conformational changes of gag hiv-1 on a tethered bilayer provide insights into viral assembly hirsh...
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Conformational changes of Gag HIV-1 on a tethered bilayer provide insights into viral assembly
Hirsh Nanda, Susan Krueger
NIST Center for Neutron Research, Gaithersburg, MD, USA
Frank Heinrich, Mathias Loesche
Carnegie Mellon, Pittsburgh, PA, USA
Siddhartha .A.K. Datta, Alan Rein
National Cancer Institute, Frederick, MD, USA
Viral Life Cycle
• HIV rapidly develops drug resistance
• Patients are given multi-drug treatments, targeting several stages of the viral life cycle
• No anti-virals target assemblyGag protein mediates viral formation
• Binds other viral components• Self-assembles on the cellular
membrane
Matrix Domain of Gag Binds the Membrane
- - - - -+ + + Matrix (MA): Targets the
membrane - basic residues interact favorably with charged lipids - myristate group inserts in bilayer
Capsid (CA): Lateral organization of Gag proteins on the membrane surface
Nucleocapsid (NC): Binds viral RNA, brings viral genome into assembling particle
PNAS (2006) v103(8) p2641
Viral Membrane:• 20% anionic lipids • Enriched in cholesterol• Specialized lipids such as PIP / celluar targeting of MA
Conformational Variability in Gag
SANS Shows Gag is Compact in Solution (Rg 35-38Å) Gag Bound to Single Stranded DNA is also compact (Rg 43-45Å)
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
Purified Gag with Yeast tRNA forms Particles too Small for a Virion (shell thickness ~70Å)
280 Å
~200 ÅPNAS 95: 7299, 1998
Immature Virion Shows Gag Extended on the Membrane
1100 Å
• Tether anchors membrane to substrate
• PEO spacer decouples bilayer from gold surface
• Fluid bilayer is highly stable• exchange of aqueous phase• proteins and other molecules can access outer leaflet
Tethered Bilayer Membranes (tBLM)Bio-mimetic environment for studying protein-lipid interactions
silicon
chromiumgold
bulk solvent
Ternary Lipid Mixture:• 30% anionic DMPS necessary to promote MA binding: lacks the myristate
• 70% DMPC & small amounts of cholesterol aided in forming complete bilayers
Basic residue patch (blue) suggests a putative binding orientation
Matrix Potein: Side View
4
6
0.1
2
4
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1
2
4
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Fresnel normalized reflectivity,
R/RF
0.250.200.150.100.05
momentum transfer, Qz (Å-1)
2
1
0
-1
Difference withneat bilayer
neat bilayer 1 µM Gag MA 10 µM Gag MA
Reflectivity Profile Shows Changes withIncreasing Concentration of MA
Comparison of Scattering Length Density fits to Reflectivity using a Box Model and Atomistic Structures
Matrix Binding to a Membrane Mimic
MALys+ Arg+
Full Length Gag on the Membrane
• Gag Bound at 1uM• Bound Gag incubated w/ TG14 (14 base DNA sequence with nM affinity at NC)
• High Salt Rinse (500mM NaCl)• Protein modeled using a multi-box model. Fitting
for all measurements and contrasts was conducted simultaneously. D2O
CM4
H2O
-0.5
0
.5
1
1.5
2
2.5
nSLD,
ρ, (10-6 Å-2)
0 50 100 150 200 250 300 , , ( )Distance from Au Surface z Å
-0.5
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1.5
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nSLD,
ρ, (10-6 Å-2)
0 50 100 150 200 250 300 , , ( )Distance from Au Surface z Å
-0.5
0
.5
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2.5
nSLD,
ρ, (10-6 Å-2)
0 50 100 150 200 250 300 , , ( )Distance from Au Surface z Å
-0.5
0
.5
1
1.5
2
2.5
nSLD,
ρ, (10-6 Å-2)
0 50 100 150 200 250 300 , , ( )Distance from Au Surface z Å
Autether
lipidheads
lipidtails
pure bilayerGag foldedGag + TG14Gag + 500mMNaCl Rinse
(1) Gag bound to the membrane adopts a compact structure (red).
(2) Introducing single stranded TG7 DNA causes an extended profiile (blue).
(3) Partial recovery of compact Gag is possible by a high salt buffer (green).
Gag Extension on the Membrane
Model for Gag Extension
RNA
MA
CA
Gag
NC
- - - - - -
- Gag is folded on the membrane or bound to viral RNA through favorable electrostatic interactions.
- However cross-linking of Gag on the membrane surface by the RNA strand causes extension.
ConclusionsMA-myr domain can target an anionic membrane with anorientation equivalent to the native MA protein
Investigate role of membrane composition and other co-factors
(ex PIP-lipids) in Gag assembly
Conformational changes in gag are observed when bound to TG14 cross linking DNA
Suggests Gag extension requires both the membrane and the cross-linking nucleic acid
Neutralized NC domain mutants to show role in binding to
charged lipid
Acknowledgements
Joseph CurtisChuck MajkrzakJoe Dura
Sidd Shenoy