contain one antigen binding Site.
  2. Syncytia-An epithelium or tissue characterized by cytoplasmic continuity, or a large mass of cytoplasm not separated into individual cells and containing many nuclei. Syncytia is the plural for syncytium.
  3. X-ray crystallography-The study of crystal structure using x-ray diffraction techniques.
  4. Retrovirus- Any virus in the family retroviridae that has rNA as its nucleic acid and uses the enzyme reverse transcriptase to copy its genome into the dNA of the host cells chromosomes.
  5. Paucity-Fewness; smallness of number; scarcity
  6. Ramachandran Spaces-A graphical representation in which the dihedral angle of rotation about the alpha-carbon to carbonyl-carbon bond in polypeptides is plotted against the dihedral angle of rotation about the alpha-carbon to nitrogen bond.
  7. Torsion/Torsion Angles- A type of mechanical stress, whereby the external forces (load) twist an object about its axis.
  8. Tropism-An involuntary orienting response; positive or negative reaction to a stimulus source.
  9. Annealing-Toughening upon slow cooling.
  10. Glycosylation-The process of adding sugar units such as in the addition of glycan chains to proteins. An occurrence where a carbohydrate is added to a protein molecule, which can occur in the golgi apparatus.


The process of adding sugar units such as in the addition of glycan chains to proteins. An occurrence where a carbohydrate is added to a protein molecule, which can occur in the golgi apparatus.

Introduction

  • HIV is a member of the lentivirus subfamily
  • gp120 binds to CD4 and another coreceptor on the cell surface
  • CXCR4 serves as the main coreceptor for T-cell
  • CCR5 serves as the major coreceptor macrophage-tropic
  • The V3 loop is one of the major immunogenic sites on the virus
  • The V3 loop becomes more sensitive with gp120-CD4 interaction
  • Work by La Rosa et al. showed that some amino acid postions in the loop are highly variable and others are highly conserved
  • V3 neutralizing antibodies may prevent binding of HIV to CD4 human cells
  • A broad double turn was found around GPGR and double bend around GRAFY
  • Aib residue was not necessary for binding or crystallization
  • One attempt to restrict V3 peptide conformation involved using hydrogen bond mimics

Results and Discussion

Structure determination and refinement
  • The crystal structures for Fab 58.2 have been determined by the molecular replacement method
    • They were isomorphous
  • The Fab-Aib142 peptide complex structure was determined
  • Two Fab-cyclic peptide structures were determined
    • Thetwo differ at only one residue in the peptide
Structure description and comparison
  • Fab 58.2 is a mouse igG1 k antibody
  • There is a tendency for increased flexibility as the H1 loop increases in length
  • The conformation of the base of the B3 loops is dictated by residues AlaH93, ArgH94, and AspH101 and resultes ion a kinked structure
  • The linear Aib142 peptide is 24 amino acids with an Aib residue replacing AlaP323
  • The peptide hasan extended conformation for residues RIHI
  • Cyclic peptide bond portions are very similar to the conformation of the linear peptide bond portions
  • The three peptides bind to the antibody in virtually the same orientations
  • A total of 124,103 and 77 van der Waals contacts gave been made between Fab and peptide for Aib142
  • A total of 11,7, and 7 hydrogen bonds and salt bridge interactions are made between peptides and Fab in the Aib142
  • The resolution of the His loop and Ser loop structure does not allow accurate placement of ordered water molecules
    • One strong peak of density in the Fab binding site of all three structures was assigned as a water position
  • The conclusions from eptiope mapping experiments of antibody 58.2 agree well with the strucutral results
  • The epitope mapping results indicate that residues GlyP391, ProP320, and ArgP322 are important for antibody binding
  • Residue GlyP319 may be important for allowing the to adjust to more than one conformation
Two different V3 conformations
  • The V3 peptide conformation, while bound to Fab 58.2, differs from the two perviously determined structures for the same or similar peptide in complex with two other Fabs
  • The peptides bound to Fabs 59.1 and 58.2 contain a beta turn followed by a double bend, but the different torsion angles for the central GPGR residues change the exact character of these bends
  • The V3 loop by itself is largely disordered in water
Correlation of structure and function
  • The V3 loop conforamtions are biologically relevant, as the antibodies bind to viral gp120 to effect their neutralization activity
  • These conformations may represent two different structural motifs that may or may not relate to viral phenotype

Biological Implications

  • HIV is a membrane bound virus with membrane embedded proteins, gp120 and gp41
  • The V3 loop is involved in virus infectivity
  • There is suggestion that structural conservation at the tip of the gp120 V3 loop is related to biological function
  • Studies have suggested that one or more amino acid residue alterations within the V3 loop leads to changes in viral tropism
  • Unanswered questions provide a direction for furthur studies with the intent to reveal the biological role of the V3 loop
  • This could open up the possibility of drug design or vaccine development

Materials and Methods

  • The linear Aib142 peptide was prepared by chemical synthesis
  • The cyclic peptides were synthesized by solid phase synthesis
  • All crystallizations were complted using the sitting drop, vapor diffusion method at 22.5 degrees Celsius
  • The Fab 58.2/Ser loop complex structure was determined by using coordinates of Fab 59.1

Figures and Tables

  • Figure 1- Shows the sequences of the different V3 loop peptides that were used in the crystallization of the peptide-Fab complexes. This includes RP70, Histidine loop, serine loop, and Aib142.
  • Table 1- Displays the X-ray diffraction data and refinement statistics for Fab58.2 complexes. This includes the peptides Aib142, His loop, and Ser loop.
  • Figure 2- Displays stereoviews of the X-ray structures of the Fab 58.2 peptide complexes. This includes stereoviews of the Fab 58.2-Aib142 complex, the Fab 58.2-His loop complex, and the Fab 58.2-Ser loop complex.
  • Figure 3- Shows a comparison of the different conformations of the H1 loops Fab 58.2, N10, and AN02. The different conformations were color coded.
  • Figure 4- Displays the electron denisty for the different V3 peptides bound to Fab 58.2.
  • Figure 5- Shows the Fab 58.2 binding pocket with the Aib142 peptide bound. Part (a) displys a representation of the antibody combining site. Part (b)displays a stereoview of the combining site.
  • Table 2- Shows the calculated Van der Waals contacts between Fab 58.2 and bound peptides.
  • Table 3- Shows the calculated hydrogen bonds and salt bridge interactions in Fab 58.2-peptide complexes.
  • Figure 6- Displays the conformations of the V3 loop, the peptides are color coded.
  • Table 4- Shows the mainchain dihedral angles for V3 peptides bound different Fabs. The Fabs included 50.1, 59.1, and 58.2


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