Determining the crystal structure of ADP-glucose pyrophosphorylase
through the purification and characterization of mutant forms
Colleen Kao, Seon-Kap Hwang, Thomas W. Okita
Abstract
If LS is absent
SS
3-PGA / PEP / F6P
S302N
LS* LS*
Oligomerization
mutant
LS* LS*
9
55
43
TEV protease
AGPase
6His
Figure 8. Purification of 6His-MBP-TEV-LS by DEAE-Sepharose FF and Talon-IMAC chromatography as
viewed by SDS-PAGE .
CE = soluble cell extract; FT = flow through; M = protein size marker; Fractions #8 through #10 had
significant amounts of TEV protease. Fractions #7 through #11 from DEAE- Sepharose FF column were
combined and then immediately purified through the TALON-IMAC column.
Figure 4. Surface Entropy Reduction predictions
Data analysis on the SERp server proposed six candidate
residues for site-directed mutagenesis.
TEV
TEV
TEV
SS
K41R, T51K,
S302N
K133A, K134A
K244A, K245A
LS
SS
SS
LS
LS
SS
SS
LS
LS
SS
SS
LS
TEV
Flow through
MUT-2
Column
K133A, K134A
K244A, K245A
E447A, E448A
K244
K245
K134
LS
Figure 9. Schematic diagram for purification of AGPase after removal of 6His-MBP with 6His-TEV protease.
The AGPase variants containing 6His-MBP-LS mutant and wildtype SS is treated with the purified 6His-TEV
protease . The protease cuts at the TEV protease cleavage site on the L subunit. The AGPase is then purified
by using a IMAC chromatography in which 6His-MBP and 6His-TEV protease are trapped .
K133
E448 E447
Figure 5. In vitro site-directed mutagenesis was performed to substitute six selected residues with alanine on
the surface of the potato tuber AGPase.
Two L subunit mutants (MUT-1 and MUT-2) were generated by site-directed mutagenesis. LS, L subunit; SS, S
subunit.
Control
MBP Tag
G
Wild type
Wild type
MBP Tag
MUT-1
MUT-1
MUT-2
MBP Tag
MUT-2
K133A, K134A
K244A, K245A
E447A, E448A
1
2
3
4
5
6
6xhis tag
TEV cleavage site
95
MBP-LS
MBP-GFP (Lanes 3 & 9)
55
43
An algorithm for Surface Entrophy Reduction predicted 6 candidate amino acid
residues for substitution to improve crystallization of AGPase LS protein (Fig. 5).
The L subunits containing all or part of the substitutions resulted in partial loss
of activity on the basis of glycogen production in E. coli cells. Since the mutant
forms of the AGPase heterotetramer exhibited very low solubility after
heterologous expression of the proteins in E. coli, we modified the L subunit to
obtain more soluble proteins. The attachment 6His-MBP tag to the L subunit
mutants of the potato tuber AGPase did not appear to reduce or disable the
activity of the enzyme on the basis of glycogen production (Fig. 6). Significant
amount of AGPase protein was observed in soluble fractions (Fig. 7).
Purification conditions of the MBP-tagged AGPase proteins are currently being
optimized. In addition, 6His-tagged TEV protease were purified to near
homogeneity for this study. The TEV protease along with the MBP tag could be
easily removed from the AGPase solution after digestion of the MBP-tagged
AGPase by trapping those proteins by using IMAC chromatography (Fig. 9).
•
Future Work
Acknowledgements
170
130
72
Conclusions
• Purify a large amount of 6His-MBP-AGPase proteins to near homogeneity.
Cleave the 6His-MBP tag off with TEV protease to further purify AGPase
protein (Fig. 9)
• Optimize crystallization conditions for the AGPase proteins
MBP-MUT2 + wt SS
MBP-MUT1 + wt SS
MBP-wtLS + wt SS
MBP-GFP + wtSS
Soluble proteins
M
MBP-MUT2 + wt SS
MBP-MUT1 + wt SS
MBP-wtLS + wt SS
M
MBP-GFP + wtSS
Total proteins
IMAC
Figure 3. Subunit oligomerization of the potato tuber
AGPase
(kDa)
Figure 2. Overview of methodology
The proteins were purified on a DEAE-Sepharose FF Column, a TALON-IMAC column, and finally a POROS 20
HQ column. To complete purification of the protein, the 6xHis -MBP tag is removed by treating the enzyme
with TEV protease followed by purification on TALON-IMAC.
8
72
26
MBP Tag
Starch or
glycogen
synthases
Cleave 6his-MBP tag
and purify AGPase
through a TALONIMAC
7
95
34
Figure 6. Iodine staining of E. coli cells co-expressing wildtype S subunit in combination with wildtype or mutant
L subunits of the potato tuber AGPase with or without a 6His-MBP-TEV protease cleavage site tag.
No significant differences in glycogen accumulation (AGPase activity) was detected in cells expressing AGPases
with and without 6His-MBP-TEV protease cleavage site tag.
Figure 1. AGPase in -1,4-polyglucans synthesis
Schematic diagram of biosynthetic pathway of -1,4-polyglucans such as starch and glycogen. AGPase
catalyses the synthesis of ADP-glucose which is a precursor for starch and glycogen synthases.
6
5
4
3
2
MW
26
M
170
130
26
ADP-glucose
Purify proteins using
three chromatography
columns
24
34
-1,4-glucann+1
Harvest and lyse cells
and collect soluble
proteins
22
43
MUT-1
K41R, T51K, S302N
K133A, K134A
K244A, K245A
-1,4-glucann
Grow E. coli cells
(glgC-) transformed
with plasmid DNAs
expressing mutant
forms of AGPase
20
55
(kDa)
MUT-1
PPi 2Pi
activation
inhibition
18
SS
MUT-2
Glucose 1-P
16
72
MBP Tag
ATP
(kDa)
95
Site-directed mutagenesis of
AGPase large subunit
Plants and bacteria utilize the enzyme ADP-glucose pyrophosphorylase
(AGPase) as the first step in the production of their carbon and energy 1,4-polyglucan reserves (Fig. 1). AGPases from higher plants function as
heterotetrameric structures consisting of cooperating two large and two
small subunits (Hwang et al., 2008; Fig. 3). The crystal structure of the
native enzyme has not been elucidated, although a homotetrameric form
consisting of the potato tuber AGPase small subunits has been determined
(Jin et al., 2005). In order to improve crystallization of the native enzyme,
we substituted several amino acid residues of the L subunits with alanine
based on Surface Entrophy Reduction concepts (Goldschmidt et a., 2007;
Fig. 4 and 5). However, we encountered a problem in protein solubility for
the mutant enzymes. In contrast to the wildtype AGPase, the mutant
proteins containing the mutant L subunits and wildtype S subunits became
insoluble after expression. Thus, to increase solubility of the mutated
AGPase protein, we attached a 6xHis tagged maltose binding protein (6XHis-MBP) containing a tobacco etch virus (TEV) proteinase cleavage site to
the amino terminus of the L subunit (Fig. 6). This strategy will allow us to
purify large quantities of AGPase protein variants without any affinity tags,
a condition required for identifying the optima conditions for crystallization
of AGPase proteins (Fig. 7).
AGPases
14
SS
Background
Background
Photosynthesis
Imidazole (0 to 0.1 M)
170
130
SS
TALON-Immobilized Metal
Affinity Column Chromatography
NaCl (0 > 0.5 M)
12
LS
10
SS
8
SS
FT
Natural
Oligomerization
LS
CE
ADP-glucose pyrophosphorylase (AGPase) is a regulatory enzyme involved
in the production of α-glucan reserves, glycogen in bacteria and starch in
higher plants (Fig. 1). Unlike the simple single subunit tetramer
arrangement of the bacterial AGPase, the higher plant enzyme is
composed of two large (L) subunits and two small (S) subunits (Fig. 3).
Studies of mutant forms of either the large or small subunits or in
combination indicate that the two types of subunits work cohesively
instead of separately to regulate α-glucan production. Despite
considerable effort, attempts to obtain crystals and, in turn, the 3dimensional structure of the higher plant enzyme have failed. Such
knowledge, if available, would enable us to rationally manipulate the
catalytic activity for enhanced plant productivity. Several plasmid DNAs
coding for mutant AGPase enzymes, which may be more amenable to
crystallization, are available (Fig. 6). Here I will show results of my efforts
to express these enzymes in Escherichia coli and the purification of these
enzymes to obtain a highly purified preparation, a condition required for
crystallization studies.
DEAE-Sepharose FF
Ion Exchange Column Chromatography
SS
This project could not have been possible without support from Dr. Seon-Kap Hwang, Dr.
Thomas Okita, the rest of the Okita Lab, along with Washington State University, and the
encouragement of Dr. Christopher Meyer, Dr. Chandra Srinivasan and the Srinivasan Lab. This
work was supported by the National Science Foundation Plant Genome Grant DBI-0605016.
34
26
Figure 7. Induction of large quantities of soluble MBP-tagged AGPase large subunit (MBP-LS) as viewed by
SDS-polyacrylamide gel electrophoresis.
M = protein size marker; MBP-tagged green fluorescence protein (GFP) along with wild type SS were used as a
control.
References
Ballicora et a. (2005) Resurrecting the ancestral enzymatic role of a modulatory subunit. J. biol. Chem.
280(11):10189-10195
Goldschmidt, L. et al. (2007) Toward rational protein crystallization: A Web server for the design
of crystallizable protein variants. Protein Sci. 16(8): 1569–1576
Hwang, S. K. et al. (2008) Direct appraisal of the potato tuber ADP-glucose pyrophosphorylase large
subunit in enzyme function by study of a novel mutant form. J. Biol. Chem. 283(11):6640-6647
Jin et al. (2005) Crystal structure of potato tuber ADP-glucose pyrophosphorylase. EMBO J. 24(4): 694-704