J'aime C. Moehlman's Week 7: Difference between revisions
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*They used a synthetic hydrogen bond to mimic incorporating a hydrazone bond-- designed to replace CO-HN hydrogen bond. | *They used a synthetic hydrogen bond to mimic incorporating a hydrazone bond-- designed to replace CO-HN hydrogen bond. | ||
*The Fab 58.2 crystal structure shows that it is a highly neutralizing antibody that neutralizes both T-tropic and M-tropic viral strains. | *The Fab 58.2 crystal structure shows that it is a highly neutralizing antibody that neutralizes both T-tropic and M-tropic viral strains. | ||
====Results and Discussion==== | |||
'''Structure determination and refinement''' | '''Structure determination and refinement''' | ||
* | *The crystal structures for Fab 58.2 in complex with one linear and two hydrazone- linked cyclic peptide have been determined to 2.0 A and 2.8 A. | ||
*Structures were determined by the molecular replacement method using previously determined Fab structures. | |||
*All structures were refined with the X-PLOR simulated annealing refinement protocol. | |||
*Analysis of the Ramachandran plot by PROCHECK shows that 89% of the residues are in most favored positions with three residues in disallowed regions. | |||
**Disallowed residues include two residues in a disordered loop in the constant heavy chain which almost always displays high displacement parameters in other antibody structure determinations. | |||
*The Ser loop complex has 87% of the residues in most favored regions with five residues in disallowed regions. | |||
'''Structure description and comparisons''' | '''Structure description and comparisons''' | ||
*Fab 58.2 is a mouse antibody, the CDR loops L1, L2, L3, and H2 belong to canonical classes 5,1,1, and 1. | |||
*The linear Aib142 peptide has an extended conformation for residues RIHI, which connect to a type I turn around residues GPGR. | |||
*The Fab uses five of its six CDR loops to bind peptide; CDR L2 is not used and CDR H1 makes only minor contacts. | |||
*A total of 124, 103, and 77 van der Waals contacts are made between the Fab and peptide fir the Aib142. | |||
*The resolution of the His loop and Ser loop structures is insufficient to 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 epitope specificity for 58.2 was determined previously by screening a peptide display libray expressing 1.5*10^8 unique 20 amino acid peptide sequences. | |||
*The epitope mapping results showed what epitopes could be replaced with amino acids, and which they could not be. | |||
*These epitopes also helped with specific things such as; antibody binding and determining the conformation of the tip of the V3 loop. | |||
'''Two different V3 conformations''' | '''Two different V3 conformations''' | ||
* | |||
'''Correlation of structure with function''' | '''Correlation of structure with function''' | ||
Revision as of 22:35, 7 March 2010
Terms and Definitions
- Syncytia:
- Fab fragments:
- X-ray Crystallography:
- Tropism:
- Ternary (complex):
- Ramachandran:
- Isomorphously:
- Hydrazone:
- Annealing:
- Canonical:
Article Outline
Dual conformations for the HIV-1 gp120 V3 loop in complexes with different neutralizing Fabs
- Stanfield et al. (1999)
Introduction
- HIV-1 is a member of the lentivirus subfamily of retroviruses.
- Beta- chemokine receptors have been implicated as the viral secondary receptors.
- CXCR4 acrs as the primary receptor for T cell tropic SI isolates.
- The V3 region is a disulfide look of about 40 amino acids.
- The V3 region is one of the major immunogenic sites.
- Exposure of the V3 loop to the gp120 protein varies depending on the viral isolate type and increases during interactions with CD4.
- Sequence changes in V3 can alter ciral cell tropism, antibody neutralization, neutralization of soluble CD4, syncytium formation, and chemokine receptor usage.
- T- tropic V3 sequences are usually more basic in charge than M-tropic sequences through accumulation of positively charged residues.
- Some amino acid positions in the loop are highly variable in amino acid composition.
- Their investigation was conducted by studying neutralizing antibodies and their complexes with V3 peptides to determine the tertiary conformation of the V3 loop.
- The researchers believe that they can explain coreceptor usage and the changes that take place in the virus upon conversion from a primary M-tropic isolate to the T-tropic strains associated with disease progression.
- Fab 50.1 and 59.1 are the two key structures of antibody fab fragments:
- Fab 50.1 is thought to be highly specific for the MN viral strain
- Fab 59.1 is thought to strongly neutralize IIIB and weakly neutralizes MN.
- In the 59.1 peptide complex 10 amino acid residues were visible and five of these residues had structural counterparts in the 50.1 complex.
- The sidechain of the Arg residue is bound in a deep, negatively charged pocket on the antibody surface.
- Torsion angles are discussed throughout their research.
- They used a synthetic hydrogen bond to mimic incorporating a hydrazone bond-- designed to replace CO-HN hydrogen bond.
- The Fab 58.2 crystal structure shows that it is a highly neutralizing antibody that neutralizes both T-tropic and M-tropic viral strains.
Results and Discussion
Structure determination and refinement
- The crystal structures for Fab 58.2 in complex with one linear and two hydrazone- linked cyclic peptide have been determined to 2.0 A and 2.8 A.
- Structures were determined by the molecular replacement method using previously determined Fab structures.
- All structures were refined with the X-PLOR simulated annealing refinement protocol.
- Analysis of the Ramachandran plot by PROCHECK shows that 89% of the residues are in most favored positions with three residues in disallowed regions.
- Disallowed residues include two residues in a disordered loop in the constant heavy chain which almost always displays high displacement parameters in other antibody structure determinations.
- The Ser loop complex has 87% of the residues in most favored regions with five residues in disallowed regions.
Structure description and comparisons
- Fab 58.2 is a mouse antibody, the CDR loops L1, L2, L3, and H2 belong to canonical classes 5,1,1, and 1.
- The linear Aib142 peptide has an extended conformation for residues RIHI, which connect to a type I turn around residues GPGR.
- The Fab uses five of its six CDR loops to bind peptide; CDR L2 is not used and CDR H1 makes only minor contacts.
- A total of 124, 103, and 77 van der Waals contacts are made between the Fab and peptide fir the Aib142.
- The resolution of the His loop and Ser loop structures is insufficient to 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 epitope specificity for 58.2 was determined previously by screening a peptide display libray expressing 1.5*10^8 unique 20 amino acid peptide sequences.
- The epitope mapping results showed what epitopes could be replaced with amino acids, and which they could not be.
- These epitopes also helped with specific things such as; antibody binding and determining the conformation of the tip of the V3 loop.
Two different V3 conformations
Correlation of structure with function
