Structural dynamics of proton pumps

• Research unit: EU RFP
• Project number: 98.0231

Key words (English)

AQUAPORINS; AQUAGLYCEROPORINS; 2D CRYSTALLIZATION; ELECTRON CRYSTALLOGRAPHY; AFM

Alternative project number (English)

EU project number: BIO-4-98-0415

Research programs (English)

EU-programme: 4. Frame Research Programme - 4.1 Biotechnology

Short description (English)

See abstract

Partners and International Organizations (English)

Coordinator: ESRF (F)

Abstract (English)

Molecular channels allow living cells to adapt to osmotic variations. Such channels are present in animals, plants, algae, fungi and bacteria and form the aquaporin super family of proteins. They block the passage of ions, but allow water and small hydrophilic solutes to pass unhindered.
We have aligned 164 different sequences of the aquaporin super family to reveal the distinct AQP and GLP cluster, altogether comprised of 46 subtypes. The core architecture derived from these aligned sequences consists of six transmembrane helices, two long functional loops, and three interlinking loops of various lengths, constituting a minimum of 208 residues. The NPA motifs of the functional loops are considered to be the water channel signature. In addition, helices 1 and 4 possess the remarkable pattern ExxxTxxL/F in their N-terminal half, while helices 3 and 6 show a distinct helical periodicity along their entire length. Besides the NPA motifs many other residues are conserved in the functional loops, indicating their significance. His74 in loop B and Arg195 in loop E are the conserved positively charged buried residues that may form ion pairs with the buried Glu in helices 1 and 4. Conserved GlylAla indicate sites of helix-helix interaction.
Aquaporin- 1 (AQP1) was isolated from the red blood cell. We have produced tetragonal 2D crystals exhibiting 96 A wide unit cells. Cryo-electron crystallography has provided a 3D structure at 4.5 A resolution which shows six membrane spanning helices that surround a central density. The latter consists of two short helices projecting outwards from the center of the monomer, connected to adjacent helices by loop regions thus forming the water channel.
The lens major intrinsic protein (MIP) is known to function as a water and solute channel. However, MIP has also been reported to occur in close membrane contacts between lens fiber cells indicating a dual function. We have isolated MIP tetramers from sheep lens fiber cells and reconstituted them into highly ordered tetragonal 2D crystals with square unit cells of a side length of 64 A. Projection maps of freeze-dried MIP exhibited a resolution of 8A and were similar to maps of AQP1.
Negative stain electron microscopy and STEM demonstrated the glycerol facilitator protein (GlpF) from E. coli to exist as a tetramer. GlpF tetramers assembled to similar 2D crystals as AQPl when reconstituted with lipids.

References in databases (English)

Swiss Database: Euro-DB of the
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CH-3003 Berne, Switzerland
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Swiss Project-Number: 98.0231