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Profile of two carboxyosome
subunits of Synechocystis
Todd O. Yeates
Nam Tonthat
Goal

To profile two carboxysome hexamers of
Synechocystis, which were recently
structurally solved by the Yeates Lab.
Carboxysomes







Inclusion bodies
Present in cyanobacteria and many
chemoautotrophs
Filled with RuBisCO
RuBisCO fixes inorganic carbons (HCO3-,
CO2) to an organic form (PGA)
RuBisCO lacks specificity for CO2 and O2
RuBisCO is less efficient, compared to
other typical enzymes
Working hypothesis:

Carbon fixation can be optimized by localizing
RuBisCO in a CO2 rich environment
*Badger, Murray. Price, Dean. 2003
Carboxysome shells

Shell subunits are small,
homologous proteins which
form hexamers or pentamers


Hexamers/Pentamers form
sheets
Two such subunits in
Synechocystis:

ccmk2 and ccmk4
 A: ccmk2
 B: ccmk4
*Kerfeld, Sawaya, Tanka, Phillips, Beeby, Yeates 2005
ccmk2 & ccmk4


Overall folding of
ccmk2 & ccmk4 is
practically identical
Major structural
difference lies in the
orientation of the
fourth helix


Pro97 (ccmk4)
Glu97 (ccmk2)
*Kerfeld, Sawaya, Tanka, Phillips, Beeby, Yeates 2005
Red=>ccmk2
Blue=>ccmk4
Metabolites




Important metabolites: O2, CO2, HCO3RuBisCO is unable to distinguish between CO2 and O2
In Synechocystis and several other cyanobacteria:
 several mechanisms of transporting HCO3 and CO2
have been uncovered*
 HCO3 is the form accumulated in the cytoplasm*
 Mass spectrometry work has shown Carbonic
Anhydrase activity in carboxysome **
Carbonic Anhydrase is the enzyme used to convert
HCO3- to CO2
* Price and Badger 1989
**So and Espie1998
Hypothesis

The pore created by the ccmk2/ccmk4
hexamer serves as a gateway for molecules
to move through.
Steps for radius calculation
1.
2.
3.
4.
Starting with a position
inside the pore at a given
end
Find the largest sphere
that will fit within the
space bounded by
surrounding atoms
Exhaustively explore all
surrounding positions in
plane
Move through the pore
iteratively toward the
other side
Y
Z
position on z axis
-23
-21
-19
-17
-15
-13
-11
2.69 Å
-8.5
-6.4
-4.2
-2.1
0.01
8
7
6
5
4
3
2
1
0
2.14
4.26
6.39
8.51
10.6
12.8
14.9
17
radius in angstrom
Pore radius:
ccmk2
pore radius of ccmk2
3.39 Å
Y
Z
position on z axis
-17
-15
-13
-12
-10
-8.5
-6.8
-5.2
-3.6
1.77 Å
-2
-0.3
1.28
2.91
4.53
6
4
6.16
10
8
7.78
9.41
11
12.7
14.3
15.9
radius in angstrom
Pore radius:
ccmk4
pore radius of ccmk4
16
14
12
2.29 Å
2
0
Electrostatics calculation



“Ezprot”
 program written by Frank Petit
 wrapper for UHBD (University of Houston Brownian
Dynamics)
Used the Poisson Boltzmann equation to calculate the
potential
Describe electrostatic potential due to:



Non-homogeneous dielectric
Mobile counterions
“Fixed” (biomolecular) charge distribution
Closer look at the pore: ccmk2

Residues of the ccmk2 pore:

LYS36 ILE37 GLY38 SER39
*Kerfeld, Sawaya, Tanka, Phillips, Beeby, Yeates 2005
potential (mol/kcal*e)
-2
-4
-6
position on z axis
-25
-22
-19
-16
-13
-10
-7.3
-4.3
-1.3
1.69
4.68
7.68
10.7
13.7
16.7
-25
-23
-22
-20
-19
-17
-16
-14
-13
-11
-10
-8.5
-7
-5.5
-4
-2.5
-1
0.51
2.01
3.51
5.01
6.51
8.01
9.51
11
12.5
14
15.5
17
radius in angstrom
Charge distribution of the pore:
ccmk2
pore radius of ccmk2
8
7
6
5
4
3
2
1
0
position on z axis
potential of ccmk2 pore
10
8
6
4
2
0
Closer look at the pore: ccmk4

Residues of the ccmk4 pore:

ARG38 ALA39 GLY40 SER41
*Kerfeld, Sawaya, Tanka, Phillips, Beeby, Yeates 2005
-2
position on z axis
-8.51
-6.98
-5.46
-3.93
-2.41
-0.88
0.64
2.17
3.69
5.22
6.74
8.26
9.79
11.31
12.84
14.36
15.89
potential (kcal/mol*e)
position on z axis
potential of ccmk4 pore
12
10
8
6
4
2
0
-8.1
-7.1
-6.1
-5.1
-4.1
-3.1
-2.1
-1.1
-0.1
0.91
1.91
2.91
3.91
4.91
5.91
6.91
7.91
8.91
9.91
10.9
11.9
12.9
13.9
14.9
15.9
radius in angstrom
Charge distribution of the pore:
ccmk4
pore radius of ccmk4
9
8
7
6
5
4
3
2
1
0
Concluding remarks

It would be reasonable to speculate that the
pore is indeed a semi-selective gateway for
molecules to diffuse in and out based on


What we know about the components that are
inside and around the carboxysome
Information about the size and charge of the pore
Future work

There is still much to be done to fully elucidate the
bacterial microcompartment’s role in the carbon
dioxide concentrating mechanism



Internal organization of the carboxysome
Sequence of events that lead to the formation of the
carboxysome
Help to understand other microcomparments:*


Salmonella typhimurium, Klebsiella oxytoca, Escherichia coli
Presence of inclusion bodies when grown under anaerobic
conditions with either ethanolamine or propanediol as the
energy source
*Stojiljkovic I, Baumler AJ, Heffron F. 1995
Thank-you

SoCalBSI

Yeates Lab:


Dr. Yeates
Yingssu Tsai
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