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

Figures & data

Figure 1.  (A) SDS-gel of the purified G_1–59 (lane 1) and a protein ladder (lane 2) with molecular weights as indicated. (B) 2D ¹H-¹⁵N-HSQC spectrum of G_1–59 in 25 mM sodium phosphate buffer (pH 6.5) at 288 K. Backbone and amide assignments (N and Q) are shown for each residue. Signals from side-chain NH₂ groups are connected by horizontal lines.

Figure 2.  (A) Sections from the overlaid 2D ¹H-¹⁵N-HSQC spectra of G_1–59 in the absence (red) and presence (green) of 1.5 equivalent of unlabeled E_18–39. (B) Combined amide (¹H) and nitrogen (¹⁵N) chemical shift changes ([(Δ¹H_N)²+(Δ¹⁵N_ppm/6.51)²]^0.5) for G_1–59 and E_18–38 binding as a function of the amino acid sequence.

Figure 3.  G_1–59/E_18–38–binding studied by fluorescence correlation spectroscopy. (A) Normalized autocorrelation functions of Atto655 labeled peptide E_18–38 by increasing the quantity of subunit G_1–59. (B) Concentration-dependent binding of E_18–38 to subunit G_1–59. The binding constant was calculated by determining the relative bound fraction using a standard autocorrelation two diffusion coefficients normalized triplet model (FCS-LSM software, ConfoCor 3, Zeiss). Best fits yielding the binding constants are represented as fitted line by a non-linear, asymptotic curve fit (see the text).

Figure 4.  (A) Far UV-CD spectrum of the peptide E_18–38. (B) HN-HN region of the NOESY spectra of E_18–38. (C) Connectivity diagram of E_18–38. The line thickness reflects the intensities of the NOE connectivities.

Figure 5.  Cartoon representation for the NMR structure of the peptide E_18–38. (A) Superposition of the five lowest energy structures, (B) average structure showing the angle between the helices and (C) average structure indicating the bend angle of the C-terminal helix with respect the N-terminal helix.

Table I.  Summary of structural statistics for five selected structures of E_18–38.

Structural statistics for E18–38
Distance restraints
Total	180
Intraresidue (i–j = 0)	69
Sequential (∣i-j∣ = 1)	72
Medium-range (2 ≤ ∣i − j∣ ≤ 4)	39
Long-range (∣i − j∣≥5)	0
Average number of violations
Distance violations > 5 Å	0
Ramachandran plot^2 (%)
Residues in most favoured regions	94.4
Residues in additionally allowed regions	5.6
Residues in generously allowed regions	0.0
Residues in disallowed regions	0.0
Average RMSD to Mean (Å)
Backbone (C^α, C′, and N)	0.825±0.29

Figure 6.  The structure of the modeled segment G_1–59 (green). The residues shown in stick representation encircled with dotted spheres are the ones that are shown to have binding (M₁, V₂, S₃ and K₅; V₂₂, S₂₃, K₂₄, A₂₅ and R₂₆) with the E_18–38 peptide. Residues shown in line representation are the ones proposed to be in the interacting region in the docked complex.

Figure S1.  (A) Overlaid ¹H/¹⁵N-HSQC spectra of G_1–59 in the absence (red) and presence (blue) of 1.5 equivalents of E_39–233 in 25 mM sodium phosphate buffer (pH 6.8) at 288 K. (B) Overlaid ¹H/¹⁵N-HSQC spectra of G_61–114 in the absence (red) and presence (blue) of 1.5 equivalents of E_39–233 in 25 mM sodium phosphate buffer (pH 6.8) at 288 K. (C) Overlaid ¹H/¹⁵N-HSQC spectra of G_1–59 in the absence (red) and presence (yellow) of 1.5 equivalents of E_1–18 in 25 mM sodium phosphate buffer (pH 6.8) at 288 K.

Figure S2.  The molecular surface showing the electrostatic potential of the peptide E_18–38 drawn in pyMol (DeLano WL [2001] The pyMol Molecular Graphics SystemSan Carlos, CA: DeLano Scientific) that uses Poisson-Boltzmann equation, where the positive potentials are drawn in blue and negative in red. (A) Front view, (B) Back view.