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

RL Stanfield, E Cabezas, AC Satterthwait, EA Stura, AT Profy, IA Wilson, Dual conformations for the HIV-1 gp120 V3 loop in complexes with different neutralizing Fabs, Structure, Volume 7, Issue 2, 15 February 1999,

Pages 131-142

Chris Rhodes and Alex CardenasLoyola Marymount University Department of Biology

BIOL 398 10/19/11

Outline• The V3 peptide loop of gp120 of HIV-1 viruses has many effects

on viral interactions and may affect antibody binding ability.

• Previous studies show conserved GPGR residues in V3 sequence variants indicating functional significance.

• The crystal structure of Fab 58.2-Peptide complex are found to show differences in GPGR β-turn conformation when compared to previous studies.

• The effects of different V3 loop conformations could lead to changes in biological functions and interactions of gp120.

• Future experiments should focus on determining the structure of intact gp120 in order to provide more definite conclusions.

Gp120 plays an essential role in HIV-1 viral infection

• gp120 is a protein complex found on the exterior of HIV-1 viral coats

• gp120 facilitates viral-CD4 receptor binding essential for infection

• The V3 peptide domain is located within gp120

– ~40 amino acid sequence

– High sequence diversity among viral variants.

The V3 peptide loop of gp120 of HIV-1 viruses may affect antibody binding and gp120 functionality

• In previous experiments changes in the sequence of the V3 domain have shown multiple effects on:

– Viral Tropism

– Antibody binding ability

– Syncytium-Formation

– Chemokine Receptor usage

Previous studies show conserved GPGR residues indicating functional significance

• Studies by La Rosa et al. (1990) show conserved region among V3 amino acid sequence variations.

• GPGR residues near tip of loop are highly conserved

• “Stem” amino acids are highly variable

• Results agree with previous Stanfield studies of crystal structures.– Fab 50.1-V3 and 59.1-V3 complexes show conserved GPGR

type II β-turn conformation

• High conservation of GPGR indicates GPGR is required for functionality

Experiment analyzes Aib142,His-Loop, and Ser-Loop crystallized in complex with the Fab 58.2

• Peptide Synthesis:– Aib142: Chemical Synthesis

– His and Ser loops: Solid phase synthesis

• Crystallization:– Sitting drop, vapor diffusion method at 22.5 degrees

Celsius

• Determination of Structure:– X-PLOR computer program– PC refinement– Modified Harada translation function

Outline• The V3 peptide loop of gp120 of HIV-1 viruses has many effects

on viral interactions and may affect antibody binding ability.

• Previous studies show conserved GPGR residues in V3 sequence variants indicating functional significance.

• The crystal structure of Fab 58.2-Peptide complexes are found to show differences in GPGR β-turn conformation when compared to previous studies.

• The effects of different V3 loop conformations could lead to changes in biological functions and interactions of gp120.

• Future experiments should focus on determining the structure of intact gp120 in order to provide more definite conclusions.

Residues and 2-D Structure of Experimental Peptide Sequences

• Amino Acid Sequences of 3 Experimental peptides

•J-Z Hydrazone linkage shown

•Aib142 replaces Ala142 to stabilize peptide structure

Data and Statistics gathered from X-ray Diffraction of Fab 58.2-Peptide Complexes

Crystallized structure of Fab 58.2-Peptide complexes

A) Fab 58.2-Aib142 Complex

B) Fab 58.2-HisLoop Complex

C) Fab 58.2-SerLoop Complex

* Fab 58.2 shown as blue and cyan* Binding peptides shown in red

H1 Loop structure of Fab 58.2 and two other H1 loops of similar length

• Red: Fab 58.2 H1 loop

• Blue: AN02 H1 loop

• Yellow: N10 H1 loop

•Fab 58.2 H1 loop differs from expected structure shown by AN02

•H1 loop is used when binding peptide but makes only minor contacts

Electron Density Maps of Peptides bound to Fab 58.2

A) GPGR β-Turn of Aib142 peptide

B) RAibFY residues of Aib142 peptide

C) Complete His-Loop peptide

D) Complete Ser-Loop peptide

• J-Z Hydrazone linkage of His and Ser loops not shown

Experimental Peptides Bind to Fab 58.2 in Essentially Identical Manners

A) Fab 58.2-Aib142 Complex• Red Regions: (-) Charge

• Blue Regions: (+) Charge

• Aib142 binds to dense negatively charged pocket

B) Fab 58.2 contacts to Aib142

• Yellow Structure: Aib142

• Blue and Cyan: Fab 58.2

• Experimental peptides all bind in the same pocket

• Each peptide has specific and distinct contacts with 58.2

Residue Contacts Between Fab 58.2 and Bound Peptides

Common Contacts Among all Peptides

Contacts Specific to Aib142

Contacts Specific to His-Loop

Contacts Specific to Ser-Loop

Hydrogen Bonds and Salt Bridge Interactions in Fab 58.2-Peptide Complexes

Bond Lengths (Å)Interacting Peptide Residue Interacting Fab 58.2 Residue

Fab 58.2 Peptides are found to show differences in GPGR β-turn conformation when compared to

previous studies• Purple: Fab 59.1-Peptide

• Yellow: Fab 50.1-Peptide

• Blue: Fab 58.2-Aib142 Peptide

• Green: Fab 58.2-HisLoop Peptide

• Fab 59.1 and 50.1 Peptides show type II β-turn GPGR conformation

• Fab 58.2 Peptides show type I β-turn GPGR conformation

Differences in GPGR conformations are seen through bond angles.

• Fab 50.1 and 59.1 peptides share fairly similar bond angles for GPGR residues

• Fab 58.2-Aib142 peptide GPGR residue angles differ distinctly from 50.1 and 59.1 peptide angles.

• Differences in bond angles corresponds to differences seen in peptide conformation.

Outline• The V3 peptide loop of gp120 of HIV-1 viruses has many effects

on viral interactions and may affect antibody binding ability.

• Previous studies show conserved GPGR residues in V3 sequence variants indicating functional significance.

• The crystal structure of Fab 58.2-Peptide complex are found to show differences in GPGR β-turn conformation when compared to previous studies.

• The effects of different V3 loop conformations could lead to changes in biological functions and interactions of gp120.

• Future experiments should focus on determining the structure of intact gp120 in order to provide more definite conclusions.

The effects of different V3 loop conformations could lead to changes in biological functions of gp120.

• The GPGR region of the V3 loop can be considered biologically relevant to gp120 functionality.

• Based on epitope mapping GlyP319, ProP320 and ArgP322 have been found to affect antibody binding affinity to gp120.

• GPGR has been shown to adopt different conformations based on environment and binding partner.

• These variations may relate to the binding potential of the V3 peptide in the gp120 complex and thus gp120 functionality.

Future Experiments Should Focus on Crystallization of Intact gp120 Complex

• To date (1999) the intact structure of the gp120 complex has not been studied.

• Can’t show conclusive findings about V3-gp120 functionality without studying the two in complex

• Future experiments:

– Structure of intact gp120 complex

– Structural studies of complete V3 peptides

– Determining effects of antibodies on V3 conformation in gp120 complex

Summary• The V3 domain of gp120 of HIV-1 viruses has multiple effects

on viral-CD4 receptor interactions

• Specifically the GPGR tip region of the V3 loop has been suspected for functional significance

• The β-turn conformations adopted by the GPGR residues are shown to change with binding partner and environment

• The various conformations of the GPGR residues of the V3 loop could affect gp120 functionality

• In order to properly study V3-gp120 functionality, future studies should research the two in complex

Acknowledgements

Kam D. Dahlquist, Ph.D

Stanfield et al. (1999)