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Crystal structure of subunits D and F in complex gives insight into energy transmission of the eukaryotic V-ATPase from Saccharomyces cerevisiae.

ABSTRACT: Eukaryotic V1VO-ATPases hydrolyze ATP in the V1 domain coupled to ion pumping in VO. A unique mode of regulation of V-ATPases is the reversible disassembly of V1 and VO, which reduces ATPase activity and causes silencing of ion conduction. The subunits D and F are proposed to be key in these enzymatic processes. Here, we describe the structures of two conformations of the subunit DF assembly of Saccharomyces cerevisiae (ScDF) V-ATPase at 3.1 Å resolution. Subunit D (ScD) consists of a long pair of ?-helices connected by a short helix ((79)IGYQVQE(85)) as well as a ?-hairpin region, which is flanked by two flexible loops. The long pair of helices is composed of the N-terminal ?-helix and the C-terminal helix, showing structural alterations in the two ScDF structures. The entire subunit F (ScF) consists of an N-terminal domain of four ?-strands (?1-?4) connected by four ?-helices (?1-?4). ?1 and ?2 are connected via the loop (26)GQITPETQEK(35), which is unique in eukaryotic V-ATPases. Adjacent to the N-terminal domain is a flexible loop, followed by a C-terminal ?-helix (?5). A perpendicular and extended conformation of helix ?5 was observed in the two crystal structures and in solution x-ray scattering experiments, respectively. Fitted into the nucleotide-bound A3B3 structure of the related A-ATP synthase from Enterococcus hirae, the arrangements of the ScDF molecules reflect their central function in ATPase-coupled ion conduction. Furthermore, the flexibility of the terminal helices of both subunits as well as the loop (26)GQITPETQEK(35) provides information about the regulatory step of reversible V1VO disassembly.

SUBMITTER: Balakrishna AM

PROVIDER: S-EPMC4318993 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Crystal structure of subunits D and F in complex gives insight into energy transmission of the eukaryotic V-ATPase from Saccharomyces cerevisiae.

Balakrishna Asha Manikkoth AM Basak Sandip S Manimekalai Malathy Sony Subramanian MS Grüber Gerhard G

The Journal of biological chemistry 20141212 6

Eukaryotic V1VO-ATPases hydrolyze ATP in the V1 domain coupled to ion pumping in VO. A unique mode of regulation of V-ATPases is the reversible disassembly of V1 and VO, which reduces ATPase activity and causes silencing of ion conduction. The subunits D and F are proposed to be key in these enzymatic processes. Here, we describe the structures of two conformations of the subunit DF assembly of Saccharomyces cerevisiae (ScDF) V-ATPase at 3.1 Å resolution. Subunit D (ScD) consists of a long pair ...[more]

PMID: 25505269

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Project description:BackgroundAs the third enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase (6PGDH) is the main generator of cellular NADPH. Both thioredoxin reductase and glutathione reductase require NADPH as the electron donor to reduce oxidized thioredoxin or glutathione (GSSG). Since thioredoxin and GSH are important antioxidants, it is not surprising that 6PGDH plays a critical role in protecting cells from oxidative stress. Furthermore the activity of 6PGDH is associated with several human disorders including cancer and Alzheimer's disease. The 3D structural investigation would be very valuable in designing small molecules that target this enzyme for potential therapeutic applications.ResultsThe crystal structure of 6-phosphogluconate dehydrogenase (6PGDH/Gnd1) from Saccharomyces cerevisiae has been determined at 2.37 A resolution by molecular replacement. The overall structure of Gnd1 is a homodimer with three domains for each monomer, a Rossmann fold NADP+ binding domain, an all-alpha helical domain contributing the majority to hydrophobic interaction between the two subunits and a small C-terminal domain penetrating the other subunit. In addition, two citrate molecules occupied the 6PG binding pocket of each monomer. The intact Gnd1 had a Km of 50 +/- 9 microM for 6-phosphogluconate and of 35 +/- 6 microM for NADP+ at pH 7.5. But the truncated mutants without the C-terminal 35, 39 or 53 residues of Gnd1 completely lost their 6PGDH activity, despite remaining the homodimer in solution.ConclusionThe overall tertiary structure of Gnd1 is similar to those of 6PGDH from other species. The substrate and coenzyme binding sites are well conserved, either from the primary sequence alignment, or from the 3D structural superposition. Enzymatic activity assays suggest a sequential mechanism of catalysis, which is in agreement with previous studies. The C-terminal domain of Gnd1 functions as a hook to further tighten the dimer, but it is not necessary for the dimerization. This domain also works as a lid on the substrate binding pocket to control the binding of substrate and the release of product, so it is indispensable for the 6PGDH activity. Moreover, the co-crystallized citrate molecules, which mimic the binding mode of the substrate 6-phosphogluconate, provided us a novel strategy to design the 6PDGH inhibitors.

Dataset Information

Crystal structure of subunits D and F in complex gives insight into energy transmission of the eukaryotic V-ATPase from Saccharomyces cerevisiae.

Publications

Crystal structure of subunits D and F in complex gives insight into energy transmission of the eukaryotic V-ATPase from Saccharomyces cerevisiae.

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