Crystal structure of the regulatory subunit H of the V-type ATPase of Saccharomyces cerevisiae - PubMed (original) (raw)

Crystal structure of the regulatory subunit H of the V-type ATPase of Saccharomyces cerevisiae

M Sagermann et al. Proc Natl Acad Sci U S A. 2001.

Abstract

In contrast to the F-type ATPases, which use a proton gradient to generate ATP, the V-type enzymes use ATP to actively transport protons into organelles and extracellular compartments. We describe here the structure of the H-subunit (also called Vma13p) of the yeast enzyme. This is the first structure of any component of a V-type ATPase. The H-subunit is not required for assembly but plays an essential regulatory role. Despite the lack of any apparent sequence homology the structure contains five motifs similar to the so-called HEAT or armadillo repeats seen in the importins. A groove, which is occupied in the importins by the peptide that targets proteins for import into the nucleus, is occupied here by the 10 amino-terminal residues of subunit H itself. The structural similarity suggests how subunit H may interact with the ATPase itself or with other proteins. A cleft between the amino- and carboxyl-terminal domains also suggests another possible site of interaction with other factors.

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Figures

Figure 1

Figure 1

Schematic overview of the V-type ATPases [adapted from Wilkens et al. (6)]. Subunit H, also named Vma13p, is highlighted in green and only one subunit is shown.

Figure 2

Figure 2

(A) Stereo ribbon diagram showing the overall structure of subunit H. The amino-terminal domain is highlighted in yellow and the C-terminal domain in green. The amino-terminal peptide including residues 2–10 is colored in red. This and other related figures were prepared with

bobscript

(41),

molscript

(42), and

raster3d

(43). (B) Stereo diagram showing the electron density map with experimental phases in the region between helices α7, α9, and α12. The resolution is 3.5 Å, and the map is contoured at 1.1 σ. (C) Electron density for residues 2–10. The coefficients are (2 _F_o−_F_c), phases are from the refined structure, the resolution is 2.9 Å, and the map is contoured at 1.2 σ. For clarity, only parts of the contacting helices α1, α8, and α11 are shown.

Figure 3

Figure 3

Superposition of subunit H (green) and residues 87–329 of karyopherin α (PDB code 1EE4) (yellow) using the overlay matrix determined by

dali

. The parts of subunit H that were not included in the superposition are colored gray. The amino-terminal peptide of subunit H (residues 2–10) is highlighted in red. The NLS peptide bound to karyopherin α is shown in blue.

Figure 4

Figure 4

Hidden Markov sequence alignment of subunit H from yeast with related sequences from Homo sapiens, Drosophila melanogaster, Arabidopsis thaliana, and Caenorhabditis elegans. Sequence identification numbers from the National Center for Biotechnology Information are gi/6325293, gi/6563196, gi/7801655, gi/10728827, and gi/1086810. The secondary structure of yeast subunit H is shown below the alignment. The regions labeled coil1 and coil2 are disordered and could not be reliably determined. The sequences that form structural repeats are underlined in orange and are labeled repeat 1 through repeat 5. Residues in the alignment that are identical are shown in red boxes; those that are similar are shown in yellow boxes. The sequence highlighted in the pink box corresponds to the alternatively spliced sequence found in mammalian subunit H sequences. The figure was prepared with the program

alscript

(44).

Figure 5

Figure 5

grasp

representation (38) of the molecular surface of subunit H showing its electrostatic potential. The amino-terminal residues 2–10 were omitted from the calculation and are displayed in white. Negative surface charge is shown in red, positive in blue. An accumulation of negative charge occurs between the two domains of the protein.

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