Architecture of Megadalton Respiratory Proteins from Annelids

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Architecture of Megadalton Respiratory Proteins
from Annelids
Bill Royer University of Massachusetts Worcester
(not Amherst)
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Diversity in annelid respiratory proteins
Riftia pachyptila C1 hemoglobin (24 subunits)
Lumbricus terrestris Erythrocruorin (180 subunits)
Arenicola marina Erythrocruorin (180 subunits)
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How do over 150 subunits assemble into a
megadalton particle with discrete size?
How is the hierarchical symmetry specified?
How are hemoglobin subunits arranged to allow
cooperative oxygen binding and other allosteric
properties?
Image reconstruction by cryo EM of Lumbricus
Erythrocruorin, courtesy of Marin van Heel,
Imperial College
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Lumbricus terrestris Erythrocruorin
P Dyad
Hb a cyan (36 copies) Hb b gold (36) Hb c
blue (36) Hb d dark violet (36) Linkers red
gray (12 copies each of three types)
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Lumbricus erythrocruorin linker subunits display
similar, but not identical tertiary structures
C-terminal b-barrel
LDL-A module
C
Short cc
C
C
Long coiled-coil helix
N
N
N
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Assembly of heterotrimeric linker complex
C-term b-barrel
Short cc
LDL-A module
Long coiled-coil helix
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Central linker complex is assembled from twelve
heterotrimers forming the scaffold for binding
12 hemoglobin dodecamers
Central linker complex
L1
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Lumbricus erythrocruorin one-twelfth protomer
Hemoglobin Dodecamer
Linker heterotrimer
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Lumbricus hemoglobin subunits display similar Mb
folded tertiary structures
Hb chain b
Hb chain a
Sperm whale myoglobin (Structure by John Kendrew,
illustration by Irving Geis)
Hb chain c
Hb chain d
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Arrangement of the hemoglobin subunits in
Lumbricus erythrocurorin resembles those of other
cooperative invertebrate hemoglobins
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Erythrocruorin from the marine polychaete
Arenicola marina displays an alternate
architecture by cryo-EM, in which the upper and
lower hexagonal rings are eclipsed.
What structural features dictate the alternate
architecture between type I and type II
erythrocruorins?
L. Jouan et al. (2001) J. Mol. Biol. 305, 757-771
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Arenicola marina Erythrocruorin at 6.2Å resolution
P Dyad
Hemoglobin dodecamer
Local 3-fold
Linker trimer
Linker trimer
Local 3-fold
Hb dodecamer
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6.2Å resolution maps for Arenicola and Lumbricus
erythrocruorins
hemoglobin subunits
Top View clearly shows eclipsed arrangement of
upper and lower halves for hemoglobin dodecamers
in Arenicola erythrocruorin.
Bottom View note the rotation of layers and
closer approach in Lumbricus erythrocruorin
Arenicola marina dodecamers
Lumbricus terrestris dodecamers
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Central Complex of 36 linker subunits
Lumbricus terrestris
Arenicola marina
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One-twelfth protomer structures
Arenicola marina
Lumbricus terrestris
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Alignment of annelid linker coiled coil sequences
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Riftia pachyptila Hemoglobins
Riftia Pachyptila, a giant vestimentiferan
tubeworm, lives around deep-sea thermal vents.
The hydrothermal solution at the base of their
tubes is rich in H2S and CO2, whereas their
plumes reach into oxygenated water. Their
hemoglobins transport both oxygen and sulfide to
chemoautotrophic bacteria residing in the worms.
Riftia hemoglobins Vascular Hbs V1
3.6 x 106 Da V2 - 4.0 x
105 Da Coelomic Hb C1 4.0 x 105 Da
How are the same hemoglobin subunits assembled
into both 3,600 kDa and 400 kDa complexes?
How do these hemoglobins simultaneously bind
oxygen and sulfide?
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3-fold
Riftia pachyptila C1 hemoglobin
3-fold
B2
B2
B1
A1
A1
A1
A1
B1
Assembles 24 hemoglobin subunits into a hollow
spherical shape with D3 symmetry
B2
B2
2-fold
2-fold
2-fold
2-fold
A2
A2
A2
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Lumbricus erythrocruorin one-twelfth subunit
Riftia C1 hemoglobin
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Lumbricus erythrocruorin one-twelfth subunit
Riftia C1 hemoglobin
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Twelve Zn ions bind near the three-fold axis in
Riftia hemoglobin
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Lumbricus terrestris Erythrocruorin (180 subunits)
Riftia pachyptila C1 Hb (24 subunits)
Annelida
Homo sapiens HbA (tetramer)
Echiura
Urechis caupo Hb (tetramer)
Caudina arenicola HbD (dimer)
Chordata
Echinodermata
Mollusca
Petromyzon marinus HbV (deoxy dimer)
Protostomes
Deuterostomes
S. inaequivalvis HbI (dimer)
Scapharca inaequivalvis HbII (tetramer)
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Lumbricus Erythrocruorin Kristen Strand
Hitesh Sharma Balaji Bhyravbhatla Arenicola
Erythorcruorin Michael Omartian James
Knapp Riftia Hemoglobin
Jason Flores Pennsylvania State University (Now
UNC, Charlotte)
- NIH
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Lumbricus Erythrocruorin is comprised of both
hemoglobin and non-hemoglobin linker subnits
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Lumbricus Erythrocruorin Structure Determination
Models (26Å) from Marin van Heel image
reconstruction
(24-fold averaging)
7.25Å map with clear helices R0.234
Iterative symmetry averaging (26Å)
Low Salt Crystals Triclinic P1 a188Å b266Å
c446Å a89.4 b97.6 g92.3 2 molecules per
a.u.
Phase extension (with improvement of
non-crystallographic operators and molecular
masks)
Phase High Salt Crystal Data at 7.25Å (R0.473)
Models (26Å)
Multiple crystal averaging R0.266, 0.254 (Mb
folds identified)
Unsuccessful de novo phasing
High Salt Crystals Orthorhombic C2221 a502Å
b298Å c350Å abg90 1/2 molecule per a.u.
(6-fold averaging)
Phase Extension in High Salt Crystals
using six-fold averaging (with improvement of
non-crystallographic operators and molecular
masks)
Multi-domain averaging (18-fold for Hb
subunits 6-fold for linker subunits)
Improved mask for linker subunits
Final 5.5Å electron density map (R0.219)
Interpretable 5.5Å map
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L2
L3
L1
One-half of central complex of 36 linker chains
Heterotrimeric Linker complex
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Subunit interface in EF dimers
Lumbricus bc dimer
Scapharca HbI homodimer
F3 Lys
E7 His
F4 Phe
F4 Phe
E7 His
F3 Lys
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Subunit interface in EF dimers
Lumbricus ad dimer
Caudina chain D homodimer
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Lumbricus hemoglobin dimers
Scapharca HbI dimer
bc dimer
ad dimer
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Sequences of E and F helices Lumbricus Hb
chains, dimeric Caudina HbD and dimeric Scapharca
HbI
I----------------- E
-----------------I I-------------- F
----------------I Lum a S G E F K S H L V R
V A N G L D L L I N L L D D T L V L Q S H L G H L
A D Q H I Q R Lum b H P A F I A H A E R V L
G G L D I A I S T L D Q P A T L K E E L D H L Q V
Q H E G R Lum c G P K F S A H A L R I L N G
L D L A I N L L D D P P A L D A A L D H L A H Q H
E V R Lum d1 S P E F G A H S Q R V L S G L D
I T I S M L D T P D M L A A Q L A H L K V Q H V E
R Caudina S R Q M H A H A I R V S A L M T T Y
I D E M D T - E V L P E L L A T L T R T H D K N
Scapharca N D K L R G H S I T L M Y A L Q N F I
D Q L D N P D D L V C V V E K F A V N H I T R H
- Distal Histidine H - Proximal Histidine X -
Key residues in Scapharca and Caudina dimeric
interfaces Lum a,b,c K. Fushitani, M.S.A.
Matsuura A.F. Riggs (1988) J. Biol. Chem. 263,
6502-6517 Lum d1 Q. Xieet al.(1997) Biochem.
Biophys. Acta 1337, 241-247 Caudina (HbD) F.
Mauri et al.(1991) Biochem. Biophys. Acta 1078,
63-67 Scapharca (HbI) R. Petruzzelli et al.
(1985) FEBS Letters 184, 328-332
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Lumbricus Erythrocruorin one-twelfth subunit
Hemoglobin dodecamer
Heterotrimeric linker complex
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Disulfide linkages in abc hemoglobin trimer