P-type ATPases studied by electron microscopy and two-dimensional crystallization
Author: Xian, Kenneth Yijing
Date: 1999-12-15
Location: Hörsalen plan 4, Novum
Time: 10.00
Department: Biovetenskaper och näringslära / Biosciences and Nutrition
Abstract
Electron microscopy and two-dimensional crystallography have been used to
study the molecular structures of the porcine renal Na+,K+-ATPase and the
porcine gastric H+,K+-ATPase. In addition, morphological changes have
been observed with electron cryomicroscopy from tubulovesicles in porcine
gastric mucosae.
The Na+,K+-ATPase and the H+,K+-ATPase belong to the P-type ATPase
family, which also includes the sarcoplasmic reticulum Ca 2+-ATPase and
the Neurospora crassa H+-ATPase, whose structures have both been
determined at 8 Å resolution. In addition to the a subunit that is found
in most eukaryotic ATPases, the H+,K+-ATPase and the H+,K+-ATPase have a
ß subunit. P-type ATPases actively translocate cations across biological
membranes using the free energy released from ATP hydrolysis. During its
activity cycle, an ATPase undergoes conformational changes thereby
transducing phosphorylation energy to the ion transport process. The two
major conformations are El and E2, corresponding to different cation
binding states. Some ATPases can be stabilized in an E2 conformation by
vanadate and magnesium, and form two-dimensional crystals.
In this work, two-dimensional Na+,K+-ATPase crystals with monoclinic p2
symmetry were induced by vanadate and magnesium. Images were recorded
from these crystals in the frozen hydrated state. The unit cell
parameters were a = 146.5 Å, b = 51.6 Å, and [gamma] = 97.1o,
accommodating 2 protein protomers related by a two-fold axis. The
untilted images contained structural information to better than 10 Å. A
three-dimensional model was reconstructed at 14 Å resolution from
projections tilted to up to 45'. The molecule had a large and compact
cytoplasmic domain; its length along the z-direction, which is
perpendicular to the membrane, was 140 Å.
Vanadate and magnesium also induced the formation of two-dimensional
crystals of the H+,K+-ATPase, with either p4 or p2 symmetry. From the
negatively stained crystals with p4 symmetry, a three-dimensional model
was obtained at 18 Å resolution. The unit cell parameters were a = b =
123 Å, [gamma] = 90o, containing 4 protein protomers. The model included
only the cytoplasmic domain and part of the stalk/transmembrane region.
It had a length of ~70 Å along the z-direction. An improved model was
later determined at 18 Å from negatively stained crystals with p2
symmetry. The unit cell had parameters a = 135 Å, b = 121 Å, [gamma] =
95.6o, in which 4 protomers were found. Information from the whole
protein molecule was preserved. The length along the z-direction was 140
Å and the molecule could be fitted into a box of 55 Å x 76 Å x 140 Å. The
cytoplasmic domain, ~80 Å in length, appeared rather compact.
The structures of both the Na+,K+-ATPase and the H+,K+-ATPase were
comparable with those of the Ca2+-ATPase and the H+-ATPase, indicating
that these proteins may share similar structural design.
Additionally, large numbers of tubulovesicles, with both unilamellar and
multilamellar forms, were observed from frozen hydrated H+,K+-ATPase-enriched
membranes. This seems to support the "membrane fusion" hypothesis of
hydrochloric acid secretion cycle and illustrates morphological changes
within the parietal cells.
List of papers:
I. Hebert H, Xian Y, Hacksell I, Mardh S (1992). "Two-dimensional crystals of membrane-bound gastric H,K-ATPase. " FEBS Lett 299(2): 159-162
Pubmed
II. Xian Y, Hebert H (1997). "Three-dimensional structure of the porcine gastric H,K-ATPase from negatively stained crystals. " J Struct Biol 118(3): 169-77
Pubmed
III. Xian Y, Hacksell I, Hebert H (1999). "The Morphology of the H,K ATPase-enriched membranes from pig gastric mucosal cells as observed by cryo elctron microscopy." (Submitted)
IV. Xian Y, Hebert H (1999). "An improved three-dimensioal model of the procine gastric H(+),K(+)-ATPase from negatively stained crystals." (Submitted)
V. Hebert H, Xian Y, Thomsen K, Maunsbach AB (1999). "Cryo electron microscopy of two- dimensional crystals of renal Na,K-ATPase." (In Print)
I. Hebert H, Xian Y, Hacksell I, Mardh S (1992). "Two-dimensional crystals of membrane-bound gastric H,K-ATPase. " FEBS Lett 299(2): 159-162
Pubmed
II. Xian Y, Hebert H (1997). "Three-dimensional structure of the porcine gastric H,K-ATPase from negatively stained crystals. " J Struct Biol 118(3): 169-77
Pubmed
III. Xian Y, Hacksell I, Hebert H (1999). "The Morphology of the H,K ATPase-enriched membranes from pig gastric mucosal cells as observed by cryo elctron microscopy." (Submitted)
IV. Xian Y, Hebert H (1999). "An improved three-dimensioal model of the procine gastric H(+),K(+)-ATPase from negatively stained crystals." (Submitted)
V. Hebert H, Xian Y, Thomsen K, Maunsbach AB (1999). "Cryo electron microscopy of two- dimensional crystals of renal Na,K-ATPase." (In Print)
Issue date: 1999-11-24
Publication year: 1999
ISBN: 91-628-3778-8
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