Tom Walz
Department of Cell Biology
Harvard Medical School
Electron Crystallography Workshop
UC Davis
August 2006
The aquaporin family of water pores
The occular lens
Lens has to be
transparent
Lens must be able
to accommodate
Capsule
Degrading
nuclei
Cortex
Core
Lens
Lens
fiber
cells
Cornea
Conjuctiva
Epithelium
AQP0 forms thin junctions in vivo
0.2 µm
Adapted from:
Paul & Goodenough (1983)
J. Cell Biol. 96: 625-632
Adapted from:
Zampighi et al. (1982)
J. Cell Biol. 93: 175-189
Purification of AQP0 from the lens
cortex core
Cortex
97
67
Core
45
31
full-length
cleaved
21
Solubilization in 1% DM
Anion exchange (MonoQ)
Gel filtration (S12)
14
2D crystals of cortical AQP0
200 nm
AQP0 2D crystal
in negative stain
CTF plot of glucoseembedded 2D crystal
Projection map at 4 Å resolution
AQP0
AQP1
How to get double-layered
2D crystals of AQP0 ?
AQP0 in the lens cortex
is full-length
Some AQP0 in the lens core
is C-terminaly cleaved
AND
AND
Cortical AQP0 forms
single-layered crystals
AQP0 membrane junctions
are more frequent in the core
Cleavage of AQP0 may induce
the formation of membrane junctions
Chymotrypsin treatment of
reconstituted AQP0
reconstituted cortical AQP0
chymotrypsin
treatment
membranes after cleavage
Cleavage of AQP0 does indeed induce
the formation of membrane junctions
Purification of AQP0 from the lens
cortex core
Cortex
97
67
Core
45
31
full-length
cleaved
21
Solubilization in 1% DM
Anion exchange (MonoQ)
Gel filtration (S12)
14
2D crystals of core AQP0
1 µm
AQP0 2D crystal
in negative stain
CTF plot of glucoseembedded 2D crystal
Projection maps of AQP0 2D crystals
p422
p4
Double-layered crystal
Single-layered crystal
Electron diffraction
of double-layered MIP 2D crystals
0 degree
70 degree
AQP0-mediated membrane junction
The water pores in AQP1 and AQP0
AQP1
AQP0
Electron
diffraction
at liquid He
temperature
The 1.9 Å
density map
The AQP0 water pore
at 1.9 Å resolution
CS-I
Arg 187
Phe 48
NPAE
Asn 164
NPAB
Asn 68
His 66
CS-II
Tyr 149
Phe 75
The water pores in the
1.9 Å EM and 2.2 Å X-ray structure
1.9 Å EM structure
2.2 Å X-ray structure
Conformational switch of loop A
upon proteolytic cleavage
2.2 Å X-ray structure
1.9 Å EM structure
Conformational switch of loop A
upon proteolytic cleavage
2.2 Å X-ray structure
Conformational switch of loop A
upon proteolytic cleavage
2.2 Å X-ray structure
1.9 Å EM structure
Conformational switch of loop A
upon proteolytic cleavage
2.2 Å X-ray structure
1.9 Å EM structure
Constriction site I - the ar/R site
AQP0
Ala 181
His 172
Arg 187
Phe 48
AQP1
Cys 191
His 182
Arg 197
Phe 58
Molecular dynamics using AQPZ
UP
Arg 189
DOWN
Arg 189
Adapted from:
Wang et al. (2005) Structure 13: 1107-1118
Arg 187 and Met 176 in AQP0
2.2 Å X-ray structure
1.9 Å EM structure
Arg 187 and Met 176 in AQP0
2.2 Å X-ray structure
1.9 Å EM structure
The packing of AQP0 in the 2D crystals
The packing of AQP0 in the 2D crystals
The packing of AQP0 in the 2D crystals
The lipids surrounding an AQP0 monomer
Protein-lipid interactions
PC 1
PC 5
PC 6
Harvard
Medical School
Tamir Gonen
Yifan Cheng
Stephen Harrison
Piotr Sliz
University of
Auckland
Joerg Kistler
Kyoto University
Yoshinori Fujiyoshi
Yoko Hiroaki