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ABSTRACT: The subunit ε of bacterial F(1)F(O) ATP synthases plays an important regulatory role in coupling and catalysis via conformational transitions of its C-terminal domain. Here we present the first low resolution solution structure of ε of Mycobacterium tuberculosis (Mtε) F(1)F(O) ATP synthase and the NMR structure of its C-terminal segment (Mtε(103-120)). The overall length of Mtε (61.6 Å) is significantly shorter compared to forms of the subunit in other bacteria, reflecting a shorter C-terminal sequence, proposed to be important in coupling processes via the catalytic β subunit. The C-terminal segment displays α-helical structure and highly positive surface charge due to the presence of arginine residues. Using NMR-, fluorescence spectroscopy and mutagenesis, we demonstrate that the new TB drug candidate TMC207, proposed to bind to the proton translocating c-ring, does also bind to Mtε. A model for the interaction of TMC207 with both, ε and the c-ring is presented, suggesting that TMC207 forms a wedge between the two rotating subunits by interacting with the residues W15 and F50 of ε and the c-ring, respectively. T19 and R37 of ε provide the necessary polar interactions with the drug molecule. This new model of the mechanism of TMC207 provides the basis for the design of new drugs, targeting the F(1)F(O) ATP synthase in M. tuberculosis.
Antimicrobial Agents and Chemotherapy 10/2012; · 4.84 Impact Factor
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ABSTRACT: Subunit E of the vacuolar ATPase (V-ATPase) contains an N-terminal extended α helix (Rishikesan et al. J Bioenerg Biomembr
43:187–193, 2011) and a globular C-terminal part that is predicted to consist of a mixture of α-helices and β-sheets (Grüber et al. Biochem
Biophys Res Comm 298:383–391, 2002). Here we describe the production, purification and 2D structure of the C-terminal segment E133-222 of subunit E from Saccharamyces cerevisiae V-ATPase in solution based on the secondary structure calculation from NMR spectroscopy studies. E133-222 consists of four β-strands, formed by the amino acids from K136-V139, E170-V173, G186-V189, D195-E198 and two α-helices,
composed of the residues from R144-A164 and T202-I218. The sheets and helices are arranged as β1:α1:β2:β3:β4:α2, which are
connected by flexible loop regions. These new structural details of subunit E are discussed in the light of the structural
arrangements of this subunit inside the V1- and V1VO ATPase.
KeywordsV1VO ATPase–V-ATPase–Subunit E–Vma4p–
Saccharomyces cerevisiae
–NMR spectroscopy
Journal of Bioenergetics 05/2012; 43(5):447-455. · 2.81 Impact Factor
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ABSTRACT: The N-terminus of V-ATPase subunit E has been shown to associate with the subunits C, G and H, respectively. To understand
the assembly of E with its neighboring subunits as well as its N-terminal structure, the N-terminal region, E1-69, of the Saccharomyces cerevisiae V-ATPase subunit E was expressed and purified. The solution structure of E1-69 was determined by NMR spectroscopy. The protein is 90.3 Å in length and forms an á-helix between the residues 12–68. The
molecule is amphipathic with hydrophobic residues at the N-terminus, predicted to interact with subunit C. The polar epitopes
of E1–69 are discussed as areas interacting with subunits G and H.
KeywordsVacuolar-type ATPase–V1VO ATPase–V-ATPase–Subunit E–Vma4p–
Saccharomyces cerevisiae
–NMR
Journal of Bioenergetics 04/2012; 43(2):187-193. · 2.81 Impact Factor
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ABSTRACT: Subunit E of the vacuolar ATPase (V-ATPase) contains an N-terminal extended α helix (Rishikesan et al. J Bioenerg Biomembr 43:187-193, 2011) and a globular C-terminal part that is predicted to consist of a mixture of α-helices and β-sheets (Grüber et al. Biochem Biophys Res Comm 298:383-391, 2002). Here we describe the production, purification and 2D structure of the C-terminal segment E₁₃₃₋₂₂₂ of subunit E from Saccharamyces cerevisiae V-ATPase in solution based on the secondary structure calculation from NMR spectroscopy studies. E₁₃₃₋₂₂₂ consists of four β-strands, formed by the amino acids from K136-V139, E170-V173, G186-V189, D195-E198 and two α-helices, composed of the residues from R144-A164 and T202-I218. The sheets and helices are arranged as β1:α1:β2:β3:β4:α2, which are connected by flexible loop regions. These new structural details of subunit E are discussed in the light of the structural arrangements of this subunit inside the V₁- and V₁V₀ ATPase.
Journal of Bioenergetics 08/2011; 43(5):447-55. · 2.81 Impact Factor
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ABSTRACT: The N-terminus of V-ATPase subunit E has been shown to associate with the subunits C, G and H, respectively. To understand the assembly of E with its neighboring subunits as well as its N-terminal structure, the N-terminal region, E(1-69), of the Saccharomyces cerevisiae V-ATPase subunit E was expressed and purified. The solution structure of E(1-69) was determined by NMR spectroscopy. The protein is 90.3 Å in length and forms an á-helix between the residues 12-68. The molecule is amphipathic with hydrophobic residues at the N-terminus, predicted to interact with subunit C. The polar epitopes of E(1-69) are discussed as areas interacting with subunits G and H.
Journal of Bioenergetics 03/2011; 43(2):187-93. · 2.81 Impact Factor
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ABSTRACT: Subunit G is an essential stalk subunit of the eukaryotic proton pump V(1)V(O) ATPase. Previously the structure of the N-terminal region, G(1)(-)(59), of the 13kDa subunit G was solved at higher resolution. Here solution NMR was performed to determine the structure of the recombinant C-terminal region (G(61)(-)(101)) of subunit G of the Saccharomyces cerevisiae V(1)V(O) ATPase. The protein forms an extended alpha-helix between residues 64 and 100, whereby the first five- and the last residues of G(61)(-)(101) are flexible. The surface charge distribution of G(61)(-)(101) reveals an amphiphilic character at the C-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The hydrophobic surface pattern is mainly formed by alanine residues. The alanine residues 72, 74 and 81 were exchanged by a single cysteine in the entire subunit G. Cysteines at positions 72 and 81 showed disulfide formation. In contrast, no crosslink could be formed for the mutant Ala74Cys. Together with the recently determined NMR solution structure of G(1)(-)(59), the presented solution structure of G(61)(-)(101) enabled us to present a first structural model of the entire subunit G of the S. cerevisiae V(1)V(O) ATPase.
Biochimica et Biophysica Acta 10/2010; 1798(10):1961-8. · 4.66 Impact Factor
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ABSTRACT: Understanding the structural traits of subunit G is essential, as it is needed for V(1)V(O) assembly and function. Here solution NMR of the recombinant N- (G(1-59)) and C-terminal segment (G(61-114)) of subunit G, has been performed in the absence and presence of subunit d of the yeast V-ATPase. The data show that G does bind to subunit d via its N-terminal part, G(1-59) only. The residues of G(1-59) involved in d binding are Gly7 to Lys34. The structure of G(1-59) has been solved, revealing an alpha-helix between residues 10 and 56, whereby the first nine- and the last three residues of G(1-59) are flexible. The surface charge distribution of G(1-59) reveals an amphiphilic character at the N-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The C-terminus exhibits a strip of negative residues. The data imply that G(1-59)-d assembly is accomplished by hydrophobic interactions and salt-bridges of the polar residues. Based on the recently determined NMR structure of segment E(18-38) of subunit E of yeast V-ATPase and the presently solved structure of G(1-59), both proteins have been docked and binding epitopes have been analyzed.
Biochimica et Biophysica Acta 02/2009; 1787(4):242-51. · 4.66 Impact Factor
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ABSTRACT: A critical point in the V(1) sector and entire V(1)V(O) complex is the interaction of stalk subunits G (Vma10p) and E (Vma4p). Previous work, using precipitation assays, has shown that both subunits form a complex. In this work, we have analysed the N-terminal segment of subunit G (G(1-59)) of the V(1)V(O) ATPase from Saccharomyces cerevisiae by using nuclear magnetic resonance (NMR) spectroscopy. Analyses of (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra of G(1-59) in the absence and presence of the N-terminal peptides E(1-18) and E(18-38) as well as the produced and purified C-terminal segment (E(39-233)) shows specific interactions only with the peptide fragment E(18-38). The binding of this peptide occurs via the residues M(1), V(2), S(3), and K(5) as well for V(22), S(23), K(24), A(25) and R(26) of G(1-59). The specific E(18-38)/G(1-59) binding has been confirmed by fluorescence correlation spectroscopy data. The E(18-38) peptide has been studied by CD spectroscopy and NMR. The 3D structure of this peptide adopts a stable helix-hinge-helix formation in solution. A model structure of the E(18-38)/G(1-59) complex reveals the orientation of E(18-38) relative to G(1-59) via salt-bridges of the polar residues and van der Waals forces at the very N-terminus of both segments.
Molecular Membrane Biology 08/2008; 25(5):400-10. · 2.86 Impact Factor
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ABSTRACT: Understanding the structural traits of subunit G is essential, as it is needed for V1VO assembly and function. Here solution NMR of the recombinant N- (G1–59) and C-terminal segment (G61–114) of subunit G, has been performed in the absence and presence of subunit d of the yeast V-ATPase. The data show that G does bind to subunit d via its N-terminal part, G1–59 only. The residues of G1–59 involved in d binding are Gly7 to Lys34. The structure of G1–59 has been solved, revealing an α-helix between residues 10 and 56, whereby the first nine- and the last three residues of G1–59 are flexible. The surface charge distribution of G1–59 reveals an amphiphilic character at the N-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The C-terminus exhibits a strip of negative residues. The data imply that G1–59–d assembly is accomplished by hydrophobic interactions and salt-bridges of the polar residues. Based on the recently determined NMR structure of segment E18–38 of subunit E of yeast V-ATPase and the presently solved structure of G1–59, both proteins have been docked and binding epitopes have been analyzed.
Biochimica et Biophysica Acta (BBA) - Bioenergetics.
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ABSTRACT: Subunit G is an essential stalk subunit of the eukaryotic proton pump V1VO ATPase. Previously the structure of the N-terminal region, G1–59, of the 13 kDa subunit G was solved at higher resolution. Here solution NMR was performed to determine the structure of the recombinant C-terminal region (G61–101) of subunit G of the Saccharomyces cerevisiae V1VO ATPase. The protein forms an extended α-helix between residues 64 and 100, whereby the first five- and the last residues of G61–101 are flexible. The surface charge distribution of G61–101 reveals an amphiphilic character at the C-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The hydrophobic surface pattern is mainly formed by alanine residues. The alanine residues 72, 74 and 81 were exchanged by a single cysteine in the entire subunit G. Cysteines at positions 72 and 81 showed disulfide formation. In contrast, no crosslink could be formed for the mutant Ala74Cys. Together with the recently determined NMR solution structure of G1–59, the presented solution structure of G61–101 enabled us to present a first structural model of the entire subunit G of the S. cerevisiae V1VO ATPase.
Biochimica et Biophysica Acta (BBA) - Biomembranes.
