Figure 1.
Schematic representation of heterotetrameric AGPase structure.
(A) Simplified two dimensional version of the heterotetrameric model of potato AGPase native structure. (B) Dimers heterotetrameric structure SS-LS.
Table 1.
Binding free energy components (kcal/mol) for each of the dimers averaged over the 200 snapshots.
Figure 2.
Snapshots of MD simulations from the final structures heterotetrameric.
AGPase. MD analyses indicate the hot-spot residues (A) in D2 and (B) in D5. LS is cyan and SS is yellow in color. Hot-spots are shown in spheres.
Table 2.
Free energy decomposition of hot spot residues in Dimer 1 (Values are in kcal/mol).
Table 3.
Free energy decomposition of hot spot residues in Dimer 2 (Values are in kcal/mol).
Figure 3.
H-bonds between the SS and LS AGPase.
(A) Snapshot showing the six H-bonds (red dashed lines) between Ile330-Ser312, Ile335-Ala317 and Ile340-Ser312 and their corresponding distances. These H-bonds are broken and reformed throughout the simulation. Ile322 is also illustrated in the picture. LS is shown cyan and SS is shown yellow in color. (B) Ribbon diagram of the interface region in the longitudinal dimer. Critical residues are highlighted.
Table 4.
ΔGbinding values of important residues in SS.
Table 5.
ΔGbinding values of important residues in single chain SS.
Figure 4.
Bacterial complementation assay using various mutants of the LS and the wildtype SS.
Iodine vapor staining of E. coli AC70R1-504 (glgC−) containing wild-type SS potato AGPase (pML10) and mutant or wild-type LS AGPase (pML7). The plate was streaked from a single colony of each strain onto a Kornberg's 2% glucose enriched plate and incubated overnight at 37°C. From A to C plates containing various mutants of the LS and the wildtype potato AGPases.
Table 6.
Functional analysis of selected hot-spot residues with comparison to backbone and total ΔGbinding energy values.
Figure 5.
Analysis of the interaction between the potato wildtype/mutants LS and the potato SS AGPase by yeast two hybrid.
The interaction between (A) SSWT and SSWT; (B) SSWT and LSWT; (C) SSWT and LSR88A; (D) SSWT and LSI330A; (E) SSWT and LSI339A,I330A vector. AH109 yeast cells expressing the designated plasmids are selected on a synthetic growth medium without Leu and Trp. Selections for interactions were carried out in the absence of Leu, Trp, and His.
Figure 6.
Heterotetrameric assembly of mutants and wildtype potato AGPases.
Western Blot analysis of various mutants of the LS and wild type SS. Top two panels belong native gels. 10 µg of total protein from crude extract were loaded on 3–13% native gradient gel and followed by western blot using anti-LS and anti-SS antibodies. Bottom two panels show western blot results from 10% SDS-PAGE using anti-LS and anti-SS antibodies.
Figure 7.
Presences of water molecule between the H-bonds.
Hydrogen bonding network between SS-I341 and LS-I340 provided by two water molecules trapped between the interfaces of D1.
Figure 8.
Aligment of the potato large subunit AGPase with various plant LS and with potato SS.
(A) Primary amino acids sequences alignment of various LS AGPase. Boxed amino acids play direct role with interaction of the SS AGPase. OS, Oryaza sativa; Hv, Hordeum vulgare; Pv, Phaseolus vulgaris; St, Solanum tuberosum, At, Arabidopsis thaliana; and Zm, Zea mays. (B) Comparison of primary amino acid sequence alignment of the potato AGPase LS and SS. Box indicated conserved amino acid residues that are important for the subunit-subunit interaction.
Table 7.
Oligonucleotide primers used for amplification of the LS cDNA and generation of site-directed mutations.