The 5A structure of heterologously expressed plant aquaporin SoPIP2;1.
SoPIP2;1 is one of the major integral proteins in spinach leaf plasma membranes. In the Xenopus oocyte expression system its water channel activity is regulated by phosphorylation at the C terminus and in the first cytosolic loop. To assess its structure, SoPIP2;1 was heterologously expressed in Pichia pastoris as a His-tagged protein and in the non-tagged form. Both forms were reconstituted into 2D crystals in the presence of lipids. Tubular crystals and double-layered crystalline sheets of non-tagged SoPIP2;1 were observed and analyzed by cryo-electron microscopy. Crystalline sheets were highly ordered and diffracted electrons to a resolution of 2.96A. High-resolution projection maps of tilted specimens provided a 3D structure at 5A resolution. Superposition of the SoPIP2;1 potential map with the atomic model of AQP1 demonstrates the generally well conserved overall structure of water channels. Differences concerning the extracellular loop A explain the particular crystal contacts between oppositely oriented membrane sheets of SoPIP2;1 2D crystals, and may have a function in rapid volume changes observed in stomatal guard cells or mesophyll protoplasts. This crystal packing arrangement provides access to the phosphorylated C terminus as well as the loop B phosphorylation site for studies of channel gating.
Kukulski
W
W
Maurice E. Müller Institute for Microscopy, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.
Schenk
A D
AD
Johanson
U
U
Braun
T
T
de Groot
B L
BL
Fotiadis
D
D
Kjellbom
P
P
Engel
A
A
D000818:Animals; D020346:Aquaporins / Q000737:chemistry*; D046529:Microscopy, Electron, Transmission; D010843:Pichia; D010940:Plant Proteins / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D018724:Spinacia oleracea; D014981:Xenopus
D020346:Aquaporins; D010940:Plant Proteins