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News and Publications
Three-dimensional Architectures of Membrane Protein Channels and
Macromolecular Complexes
A.K. Mitra, G. Ren, A. Froger, J. Hillman, J. Quispe
Our goal is to understand the structural correlates of the function
of membrane channels and macromolecular signaling at the membrane
interface. We use electron microscopy, image processing, and electron
crystallography to directly determine 3-dimensional structures of
membrane protein channels and macromolecular complexes in the lipid
bilayer.
ATOMIC MODEL OF HUMAN AQUAPORIN 1
Using a technique that preserves specimens in close to their native
state, we determined the 3-dimensional structure of aquaporin 1,
a water-selective, bidirectional, integral membrane protein channel
purified from human erythrocytes. The 3.7-Å-resolution structure
of aquaporin 1 obtained with frozen-hydrated 2-dimensional lipid-reconstituted
crystals showed that the size-selective (4.0 ± 0.5 Å)
aqueous pathway in an aquaporin 1 monomer is defined by mostly hydrophobic
amino acid residues interspersed with a few polar residues contributed
by 4 transmembrane a-helices
and 2 short a-helices.
This narrow pore is connected by wide, funnel-shaped openings at
both the extracellular and cytoplasmic faces that are lined primarily
by polar and charged residues. Thus, the strongly hydrated environment
at the cytoplasmic or extracellular entrances leading to the relatively
inert size-selective pore explains how a pathway conducive to rapid,
diffusion-limited water flow through the lipid bilayer can be generated.
The atomic structure revealed putative binding sites of a permeant
water molecule and clues for selectivity. We are using yeast-expressed
recombinant aquaporin 1 to probe the extracellular constriction
defined by a highly conserved arginine (Arg195), a cysteine (Cys189),
a conserved phenylalanine (Phe56), and a conserved histidine (His180).
The data will enable us to develop a comprehensive mechanistic model
that explains the exquisite water selectivity of the aquaporin.
ANTIGEN PRESENTATION ON THE MEMBRANE SURFACE
In collaboration with H. Celia, University of Strasbourg, France,
and L. Teyton, Department of Immunology, we established the structural
relationship of MHC molecules with the membrane bilayer, a critical
factor in the recognition of antigen by T-cell receptors at the
surface of nucleated cells. Electron crystallographic data from
highly ordered 2-dimensional crystals of histidine-tagged H-2Kb
murine class I MHC molecules generated on a lipid monolayer containing
nickel-chelated lipids were compared with the x-ray structure of
the soluble H-2Kb molecule. The results unambiguously
indicated that the MHC molecule orients with its long axis approximately
"parallel" to the membrane plane rather than normal to it, as was
generally thought. This novel orientation helps explain a number
of biophysical results and expands our knowledge of the recognition
process.
STRUCTURAL STUDIES OF PORE-FORMING SOLUBLE PROTEINS
We are using anthrax toxin and aerolysin as models for understanding
the molecular dynamics involved in membrane insertion and channel
formation. We used single-particle image analysis to reveal the
mode of binding of the lethal factor of anthrax toxin to PA63, the
toxin's central component; this binding is a crucial step in the
eventual translocation of the complex that leads to cell death.
In the case of aerolysin, we are analyzing lipid-reconstituted 2-dimensional
crystals, which are revealing the oligomeric association and the
architecture of the pore in the lipid bilayer.
PUBLICATIONS
Mitra, A.K., Ren, G., Reddy, V.S., Cheng, A., Froger. A.
The architecture of a water-selective pore in the lipid bilayer
visualized by electron crystallography in vitreous ice. Novartis
Found. Symp. 245:33, 2002.
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