Transcript Dual conformations for the HIV-1 gp120 V3 loop in

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