Allosteric action in real time: Timeresolved crystallographic studies of a cooperative dimeric hemog

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Allosteric action in real time Time-resolved
crystallographic studies of a cooperative dimeric
hemoglobin
Bill Royer and James Knapp University of
Massachusetts Worcester (not Amherst) Vukica
rajer and Reinhard Pahl BioCars, APS University
of Chicago
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Riftia pachyptila C1 Hb (24 subunits)
Homo sapiens HbA (tetramer)
Annelida
Echiura
Urechis caupo Hb (tetramer)
Caudina arenicola HbD (dimer)
Chordata
Echinodermata
Mollusca
Petromyzon marinus HbV (deoxy dimer)
Protostomes
S. inaequivalvis HbI (dimer)
Deuterostomes
Scapharca inaequivalvis HbII (tetramer)
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Scapharca homodimeric HbI
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Deoxy HbI 1.6 Å resolution
HbI-CO 1.4 Å resolution
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HbI-CO
HbI-O2
40-0.82 Å RFree 14.2 R12.4
40-0.85 Å RFree 13.5 R11.9
Fe-C-O 173.3, 173.5
Fe-O1-O2 126.2, 128.8
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Cooperative ligand binding in HbI relies on only
small subunit rotations
Unlike binding of oxygen to human hemoglobin
crystals, binding of oxygen to HbI crystals is
fully cooperative (Mozzarelli et al, 1996).
Despite rather localized structural transitions,
the R-state is estimated to bind oxygen 300 times
more tightly than the T-state.
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Key structural transitions with functional
ramifications
Heme movement
What is the cascade of structural events?
F4 Phe flipping
Are these transitions concerted or sequential?
Interface water rearrangment
Do structural kinetic intermediates facilitate R
to T transition?
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Time-resolved crystallography to obtain snapshots
along the trajectory between high-affinity and
low affinity states
BIOCARS 14-IDB, APS
Heat load shutter
ms shutter
Single bunch 150ps
X-rays (Undulator)
CCD
2 ?s chopper
e-
Cycle time 3.683 ms
Dye laser
Superbunch 500ns
Experimental Setup
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Crystals and Data Collection
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Mutation of Met 37 to Val (M37V) lowers geminate
rebinding as the ligand migrates to a B-site in
the distal pocket
Difference Fourier Fo(light)-Fo(dark) (Red
-3s, Blue 3s)
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F4
(Red -2.5s Blue 2.5s)
CD
CO
F
E7
CD1
CO
CD3
5ns
Fe
F8
F4
F
F7
(Red -2.5s Blue 2.5s)
F4
CD
E7
CD1
F
60ms
CD3
Fe
F
F8
F4
F7
Subunit A
Fo(light)-Fo(dark) - (Red -3s, Blue 3s)
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Heme A movement
Heme B movement
Models Gray HbI-CO heme Magenta deoxy
heme Yellow iron atom
Fo(light)-Fo(dark) density green 6s, blue
2.5s, red -2.5s
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Change in iron position, based on difference
refinement Distance along the parallel and
perpendicular components of the heme plane
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Integrated difference Fo(light)-Fo(dark)
electron density values
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Quaternary subunit rotation Based on subunit
rigid-body difference refinement
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F4
(Red -2.5s Blue 2.5s)
CD
CO
F
E7
CD1
CO
CD3
5ns
Fe
F8
F4
F
F7
(Red -2.5s Blue 2.5s)
F4
CD
E7
CD1
F
60ms
CD3
Fe
F
F8
F4
F7
Subunit A
Fo(light)-Fo(dark) - (Red -3s, Blue 3s)
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Mutations that increase the packing of the distal
pocket against the heme destabilize the unligated
R-state and increase oxygen affinity.
M37A 13.5 torr M37V 13.8 torr M37L 5.1
torr M37F 1.1 torr F41L 17.3 torr F41W
4.2 torr L73V 7.6 torr L73I 18.1 torr L73M
36 torr L73F 2.8 torr
F51L L54V L54F
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HbI and Allostery
MWC Equilibrium
Kinetic Transition
O2
O2
T
-2O2
O2
O2
R
JMB (1965) 12, 88-118
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Key structural transitions with functional
ramifications
What is the cascade of structural
events? Intermediate is formed rapidly (lt5ns)
upon ligand release, relaxing to T-like structure
in microsecond time domain
Heme movement
F4 Phe flipping
Are these transitions concerted or
sequential? Key allosteric changes appear to be
tightly coupled.
Interface water rearrangment
Do structural intermediates facilitate R to T
transition? Rapid disordering of water molecules
H-bonded to propionates appears to lay the
foundation for subsequent heme movement.
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University of Massachusetts, Worcester James
Knapp
Univ. of Chicago BioCARS, APS Vukica
rajer Reinhard Pahl
- NIH
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Mutation of Ile 114 to Phe prevents ligand
induced heme movement
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Allosteric Ligand Binding
Koshland
MWC Model
T
O2
O2
T
O2
O2
O2
O2
R
R
JMB (1965) 12, 88-118
Biochemistry (1966) 5, 365-385
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Results of difference Fourier refinement,
Fo(light) Fo(dark) coefficients
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Ligand migration in HbI dissociated CO moves
from distal pocket to BEG channel within 5ns
(Folight-Fodark Red -3s, Blue 3s)
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Ligand Migration in HbI
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Raising the osmotic pressure increases the oxygen
affinity of HbI
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Transitions in the distal pocket and proximal
pockets
Subunit A
Subunit B
Phe CD1
Phe CD1
Phe F4
Phe F4
Fo(light)-Fo(dark) density dark blue 3s light
blue 2s red -3s
Models Gray HbI-CO heme Magenta deoxy heme
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Ligation of HbI results in extrusion of Phe 97
from the proximal pocket
Distal His
Proximal His
Phe 97
Distal His
ligand
Proximal His
Phe 97
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Mutation of Ile 114 to Phe attenuates
ligand-linked heme movement, lowering oxygen
affinity and nearly abolishes cooperativity
Ile 114
Ile 114