Structures and regulation of coupling subunit F and the arrangement of the subunit DF-assembly in the Saccharomyces cerevisiae v1vO ATPase

• Main Author: Sandip Basak
• Other Authors: Gerhard Gruber
• Format: Theses and Dissertations
• Language: English
• Published: 2014
• Subjects: DRNTU::Science::Biological sciences::Biochemistry
• Online Access: https://hdl.handle.net/10356/55396
• Institution: Nanyang Technological University

V-ATPases play an important role in the acidification of intracellular compartments such as lysosomes, endosomes, Golgi complexes and secretary granules. The V-ATPases are composed of at least 14 separate gene products, with many of these subunits present in multiple isoforms. The proposed subunit stoichiometry of V1 is A3:B3:C:D:E3:F:G3:H1 (1). The integral VO domain contains six different subunits in a stoichiometry of a:d:c4-5:c’:c”:e. V-ATPases exist in a dynamic equilibrium between fully assembled complexes and reversibly disassembled V1 and VO subcomplexes. Depending on the energy status of the cell, this equilibrium can be rapidly shifted (2). Vacuolar ATPases use the energy derived from ATP hydrolysis, catalyzed in the A3B3 sector of the V1 ATPase to pump protons via the membrane-embedded VO sector. The energy coupling between the two sectors occurs via the so-called central stalk, to which subunit F belongs. In the present study, the low resolution structure of recombinant subunit F (VMA7p) of the eukaryotic V-ATPase from Saccharomyces cerevisiae has been analyzed by small angle X-ray scattering (SAXS). The protein is divided into a 5.5 nm long egglike shaped region, connected via a 1.5 nm linker to a hook-like segment at one end. Circular dichroism spectroscopy revealed that subunit F comprises of 43% -helix, 32% -sheet and a 25% random coil arrangement. To determine the localization of the N- and C-termini in the protein, the C-terminal truncated form of F, F1-94 was produced and analyzed by SAXS. Comparison of the F1-94 shape with the shape of the entire subunit F showed the missing hook-like region in F1-94, supported by the decreased Dmax value of F1-94 and indicating that the hook-like region consists of the C-terminal residues (3). The NMR solution structure of the C-terminal peptide, F90-116, was solved, showing an α-helical region between residues 103-113 (3). The F90-116 solution structure fitted well in the hook-like region of subunit F (3). In order to understand the structural features of F1-94 at the atomic level, X-ray crystallography was performed. The crystal structure of F1-94 reveals a Rossmann fold with alternating β-strands and α-helices (4). Elliptical shaped F1-94 has four β-strands which are surrounded by four α-helices. F1-94 contains two important loops spanning between α1-β2 (26GQITPETQEK35) and α2-β3 (60ERDDI64) which are present only in eukaryotic F subunits. Multiple sequence alignments of subunit F show that the 60ERDDI64 loop is highly conserved among the eukaryotic V-ATPases (4). NMR spectroscopy of the entire subunit F confirmed the secondary structural features of the crystallographic structure F1-94 in solution as well as the C-terminal peptide, F90-116. The heteronuclear NOE experiment shows that subunit F has a rigid core in the N-terminal domain, whereas α1 and α5 are more flexible in the solution (4). To understand the cross-talk between central stalk subunits with the neighboring subunits, a DF-heterodimer was generated. The DF-heterodimer binds to subunit d with a dissociation constant (Kd) of 52.9 µM as determined by ITC experiment (4). The DF-heterodimer yielded crystals with a dimension of 0.13 mm x 0.10 mm x 0.04 mm, which diffracted maximum to 5 Å.