Epitope structure on the hepatitis B virus capsid

1
Epitope structure on thehepatitis B virus capsid
James Conway Department of Structural
BiologyUniversity of Pittsburgh School of
Medicine
The Structure and Function of Large
Macromolecules The 38th Crystallographic Meeting
at Erice
2
HBV Assembly
3
HBV Cryo-EM
4
HBV at 9Å
5
HBV Capsid Movie
6
Dimer Structure
Conway et al, J Mol Biol 1997 Zlotnick et al,
PNAS 1997 Conway et al, PNAS 1998
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HBV - Xtal Structure
Wynn et al, Mol Cell 1999
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Fab.312 - map
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Fab 312 - model
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Conformational Epitopes
3105
F11A4
3120
312
Stain
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Fabs Targeting the Spike - 1
3105
F11A4
Control
Belnap et al, PNAS 2003
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Fabs Targeting the Spike - 2
T3
T4
3105
F11A4
13
3120  A Fab off the Spike
Conway et al, J Virol 2003
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Fab 3120 - epitope detail
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Fab 3120 Occupancy
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Summary of Fabs
F11A4
312
3105
3120
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HBV Fab 88
T4
T3
50Å
Harris et al, J Mol Biol 2006
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HBV Fab 842
T4
T3
50Å
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Fitting Fabs 88, 842
T4
T3
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Same Epitope - Different Binding Aspects
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Resolution Estimates - HBV Fab88
T4
T3
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Summary of Fabs
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HBV Fabs Conclusions

• In General
• Identify conformational epitopes by cryoEM
  modeling
• Occupancy steric blocking epitope conformation

• HBV-Specific
• Distinction between core-antigen and e-antigen -
  Accessible surfaces different - Fabs sensitive
  to small changes in structure (eg, 3120 88)
• Structural basis for literature on competition
  assays - Crowding almost any pair of Fabs will
  compete - Competition does not imply overlapping
  epitopes
• Diversity - 5 epitopes from 6 Abs gt total
  epitopes 20 - More Abs from quasi-equivalence
  and binding orientations
• Most epitopes are conformational and
  discontinuous - But not all 312 88

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Acknowledgements
LSBR/NIH Alasdair Steven, Naiqian Cheng, Norman
Watts, Audray HarrisDavid Belnap (BYU), Adam
Zlotnick (UOHSC) PEL/NIH Paul Wingfield Steve
Stahl
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Structural Biology at Pitt
Structural Biology at Pitt
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Molecular Biology Structural BiologyGraduate
Program
http//www.biophysics.pitt.edu/
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B L A C K
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HBV Fabs Conclusions

• In General
• Identify conformational epitopes by cryoEM
  modeling
• Occupancy steric blocking epitope conformation
• Diversity 4 Fabs 4 epitopes

• HBV-Specific
• Distinction between core-antigen and e-antigen -
  Accessible surfaces different - Fabs sensitive
  to small changes in structure (eg, 3120)
• Structural basis for literature on competition
  assays - Crowding almost any pair of Fabs will
  compete - Competition does not imply overlapping
  epitopes

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Acknowledgements
Adenovirus Dodecahedron P. Fuschiotti, G.
Schoehn, P. Fender, R. Ruigrok, E. Hewat, J
Chroboczek Hepatitus B Virus N. Cheng, A. Steven,
S. Stahl, P. Wingfield HK97 B. Duda, R. Hendrix,
N. Cheng, A. Steven, W. Wikoff, J. Johnson T7 M.
Cerritelli, N. Cheng, A. Steven Gifsy-2 G.
Effantin, G. Schoehn, N. Figueroa-Bossi, L.
Bossi ? M. Cerritelli, N. Cheng, A.
Steven HPV-31 P. Coursaget, A. Touze T5 G.
Effantin, P. Boulanger, L. Letellier G B. Duda,
R. Hendrix SPO1 B. Duda, R. Hendrix, W-M.
Huang HSV-1 images B. Trus, N. Cheng, A. Steven
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The Scale of Protein Structure
Assemblies
Secondary
Organelles
Atoms
Proteins
Viruses
Cells
Organisms
10Å
1-3Å
100Å
1000Å
1µm
200 - 4000Å (icosahedral) lt14000 (helical)
NMR
X-ray Crystallography
Electron Microscopy
Light Microscopy
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Virus Morphology
Spherical - Icosahedral herpes simpex
virus hepatitis A and B viruses tailed
bacteriophages
Phage T4
TMV
Marburg
Filamentous - Helical tobacco mosaic
virus filoviruses - ebola filamentous phages
Influenza B virus
HIV
Other influenza virus HIV measles VSV
Ruigrok, Schoehn Conway, Topley and Wilsons
Microbiology Microbial Infections Virology,
10th Ed. 2005
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Biophysical Methods to Study Viruses
Electron Microscopy metal shadowing
negative stain thin sections cryo-EM
tomography
Herpes simplex virus capsids (1250Å)
X-ray Crystallography
Hepatitis B virus capsid (355Å)
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CryoEM Samples Symmetry
2D crystallographymembrane proteins bacteriorh
odopsin, LHC-II, aquaporinother
proteins tubulin Helical structures microtubul
es, bacterial flagellar filament filamentous
phages, filoviridae (eg, Ebola)
ParticlesIcosahedral virus capsids/complexesLowe
r symmetry Clp-family of ATP-dependent
proteasesNo symmetry ribosome, phage
particles Non-uniform structuresTomography vir
uses, organelles, cells
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CryoEM - Why? How?
Metal Shadow
Negative-Staining
CryoEM
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CryoEM - Why? How?
Metal Shadow
Negative-Staining
CryoEM
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Methods Image reconstruction
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Analysis of Single Particles
Single Particles means many isolated, or
free-standing particlesand preferably random
views Combine different views of identical
objects Resolution will be limited by- degree
of identity- accuracy of view estimations-
completeness of dataset (views)- S/N and amount
of data- everything else! Symmetry size help,
eg, icosahedral virus capsids Contrast helps, eg
cryo-negative stain
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2. T-numbers
The icosahedron has 60-fold symmetry 5x3x2x2 The
simplest is T1 (60 subunits) More complex
structures have more subunits 60 x T Selection
rule T h2 hk k2 h,k 0,1,2 Allowed
T-numbers 1, 3, 4, 7, 9, 12, 13, 16, 19,
Special T-numbers 2, 6, ???
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T Triangulation
T 1
T 3
T 4
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T Triangulation
T 7d
T 7l
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T Triangulation
T 1
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Viruses
Ad3 DB 250Å Ad3 DF 400Å
HBV, T4, 350Å
SPO1, T16950-1080Å
HK97, T7l 480 Å
Phage G, T521500-1800 Å
Gifsy2 T7l 600 Å
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4. Some other structures under the microscope
HBV, T4350Å
Ad3 DB 250Å Ad3 DF 400Å
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T13 T52
T13 T52
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HBV 4Ir maps
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HBV N-term map
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Fab 3120 Not on the Spike
T3
T4
Conway et al, J Virol June 2003