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Spectroscopical identification of residues of subunit G of the yeast V-ATPase in its connection with subunit E

, , , , , & show all
Pages 400-410 | Received 13 Feb 2008, Published online: 09 Jul 2009
 

Abstract

A critical point in the V1 sector and entire V1VO 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 (G1–59) of the V1VO ATPase from Saccharomyces cerevisiae by using nuclear magnetic resonance (NMR) spectroscopy. Analyses of 1H-15N heteronuclear single quantum coherence (HSQC) spectra of G1–59 in the absence and presence of the N-terminal peptides E1–18 and E18–38 as well as the produced and purified C-terminal segment (E39–233) shows specific interactions only with the peptide fragment E18–38. The binding of this peptide occurs via the residues M1, V2, S3, and K5 as well for V22, S23, K24, A25 and R26 of G1–59. The specific E18–38/G1–59 binding has been confirmed by fluorescence correlation spectroscopy data. The E18–38 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 E18–38/G1–59 complex reveals the orientation of E18–38 relative to G1–59 via salt-bridges of the polar residues and van der Waals forces at the very N-terminus of both segments.

Abbreviations
CSI=

chemical shift index

DSS=

2,2-Dimethyl-2-silapentane-5-sulfonate

DTT=

dithiothreitol

EDC=

1-ethyl-3-(dimethylaminopropyl)-carbodiimide

FCS=

fluorescence correlation spectroscopy

GST=

glutathione-S-transferase

HSQC=

heteronuclear single quantum coherence

HPLC=

high pressure liquid chromatography

IPTG=

isopropyl-β-D-thio-galactoside

NMR=

nuclear magnetic resonance

NOESY=

nuclear Overhauser effect spectroscopy

NTA=

nitrilotriacetic acid

PAGE=

polyacrylamide gel electrophoresis

PCR=

polymerase chain reaction

SAXS=

small angle X-ray scattering

SDS=

sodium dodecyl sulfate

TFE=

trifluorethanol

TOCSY=

total correlation spectroscopy

Tris=

Tris-(hydroxymethyl)aminomethane

Abbreviations
CSI=

chemical shift index

DSS=

2,2-Dimethyl-2-silapentane-5-sulfonate

DTT=

dithiothreitol

EDC=

1-ethyl-3-(dimethylaminopropyl)-carbodiimide

FCS=

fluorescence correlation spectroscopy

GST=

glutathione-S-transferase

HSQC=

heteronuclear single quantum coherence

HPLC=

high pressure liquid chromatography

IPTG=

isopropyl-β-D-thio-galactoside

NMR=

nuclear magnetic resonance

NOESY=

nuclear Overhauser effect spectroscopy

NTA=

nitrilotriacetic acid

PAGE=

polyacrylamide gel electrophoresis

PCR=

polymerase chain reaction

SAXS=

small angle X-ray scattering

SDS=

sodium dodecyl sulfate

TFE=

trifluorethanol

TOCSY=

total correlation spectroscopy

Tris=

Tris-(hydroxymethyl)aminomethane

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