News and Publications

Three-Dimensional Architecture of Membrane Protein Channels and Transporters

A.K. Mitra, G. Ren, A. Froger, S. Hansen, J. Quisppe, C. Atteredge

Our research is directed at understanding the structural basis of transmembrane signaling. We use image processing and electron crystallography to reveal the 3-dimensional structures of membrane proteins in the lipid-bilayer environment. We focus on 2 classes of channels and transporters: integral membrane proteins and channel-forming soluble proteins. In collaborative studies on integral membrane proteins, we are investigating a bacterial pump of the so-called ABC superfamily with M. Hermodson, Purdue University, Lafayette, Indiana. In collaborative studies on channel-forming soluble proteins, we are examining anthrax toxin with S. Leppla, National Institutes of Health, Bethesda, Maryland, and E. Wilson, Department of Cell Biology; colicin Ia with K. Jakes, Albert Einstein College of Medicine, Bronx, New York; and the bcL2 family of proapoptotic and antiapoptotic proteins with S. Reed, The Burnham Institute, La Jolla, California.

We determined the atomic-resolution 3-dimensional structure of aquaporin 1, a water-selective bidirectional channel purified from human erythrocytes, to understand the mechanism of function of this molecule. Electron crystallographic data acquired from tilted views of highly ordered 2-dimensional crystals of aquaporin 1 in synthetic lipid bilayers preserved in vitreous ice allowed us to examine the unperturbed structure at progressively higher resolution, culminating in the delineation of an atomic model based on a 3.7-Å density map. In the 3-dimensional structure, a novel, in-plane, pseudo 2-fold axis of symmetry dictates the tertiary folding by relating the locations of the 3 transmembrane a-helices and a short, buried a-helix that characterize each of the tandemly repeated, homologous, N- and C-terminal halves of the polypeptide chain (Fig. 1).

The aqueous pathway in an aquaporin 1 monomer is characterized by a size-selective pore about 4.0 Å in diameter that spans a length of approximately 18 Å and bends by approximately 25° as it traverses the bilayer (Fig. 2). This narrow pore is outlined mostly by hydrophobic amino acid residues interspersed with short stretches of polar residues contributed by 4 of the 6 transmembrane a-helices (2 each from the 2 tandem repeats) and the 2 short a-helices. The pore is connected by wide, funnel-shaped openings at the extracellular and cytoplasmic faces, which are primarily lined by polar and charged residues. Thus, the strongly hydrated environment at the cytoplasmic or extracellular entrance leading to the relatively inert, size-selective pore generates a pathway conducive to rapid, diffusion-limited water flow. To provide a comprehensive mechanistic model that describes how the exquisite selectivity for water is achieved, we are using site-directed mutagenesis and structural studies to probe functionally important residues in the transmembrane domain of aquaporin 1.

Channel-forming soluble proteins are excellent models for understanding the dynamics of membrane-protein insertion. We are investigating the structure of the central component PA63 of anthrax toxin and its complex with lethal factor by analyzing images of both single particles and helical crystals. Our goal is to delineate the interaction involved in the binding and eventual translocation of toxic components of anthrax toxin that lead to cell death. We are doing structural studies of the membrane-integrated state of colicin Ia and bcl-Xl to gain insight into (1) the molecularity of the channel and (2) the molecular architecture of the channel and the folding pathway that leads to membrane insertion.

PUBLICATIONS
Mitra, A.K. Three-dimensional organization of the aquaporin water channel: What can structure tell us about function? Vitam. Horm. 60:133, 2000.

Mitra, A.K., Ren, G., Cheng, A., Reddy, V., Melnyk, P. Three-dimensional fold of human AQP1 water channel determined by electron cryo-crystallography of 2-dimensional crystals embedded in ice. In: Molecular Biology and Physiology of Water and Solute Transport. Hohmann, S., Nielsen, S. (Eds.). Kluwer Academic, Boston, 2000, p. 35.

Mitra, A.K., Ren, G., Cheng, A., Reddy, V.S., Melnyk, P. Three-dimensional reconstruction from electron micrographs of tilted 2-dimensional crystal: Structure of a human water channel. In: Image Reconstruction From Incomplete Data. Fiddy, M.A., Millane, R.P. (Eds.). SPIE Press, Bellingham, WA, 2000, p. 224. Vol. 4123 in Proceedings of SPIE.

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. Norvartis Found. Symp., in press.

Ren, G., Cheng, A., Reddy, V., Melnyk, P., Mitra, A.K. Three-dimensional fold of the human AQP1 water channel determined at 4 Å resolution by electron crystallography of two-dimensional crystals embedded in ice. J. Mol. Biol. 301:369, 2000.

Ren, G., Reddy, V.S., Cheng, A., Melnyk, P., Mitra, A.K. Visualization of a water-selective pore by electron crystallography in vitreous ice. Proc. Natl. Acad. Sci. U. S. A. 98:1398, 2001.

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