SUPPLEMENTARY MATERIAL
Title
Structural, Kinetic and Computational Investigation of Vitis vinifera DHDPS Reveals New Insight into
the Mechanism of Lysine-Mediated Allosteric Inhibition
Authors and Affiliations
Sarah C. Atkinson
1,2
, Con Dogovski 1,, Matthew T. Downton3, Peter E. Czabotar4, Renwick C.J.
Dobson 2,5, Juliet A. Gerrard5,6, John Wagner3 and Matthew A. Perugini1,2#
1
Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University,
Melbourne, Victoria 3086, Australia.
2
Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology
Institute, 30 Flemington Road, The University of Melbourne, Victoria 3010, Australia,
3
IBM Research Collaboratory for Life Sciences-Melbourne, Victorian Life Sciences Computation
Initiative, Carlton, VIC 3010, Australia
4
The Walter and Eliza Hall Institute of Medical Research, Parkville, 3052, Victoria, Australia
5
Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, Private
Bag 4800, Christchurch, New Zealand
6
Industrial Research Limited, PO Box 31310, Lower Hutt 5040, New Zealand
#
Address correspondence to: A/Prof Matthew Anthony Perugini. Department of Biochemistry, La Trobe
Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia. Tel: +61 3 9479 6570.
Fax: +61 3 9479 1266; E-mail: M.Perugini@latrobe.edu.au
1
Supplementary Table 1. Comparison of the ‘tight’ dimer and ‘weak’ dimer interfaces of Vv-DHDPS in
the absence (PDB ID: 3TUU) and presence of lysine (PDB ID: 4HNN)
TIGHT DIMER INTERFACE
WEAK DIMER INTERFACE
Residues
Residues
Residues
involved in
involved in
involved in
Hydrophobic
Hydrogen
Ion-Ion
contacts
bonding
interactions
1788
26
20
12
1827
29
22
13
Vv1
Residues
Residues
Residues
SISA1
involved in
involved in
involved in
(Å2)
Hydrophobic
Hydrogen
Ion-Ion
contacts
bonding
interactions
618
11
5
3
575
12
5
3
2
SISA (Å )
DHDPS
minus
lysine
plus
lysine
1
SISA
=
solvent
inaccessible
surface
area.
(http://www.ebi.ac.uk/msd-srv/prot_int/pistart).
2
Calculated
employing
PISA
[70]
analysis
Supplementary Figure 1. Lysine inhibition of Vv-DHDPS. (A) Kinetic analyses were performed by
measuring the initial velocity of Vv-DHDPS with respect to various concentrations of the substrate
ASA and inhibitor lysine (Lys). Initial velocity data were fitted to a mixed inhibition kinetic model (Eq.
2) using ENZFITTER (Biosoft software). Each data point was measured in triplicate. (B) Hill plot
using data collected at a fixed ASA concentration of 0.05 mM yielding a slope (apparent Hill
coefficient, napp) of 1.7 (Table I).
Supplementary Figure 2. Lineweaver-Burk analysis of kinetic data presented in Fig. 3 and
Supplementary Fig. 1(A). (A) Data shown in Fig. 3a (initial rate with respect to the substrate pyruvate
and inhibitor lysine) were expressed as double reciprocal plots. Global best-fits (lines) are indicated.
(B) Data shown in Supplementary Fig. 1(A) (initial rate with respect to the substrate ASA and inhibitor
lysine) were expressed as double reciprocal plots. Global best-fits (lines) are indicated.
Supplementary Figure 3. ITC isotherms showing (A) 10 mM lysine titrated into 80 μM Vv-DHDPS
(200 mM HEPES, pH 7.7). (B) 2.5 mM lysine titrated into 80 μM Vv-DHDPS (200 mM HEPES, pH
7.7).
Supplementary Figure 4. CD spectroscopy analyses of Vv-DHDPS in the absence and presence of
ligands. The mean residue ellipticity (ocm2dmol-1) is plotted as a function of wavelength (nm). Plotted
are the CD spectra of Vv-DHDPS for the unliganded (apo) enzyme (●), in the presence of 5 mM
pyruvate (◆), presence of 1 mM lysine (△), and presence of both 5 mM pyruvate & 1 mM lysine (■).
Supplementary Figure 5. Sedimentation velocity analytical ultracentrifugation analysis of Vv3
DHDPS. Absorbance (280 nm) is plotted as a function of radial position from the axis of rotation (cm)
for Vv-DHDPS (13 μM) centrifuged at 40,000 rpm in the (A) absence and (B) presence of lysine. Raw
data (open symbols) are plotted at time intervals of 10 min.
Supplementary Figure 6. Crystallization and diffraction of Vv-DHDPS crystals grown in the presence
of pyruvate and lysine. (A) Crystal of recombinant Vv-DHDPS in complex with lysine and pyruvate.
The approximate length of the crystal is 0.1 mm. (B) Single frame X-ray diffraction image from the
crystal of Vv-DHDPS shown in panel (A).
Supplementary Figure 7. X-ray structure of Vv-DHDPS in complex with lysine and pyruvate. (a)
Subunit structure of Vv-DHDPS. Active site residue Lys184 in complex with pyruvate is indicated in
yellow. (b) Overlay of Vv-DHDPS structures in the absence (PDB ID 3TUU) and presence (PDB ID:
4HNN) of lysine. Active site residues of Vv-DHDPS in the absence (blue) and presence (purple) of
lysine are indicated. The substrate, pyruvate, is shown in yellow.
Supplementary Figure 8. Omit map of the lysine-bound Vv-DHDPS structure (PDB ID 4HNN). A
close up of the allosteric cleft is shown depicting electron density due to the presence of the two bound
lysine molecules.
Supplementary Figure 9. Changes in the Vv-DHDPS allosteric site upon lysine binding. Shown are
residues (A) Trp78, (B) His 81, and (C) Tyr131 & Tyr132 in the absence (purple) and presence (cyan)
of lysine (green).
Supplementary Figure 10. Overlay of the Vv-DHDPS (purple) and E. coli DHDPS (green) allosteric
4
sites bound to lysine. Lysine is shown in orange.
5
Supplementary Figure 1
6
Supplementary Figure 2
7
Supplementary Figure 3
8
Supplementary Figure 4
9
Supplementary Figure 5
10
Supplementary Figure 6
11
Supplementary Figure 7
12
Supplementary Figure 8
13
Supplementary Figure 9
14
Supplementary Figure 10
15