Colleen Kao, Seon-Kap Hwang, Thomas W. Okitareu.mme.wsu.edu/2011/files/19.pdf · 2011. 9. 7. ·...

1
Determining the crystal structure of ADP-glucose pyrophosphorylase through the purification and characterization of mutant forms Colleen Kao, Seon-Kap Hwang, Thomas W. Okita 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 3- dimensional 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. 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 (6X- His-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). 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). 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 Acknowledgements 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. 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): 15691576 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 Abstract Background ADP-glucose Starch or glycogen synthases -1,4-glucan n Glucose 1-P PPi ATP AGPases 2Pi 3-PGA / PEP / F6P activation inhibition Photosynthesis -1,4-glucan n+1 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. LS SS LS SS SS SS SS SS S 302 N If LS is absent Oligomerization mutant Natural Oligomerization LS * LS * LS * LS * E447 E448 K133 K134 K245 K244 SS LS MUT-1 K41R, T51K, S302N K133A, K134A K244A, K245A MUT-2 K133A, K134A K244A, K245A E447A, E448A Site-directed mutagenesis of AGPase large subunit LS SS LS SS TEV LS SS LS SS LS SS LS SS TEV TEV IMAC Flow through G Wild type MBP Tag Wild type MUT-1 MBP Tag MUT-1 MUT-2 MBP Tag MUT-2 6xhis tag TEV cleavage site MBP Tag Control 1 2 3 4 5 6 MUT-1 K41R, T51K, S302N K133A, K134A K244A, K245A MUT-2 K133A, K134A K244A, K245A E447A, E448A M MBP-GFP + wtSS MBP-wtLS + wt SS MBP-MUT1 + wt SS MBP-MUT2 + wt SS M MBP-GFP + wtSS MBP-wtLS + wt SS MBP-MUT1 + wt SS MBP-MUT2 + wt SS Total proteins Soluble proteins (kDa) 170 130 95 72 55 43 34 26 MBP-LS SS Conclusions Future Work Grow E. coli cells (glgC - ) transformed with plasmid DNAs expressing mutant forms of AGPase Harvest and lyse cells and collect soluble proteins Purify proteins using three chromatography columns Cleave 6his-MBP tag and purify AGPase through a TALON- IMAC 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. Column 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 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. Figure 3. Subunit oligomerization of the potato tuber AGPase Figure 4. Surface Entropy Reduction predictions Data analysis on the SERp server proposed six candidate residues for site-directed mutagenesis. 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 . 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. TEV TEV protease MBP Tag (kDa) M 2 3 4 5 6 7 8 9 TALON-Immobilized Metal Affinity Column Chromatography Imidazole (0 to 0.1 M) CE FT 8 10 12 14 16 18 20 22 24 26 MW NaCl (0 > 0.5 M) DEAE-Sepharose FF Ion Exchange Column Chromatography 6His 170 130 95 72 55 43 34 26 170 130 95 72 55 43 34 26 (kDa) 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. AGPase MBP-GFP (Lanes 3 & 9)

Transcript of Colleen Kao, Seon-Kap Hwang, Thomas W. Okitareu.mme.wsu.edu/2011/files/19.pdf · 2011. 9. 7. ·...

Page 1: Colleen Kao, Seon-Kap Hwang, Thomas W. Okitareu.mme.wsu.edu/2011/files/19.pdf · 2011. 9. 7. · Colleen Kao, Seon-Kap Hwang, Thomas W. Okita ADP-glucose pyrophosphorylase (AGPase)

Determining the crystal structure of ADP-glucose pyrophosphorylase through the purification and characterization of mutant forms

Colleen Kao, Seon-Kap Hwang, Thomas W. Okita

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 3-dimensional 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.

Background

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 asheterotetrameric structures consisting of cooperating two large and twosmall subunits (Hwang et al., 2008; Fig. 3). The crystal structure of thenative enzyme has not been elucidated, although a homotetrameric formconsisting 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 alaninebased on Surface Entrophy Reduction concepts (Goldschmidt et a., 2007;Fig. 4 and 5). However, we encountered a problem in protein solubility forthe mutant enzymes. In contrast to the wildtype AGPase, the mutantproteins containing the mutant L subunits and wildtype S subunits becameinsoluble after expression. Thus, to increase solubility of the mutatedAGPase protein, we attached a 6xHis tagged maltose binding protein (6X-His-MBP) containing a tobacco etch virus (TEV) proteinase cleavage site tothe amino terminus of the L subunit (Fig. 6). This strategy will allow us topurify large quantities of AGPase protein variants without any affinity tags,a condition required for identifying the optima conditions for crystallizationof AGPase proteins (Fig. 7).

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).

• 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

AcknowledgementsThis 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.

ReferencesBallicora et a. (2005) Resurrecting the ancestral enzymatic role of a modulatory subunit. J. biol. Chem. 280(11):10189-10195Goldschmidt, L. et al. (2007) Toward rational protein crystallization: A Web server for the design of crystallizable protein variants. Protein Sci. 16(8): 1569–1576Hwang, 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):6640-6647Jin et al. (2005) Crystal structure of potato tuber ADP-glucose pyrophosphorylase. EMBO J. 24(4): 694-704

Abstract

Background

ADP-glucose

Starch or glycogen synthases

-1,4-glucann

Glucose 1-P

PPi

ATP

AGPases

2Pi

3-PGA / PEP / F6P

activation

inhibition

Photosynthesis

-1,4-glucann+1

Figure 1. AGPase in -1,4-polyglucans synthesisSchematic 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.

LS

SS LS

SS

SS

SS SS

SS

S302N

If LS is absent

Oligomerizationmutant

NaturalOligomerization

LS* LS*

LS*LS*

E447E448

K133

K134

K245

K244

SS

LS

MUT-1

K41R, T51K, S302N K133A, K134A K244A, K245A

MUT-2

K133A, K134A K244A, K245A E447A, E448A

Site-directed mutagenesis of AGPase large subunit

LS

SS LS

SS

TEV

LS

SS LS

SSLS

SS LS

SS

TEV

TEV IMA

C

Flow through

G

Wild type MBP Tag

Wild type

MUT-1MBP Tag

MUT-1

MUT-2MBP Tag

MUT-2

6xhis tag

TEV cleavage site

MBP Tag Control

1

2

3

4

5

6MUT-1K41R, T51K, S302N K133A, K134A K244A, K245A

MUT-2K133A, K134A K244A, K245A E447A, E448A

M MB

P-G

FP +

wtS

S

MB

P-w

tLS

+ w

tSS

MB

P-M

UT1

+ w

tSS

MB

P-M

UT2

+ w

tSS

M MB

P-G

FP +

wtS

S

MB

P-w

tLS

+ w

tSS

MB

P-M

UT1

+ w

tSS

MB

P-M

UT2

+ w

tSS

Total proteins Soluble proteins

(kDa)

170

130

95

72

55

43

34

26

MBP-LS

SS

Conclusions

Future Work

Grow E. coli cells (glgC-) transformed with plasmid DNAs expressing mutant forms of AGPase

Harvest and lyse cells and collect soluble

proteins

Purify proteins using three chromatography

columns

Cleave 6his-MBP tag and purify AGPase through a TALON-

IMAC

Figure 2. Overview of methodologyThe 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.

Column

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 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.

Figure 3. Subunit oligomerization of the potato tuber AGPase

Figure 4. Surface Entropy Reduction predictionsData analysis on the SERp server proposed six candidate residues for site-directed mutagenesis.

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 .

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.

TEV

TEV proteaseMBP Tag

(kDa) M 2 3 4 5 6 7 8 9

TALON-Immobilized MetalAffinity Column Chromatography

Imidazole (0 to 0.1 M)

CE

FT 8 10

12

14

16

18

20

22

24

26

MW

NaCl (0 > 0.5 M)

DEAE-Sepharose FF Ion Exchange Column Chromatography

6His

170 130

95

72

55

43

34

26

170 130

95

72

55

43

34

26

(kDa)

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.

AGPase

MBP-GFP (Lanes 3 & 9)