P-type ATPases studied by electron microscopy and two-dimensional crystallization

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.