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1

Structural basis for the inhibition of human alkyladenine DNA glycosylase
(AAG) by 3,
N
4
-
ethenocytosine containing DNA

Gondichatnahalli M. Lingaraju
, C. Ainsley Davis
, Jeremy W. Setser
,
Leona D.
Samson

and Catherine L. Drennan


Supplemental

Additional
details on the e
xperimental
p
rocedures

AAG protein expression and purification

Both full
-
length and

79AAG and its mutants (N169L, N169A) were expressed and
purified using a similar protocol. Each pET19b
-
PPS AAG plasmid construct was
transformed into BL21(
DE3) cells by heat shock and plated onto LB (Luria Bertani) agar
plates, supplemented with 100

g/ml of ampicillin and incubated at 37

C overnight. A
single colony was used to inoculate 250 ml of LB broth supplied with 100

g/ml of
ampicillin and grown at
37

C overnight. This starter culture was used to inoculate 6 L of
LB broth supplied with 100

g/ml of ampicillin. The culture was grown at 37

C, until the
cells reached an optical density at 600 nm (OD
600
) equal to 1.0. The cultures were cooled
to room
temperature, and protein expression was induced with 0.5 mM IPTG for 6
-
8 hrs.
The cells were centrifuged at 4

C and 4000 rpm for 30 min and resuspended in 150 ml of
Buffer A (20 mM Potassium Phosphate pH 7.0, 500 mM NaCl, 10% v/v g1ycerol, 1 mM
DTT) suppli
ed with 4 tablets of EDTA
-
free protease inhibitor mixture and frozen at
-
80

C. The cells were thawed and lysed by sonication. The cell lysate was centrifuged at
18,000 rpm for 45 min and the supernatant was loaded onto a HisPrep


FF 16/10 Ni
-
affinity colum
n (Amersham Biosciences), which was pre
-
equilibrated with 10 column
volumes (CV) of Buffer A. The first wash was done with 10 CV of Buffer A, followed by
a wash with 10 CV of Buffer A containing 40 mM imidazole. The protein was eluted by
creating a 10 CV i
midazole gradient from Buffer A and Buffer B (Buffer A supplied with

2

500 mM imidazole).
Upon elution, the N
-
terminal 10X

histidine tag was cleaved from the
protein by precision protease (GE Healthcare Biosciences) treatment at 16

C for 12
-
14
hrs. This samp
le was diluted to the final NaCl concentration of 100 mM and loaded
directly onto a HiTrap


SP FF ion
-
exchange column (Amersham Biosciences). The
unbound protein was eluted using 10 CV of Buffer C (20 mM Hepes pH 7.5, 100 mM
NaCl, 10% glycerol, 5 mM DTT).
The bound protein was eluted using a gradient
between Buffer C and Buffer D (Buffer C supplemented with 1M NaCl), and
concentrated by centrifugation at 3500 rpm at 4

C using Amicon 10
-
KDCO (kilodalton
cut off) ultrafilters. Further purification was done b
y gel filtration using Buffer E (20
mM Hepes pH 7.5, 100 mM NaCl, 10% glycerol and 5 mM DTT) and a Superdex


75
gel filtration column (Amersham Biosciences). The final purified protein in Buffer E was
almost 99% pure as evidenced by the SDS
-
PAGE analysis.
The purified protein was
concentrated using Amicon 10
-
KDCO ultrafilters and the amount of protein was
estimated using the extinction coefficient method by UV absorption at 280 nm.

Preparation of oligonucleotides and
32
P
-
labeling

DNA oligonucleotide substra
tes (Integrated DNA Technologies) were dissolved in TE
buffer (10 mM Tris
-
HCl pH 8.0 and 1 mM EDTA) and quantified by the extinction
coefficient method using UV absorption at 260 nm. For the DNA glycosylase and
binding studies, the lesion
-
containing strand

was labeled on the 5


end with
32
P
-
γATP
(Perkin Elmer) using polynucleotide kinase (PNK) (New England Biolabs) at 37

C for 30
min, followed by heat inactivation of PNK at 70

C for 15 min. Duplex oligonucleotides
were created by annealing the
32
P
-
labeled s
trand with its complementary strand. The

3

unincorporated
32
P
-
γATP was separated from
32
P
-
labeled oligonucleotides using
Sephadex
G
-
25 quick spin columns (
Amersham
-
Pharmacia)
.

Crystallization of the

79AAG
-

C:G complex

The

C:G DNA duplex was prepared by annealing the

C containing 13
-
mer
crystallization oligonucleotide (5′
-
GAC ATG

C
TT GCC T
-
3′) with
its

complementary
strand that contained G opposite

C (5′
-
GGC AA
G
CAT GTC A
-
3′). The

79AAG
-

C
complexes were prepared by mixi
ng equimolar ratios of

79AAG and

C:G 13
-
mer
DNA duplex at the final protein
-
DNA complex concentration of 0.3 mM in the complex
buffer (20 mM Hepes
-
NaOH pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 5% v/v glycerol
and 1 mM DTT). The complex was incubated on ice for

15 min and used for
crystallization. The crystals were obtained by the hanging drop vapor diffusion method,
upon mixing 1

l of complex and 1

l of the reservoir solution (100 mM sodium
cacodylate pH 6.0, 200 mM manganese chloride and 20% polyethylene gly
col (PEG)
-
3350) over 0.5 ml of the reservoir solution, followed by incubation for 2 days at 22

C.
The crystals formed as plates, which were mounted directly from the hanging drop to the
center of loops larger than the crystal size and flash frozen in liqui
d nitrogen.

Data collection and structure determination


The X
-
ray diffraction data for the

79AAG
-

C:G complex were collected at the
Advanced Light Source (Berkeley, CA) on beamline 12.3.1 at 100K to 2.2 Å resolution.
These data

were processed using Denz
o/Scalepack
(
1
)

and the data statistics are given in
Table 2. The structure of the

79AAG
-

C:G, with 2 molecules in the asymmetric unit,
was determined by molecular replacement using PHASER
(
2
)

and the AAG protein
coordinates from the

79AAG
-
pyr
:T complex structure (PDB ID 1BNK) as a search

4

model
(
3
)
. A 2m|F
o
|
-
D|F
c
| electron density map (all maps having coefficients from
sigmaA weighting) contoured at 1

, and a m|F
o
|
-
D|F
c
| electron density map contoured at
3

, calculated in the absence of DNA, sh
owed an interpretable electron density map for
the DNA backbone. Another round of refinement was performed upon partial fitting of
the DNA (7 nucleotide pairs starting at 5
'
-

4
ATG(
pyr
)TTG
10

-
3
'
and the complementary
strand 5'
-

16
CAAGCAT
22

-
3
').
The resulti
ng calculated 2m|F
o
|
-
D|F
c
| (1

) and m|F
o
|
-
D|F
c
|
(3

) electron density maps showed interpretable electron density for the

C base in the
active site pocket of AAG, as well as continuous electron density for building the
remaining DNA backbone. Upon fitting
the missing DNA portions into the electron
density, initial models were subjected to restrained refinements using Refmac 5.4
(
4, 5
)
.
Topology and parameter files for the

C lesion were generated using XPLO2D
(
6
)
. Initial
refinement included simulated annea
ling in CNS
(
7
)
. Iterative rounds of positional and B
-
factor refinement of the

79AAG
-

C complexes were performed with the guidance of
calculated 2m|F
o
|
-
D|F
c
|, m|F
o
|
-
D|F
c
| electron density maps (generated by Refmac 5.4), and
2m|F
o
|
-
D|F
c
| composite omit
-
maps (generated by CNS), using the model building
program Coot
(
8
)
. Anomalous difference maps were calculated in CNS (
7
) using the
native data (λ = 1.116 Å) and the phases from the final

79AAG
-

C:G model. Additional
rounds of refinement u
sing TLS parameters and non
-
crystallographic restraints were very
effective in improving the quality of the fit. At each stage, the progress of model building
was judged by following the change in R factors. The final model of

79AAG
-

C:G
complex converged

at an R factor of 23.9 (R
free
= 28.4) (Table 2). The final model was
evaluated using PROCHECK
(
9
)

and Rampage
(
10
)
. For residues 80
-
298 of the

79AAG
-

C:G structure, the following have no electron density and are therefore not

5

included in the model: the r
esidues 203
-
207, 265
-
268 and 295
-
298 in chain A; the
residues 205
-
206, 265
-
266 and 294
-
298 in chain B. Due to a lack of interpretable electron
density for the side chains of some residues in the structure of

79AAG
-

C:G (R201,
L249 and E253 in chain A), th
ese residues were modeled as alanines. Each protein
molecule in the asymmetric unit has a DNA 13
-
mer associated with it. One nucleotide of
each of these duplexes is disordered (A26).



















6


Supplemental Figure
Legends

Figure
S1
: Gel results of DNA glycosylase assays for truncated

79AAG on

A:T and

C:X (X=G/A/T/C) 13
-
mer DNA duplexes used for crystallization.


Figure

S2
: Comparison of the overall structures of the

79AAG
-
DNA complexes. The
superposition of AAG
-

C:G (green) and
AAG
-

A:T (gray) (PDB ID 1F4R;
(
11
)
)
complexes with the carbons of the flipped

C nucleotide colored yellow, and the Tyr162
side chain indicated with an arrow.
Regions that show differences with respect to
disordered loops are indicated by dashed ellipses.

The region 1 (left) corresponds to the
residues Gly265
-
Gly268; and region 2 (right) corresponds to the residues Leu249
-
Pro254.


Figure

S3
: Cation site in

79AAG structures
.
A
,

In the structure of the

79AAG
-

A:T
substrate complex (PDB ID 1F4R (11)), a Na
+

ion (purple sphere) is coordinated by AAG
(carbons in gray) and a water molecule (red sphere). Oxygens are in red, nitrogens in

blue, and sulfurs in yellow.
B
,

In the structure of

79AAG
-

C:G, Na
+

is replaced by the
N
-
terminal NH
3
+

of Gly80 from a symmetry

related molecule (carbons in cyan) in
interacting with the

79AAG molecule (carbons in green). All the residues in cyan,
Gly80, Pro81 and His82 (along with Met83 not shown in the figure), are part of the
precision protease cleavage site sequence and do no
t represent the wild type AAG
sequence.
Non
-
carbon atoms colored as in
A
. For both
A

and
B
, hydrogen bonds are
indicated by dashed lines with distances given in Ångströms (Å).


7


Figure S4
: Interaction of Tyr162 and putative Mn
2+

binding site in the structu
re of

79AAG
-

C:G.
A
,

2m|F
o
|
-
D|F
c
| omit electron density map contoured at 1


(gray) is
drawn around the Mn
2+

ion (orange sphere) and the DNA bases (A18 and G19) that
coordinate the metal ion. An anomalous difference electron density map calculated with
the native dataset (λ = 1.116 Å) contoured at 8


(magenta) shows a strong positive peak
for an anomalous scatte
rer such as Mn
2+

(chain A site depicted)
. Water molecules that
coordinate the Mn
2+

ion are not shown. Carbons are in yellow and non
-
carbon a
toms are
colored as in Figure S3.
B
,

Difference in the sugar pucker and conformation of DNA
backbone near the Mn
2+

i
on in the structure of

79AAG
-

C:G compared to

79AAG
-

A:T (carbons in gray) (PDB ID 1F4R
(
11
)
). All o
ther atoms are colored as in
A
.

C
,

The
Tyr162 intercalation site is near a Mn
2+

ion and Met164 (sulfur atom in yellow), which
both interact with G19 by us
ing hydrogen bonding and van der Waals interactions
respectively. Protein carbons are in green and all o
ther atoms are colored as in
A
.


Figure

S5
: AAG active site architecture. A side

(
A
)

and top (
B
)

view of

C in the

79AAG
-

C:G structure with van der W
aal
s

surfaces shown in gray spheres. DNA and
protein carbons are in yellow and green respectively. Oxygens are in red, nitrogens in
blue, sulfur in yello
w, and phosphorus in orange.
C
,

2m|F
o
|
-
D|F
c
| omit electron density
map contoured at 1


(gray) is drawn only around

C:G containing DNA and a putative
catalytic water molecule (red spher
e). Atoms are colored as in
A

and
B
.



8

Figure

S6
: A wall
-
eyed stereoview of the active site of AAG. 2m|F
o
|
-
D|F
c
| omit electron
density map contoured at 1


(gr
ay) is around

C:G containing DNA, active site protein
residues and a putative catalytic water molecule. A
toms are colored as in Figure S5
.

Supplemental References

1.

Otwinowski, Z. , and Minor, W. (1997)
Methods Enzymol.

276
, 307
-
326

2.

McCoy, A. J. , Grosse
-
Kunstleve, R. W. , Adams, P. D. , Winn, M. D. , Storoni, L. C. ,
and Read, R. J. (2007)
J. Appl. Crystallogr.

40
, 658
-
674

3.

Lau, A. Y. , Scharer, O. D. , Samson, L.D. , Verdine, G. L. , and Ellenberger, T. (1998)
Cell

95
, 249
-
258

4.

Collaborative Computational Project, Number 4. (1994)
Acta Crystallogr., Sect. D: Biol.
Crystallogr.

50
, 760
-
763

5.

Murshudov, G. N. , Vagin, A. A. , and Dodson, E. J. (1997)
Acta Crystallogr., Sect. D:
Biol. Crystallogr.

53
, 240
-
255

6.

Kleywegt, G. J. , Henrick, K. , Dodson, E. J. , and van Aalten, D. M. (2003)
Structure

11
,
1051
-
1059

7.

Brunger, A. T. , Adams, P. D. , Clore, G. M. , DeLano, W. L. , Gros, P. , Grosse
-
Kunstleve, R. W. , Jiang, J. S. , Kuszewski, J. , Nilges, M. , Pannu, N
. S. , Read, R. J. ,
Rice, L. M. , Simonson, T. , and Warren, G. L. (1998)
Acta Crystallogr., Sect. D: Biol.
Crystallogr.

54
, 905
-
921

8.

Emsley, P. , and Cowtan, K. (2004)
Acta Crystallogr., Sect. D: Biol. Crystallogr.

60
,
2126
-
2132

9.

Laskowski, R. , MacA
rthur, M.W. , Moss, D. S. , and Thornton, J. M. (1993)
J. Appl.
Crystallogr.

26
, 283
-
291

10.

Lovell, S. C. , Davis, I. W. , Arendall III , W. B. , de Bakker, P. I. W. , Word, J. M. ,
Prisant, M. G. , Richardson, J. S. , and Richardson, D. C. (2003)
Protein
s

50
, 437
-
450


11.

Lau, A. Y. , Wyatt, M. D. , Glassner, B. J. , Samson, L. D. , and Ellenberger, T. (2000)
Proc. Natl. Acad. Sci. U.S.A

97
, 13573
-
13578