Surface topographies at subnanometer-resolution reveal asymmetry and sidedness of aquaporin-1.
Aquaporin-1 (AQP1) is an abundant protein in human erythrocyte membranes which functions as a specific and constitutively active water conducting pore. Solubilized and isolated as tetramer, it forms well-ordered two-dimensional (2D) crystals when reconstituted in the presence of lipids. Several high resolution projection maps of AQP1 have been determined, but information on its three-dimensional (3D) mass distribution is sparse. Here, we present surface reliefs at 0.9 nm resolution that were calculated from freeze-dried unidirectionally metal-shadowed AQP1 crystals as well as surface topographs recorded with the atomic force microscope of native crystals in buffer solution. Our results confirm the 3D map of negatively stained AQP1 crystals, which exhibited tetramers with four major protrusions on one side and a large central cavity on the other side of the membrane. Digestion of AQP1 crystals with carboxypeptidase Y, which cleaves off a 5 kDa intracellular C-terminal fragment, led to a reduction of the major protrusions, suggesting that the central cavity of the tetramer faces the outside of the cell. To interpret the results, sequence based structure predictions served as a guide.
Walz
T
T
Maurice E. Müller-Institute Biozentrum, University of Basel, Switzerland.
Tittmann
P
P
Fuchs
K H
KH
Müller
D J
DJ
Smith
B L
BL
Agre
P
P
Gross
H
H
Engel
A
A
D051398:Aquaporin 1; D020346:Aquaporins; D001789:Blood Group Antigens; D002268:Carboxypeptidases; D043402:Cathepsin A; D003460:Crystallization; D004910:Erythrocyte Membrane / Q000737:chemistry; D006801:Humans; D007473:Ion Channels / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure*; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D011487:Protein Conformation; D013499:Surface Properties
C496020:AQP1 protein, human; D020346:Aquaporins; D001789:Blood Group Antigens; D007473:Ion Channels; D051398:Aquaporin 1; D002268:Carboxypeptidases; C468629:CTSA protein, human; D043402:Cathepsin A