Supplementary Information
Arginine 122 and Asparagine 111 Play a Pivotal Role in the
Interaction of Perfluoroalkyl Acids with Human Liver Fatty
Acid Binding Protein
Nan Sheng, Juan Li, Hui Liu, Aiqian Zhang*, and Jiayin Dai*
Overexpression and purification of wild type human liver fatty acid binding
protein (hL-FABP) and its variants. Full-length hL-FABP was cloned into the
pET-28a vector between BamH I and Xho I restriction sites. To obtain recombinant
protein consisting of 127 amino acids in accordance with natural hL-FABP, we
inserted a 5 thrombin restriction enzyme cutting site before the target gene and
mutated the original thrombin restriction enzyme cutting site (CGC to CCC) in the
pET-28a vector. The insert sequence was confirmed by DNA sequencing. The
resultant plasmid was transformed to the BL21 (DE3) strain of Escherichia coli.
Following IPTG (1 mM) induction at a cell density of 0.6, the recombined protein was
overexpressed for 18 h at 23°C. Bacterial cells were lysed by sonication in a buffer
containing 1 mM PMSF and 1 mM DTT. Affinity purification was performed on a
HisTrap HP column on an AKTA FPLC system (GE Healthcare). Fractions
containing hL-FABP were pooled and brought to 65% saturation with ammonium
sulfate at 4°C. Delipidation was achieved by HIC on a HiTrap Phenyl HP column. A
6×His tag was cleaved using thrombin protease in buffer A (50 mM
Na2HPO4/NaH2PO4, 10% Glycerin, pH 6.5) at 20°C overnight. Uncleaved protein and
thrombin protease were removed by passing through a HisTrap HP and a HiTrap
benzamidine HP column on an AKTA FPLC system (GE Healthcare). The cleaved
protein was further purified on a Superdex-75 column (GE Healthcare) in buffer B
1
(20 mM Na2HPO4/NaH2PO4, 5% glycerin, pH 7.4). The protein was concentrated
using 3 kDa Amicon® Ultra Filters (Millipore). Protein concentration was determined
by the bicinchoninic acid method.
Fluorescence binding assays. Fluorescence emission spectra were measured on a
Horiba Fluoromax-4 spectrofluorometer (Edison, NJ, USA) in buffer C (50 mM
Tris-HCl, pH 8.0) at room temperature under steady-state conditions. Fluorometric
titrations of 1,8-ANS into hL-FABP and PFAAs displacement were performed as
described previously (Carbone and Velkov, 2013), with an adjustment in protein
concentration (0.25 M). The binding of 1,8-ANS to hL-FABP was monitored by
measuring the fluorescence signal between 410 and 550 nm following excitation at
392 nm. The binding of various ligands to hL-FABP was measured by the
displacement of bound 1,8-ANS from the protein. Briefly, hL-FABP (0.25 M, 100
l) in buffer C was allowed to equilibrate with 1,8-ANS (12.5 M) for 2 min prior to
fluorescence measurements. Freshly prepared PFAA in buffer C (10 l) was added
into the hL-FABP-1,8-ANS sample (90 l) and incubated for 2 min before the
fluorescence intensity was measured. The displacement of the protein-bound probe
was calculated from the decrease in fluorescence intensity with increasing
concentrations of PFAA. The maximal fluorescence emission plot for 1,8-ANS was
conducted to obtain Kd
probe
(dissociation constant) using GraphPad Prism V5.0
(GraphPad software, San Diego, CA, USA). The competition curves for PFAAs were
fitted with a sigmoidal model (OriginLab, Northampton, MA, USA) to derive an IC50
value. The inhibition constant (Ki ligand) values for PFAAs were calculated according
to the equation:
IC50 ligand/[Probe]total = Ki ligand/Kd probe
Where Kd probe is the measured Kd for 1,8-ANS obtained in the direct binding assay,
and [Probe]total is the total concentration of 1,8-ANS.
Supplementary Tables and Figures
Table legends:
Table S1: Effect of PFAAs on the relative proportion of the secondary structure
2
components of WT hL-FABP and its four variants as determined by CDa.
Table S2: Concentrations of hL-FABPs and PFAAs for ITC experiments.
Table S3: Thermodynamic parameters for the binding of PFHxS to wild type
hL-FABP and its four variants as determined by ITC at 25°C.
Figure Legends
Figure S1. CD spectra of different concentrations of PFAA added to 5 μM WT
hL-FABP and its four variants (S39G, M74G, N111D, and R122G) respectively. (A)
PFHxA; (B) PFHxS; (C) PFNA.
Figure S2. (A) (a) Fluorescence emission spectra of 0.25 M WT hL-FABP titrated
with increasing concentrations of 1,8-ANS. Arrow indicates increasing concentrations
of 1,8-ANS (0~15 μM). (b) Linear plot of the binding curve for 1,8-ANS. (B, C)
Relative fluorescence intensity of 12.25 M 1,8-ANS in 0.25 M hL-FABP and its
variant (S39G and M74G) as a function of added PFAA concentrations. (A) PFOA; (B)
PFNA. Values represent means ± S.E., n = 3.
Figure S3. ITC profiles for the binding of PFAA to WT hL-FABP and its four variants
(S39G, M74G, N111D, and R122G) at 25°C. (A) PFHxA; (B) PFHxS; (C) PFNA.
Figure S4. Structure of the active site in LFABP (PDB ID: 3STK). Pictured are the
key amino acids surrounded by the buried active pocket within a distance of 5Å. (A),
outer binding site occupied with PFOA; (B), inner binding site occupied with PFOA.
The hydrogen bonds are shown by green dashed lines. Oxygen, fluorine, nitrogen and
carbon atoms are displayed in red, green, blue, and black, separately.
Figure S5. ITC profiles for the binding of OA to WT hL-FABP and its four variants.
3
Table S1. Effect of PFAAs on the relative proportion of the secondary structure
components of WT hL-FABP and its four variants as determined by CDa.
PFAAs (μM)
WT
PFHxA
PFOA
M74G
N111D
R122G
α-helices
β-sheet
α-helices
β-sheet
α-helices
β-sheet
α-helices
β-sheet
α-helices
β-sheet
(%)
(%)
(%)
(%)
(%)
(%)
(%)
(%)
(%)
(%)
17.1
50.3
16.9
50.4
16.6
51.2
17.5
49.1
15.3
51.5
10
17.3
50.8
16.9
50.7
16.7
50.9
17.4
49.3
15.3
51.7
50
18.2
50.0
17.3
50.7
16.9
50.8
17.6
49.3
15.3
51.4
10
17.2
50.2
17.1
50.2
16.5
51.0
17.5
48.8
14.9
51.8
50
17.2
50.3
17.1
49.8
16.4
50.8
17.6
50.3
15.2
51.6
10
17.0
48.5
17.1
50.0
16.7
50.9
17.7
48.9
15.3
51.1
50
17.1
47.1
17.3
48.9
16.8
49.7
18.0
48.4
14.9
51.3
17.7
47.6
17.8
47.2
17.4
48.4
18.5
47.2
15.5
49.5
10
17.1
49.3
17.0
49.7
16.7
50.6
17.5
48.6
15.2
51.2
50
17.6
48.3
17.6
49.1
17.0
49.4
18.1
48.7
15.1
50.5
18.4
47.4
18.3
48.0
18.0
48.3
18.5
47.1
15.1
50.8
No PFAAs
PFHxS
S39G
20
0
PFNA
20
0
a
All data were determined by adding various concentrations of PFAA (μM) to 5 μM
hL-FABP in 20 mM NaH2PO4/Na2HPO4 (pH 7.4).
4
Table S2. Concentrations of hL-FABPs and PFAAs for ITC experiments.
Complex
PFOA: hL-FABP
PFNA: hL-FABP
Protein
Ligand
(μM)
(mM)
WT/S39G/M74G
100
1.3
13
R122G
100
0.65
6.5
N111D
50
2
40
WT/S39G/M74G
100
1.3
13
R122G
100
0.65
6.5
N111D
25
1
40
50
0.65
13
50
0.75
15
100
1.3
13
Proteins
[PFOA]/[ protein]
WT/S39G/M74G/
PFHxA: hL-FABP
N111D/R122G
WT/S39G/M74G/
PFHxS: hL-FABP
N111D/R122G
OA: hL-FABP
WT
5
Table S3. Thermodynamic parameters for the binding of PFHxS to wild type
hL-FABP and its four variants as determined by ITC at 25°C.
N (sites)
PFHxS
Ka (M-1)
Kd (M)
ΔH (kJ/mol)
ΔS (J/mol/deg)
ΔG (kJ/mol)
WT
0.987 ± 0.056
(3.01 ± 0.42) × 104
33.22
-44.38 ± 3.66
-63.1
-42.80 ± 5.24
S39G
1.02 ± 0.112
(2.05 ± 0.29) × 104
48.78
-73.62 ± 12.01
-164
-69.52 ± 16.11
M74G
1.12 ± 0.060
(3.00 ± 0.61) × 104
33.33
-4.37 ± 0.38
5.83
-4.52 ± 0.24
N111D
0.925 ± 0.081
(3.71 ±0. 57) × 104
26.95
-12.47 ± 1.47
47.7
-13.67 ± 0.28
R122G
0.421 ± 0.101
(1.63 ± 0.30) × 104
61.35
-101.70 ± 35.85
-260
-95.2 ± 41.35
6
A
S39G
WT hL-FABP
20
20
control
10 M PFHxA
50 M PFHxA
100 M PFHxA
15
15
control
10 M PFHxA
50 M PFHxA
100 M PFHxA
10
5
0
CD (mdeg)
10
CD (mdeg)
CD (mdeg)
10
M74G
control
10 M PFHxA
50 M PFHxA
100 M PFHxA
5
0
5
0
-5
-5
-5
-10
-10
200
210
220
230
240
200
250
N111D
20
230
240
200
250
210
220
230
240
250
wavelength (nm)
control
10 M PFHxA
50 M PFHxA
100 M PFHxA
15
10
CD (mdeg)
10
CD (mdeg)
220
R122G
control
10 M PFHxA
50 M PFHxA
100 M PFHxA
15
210
wavelength (nm)
wavelength (nm)
5
0
5
0
-5
-5
-10
-10
200
210
220
230
240
200
250
wavelength (nm)
210
220
230
240
250
wavelength (nm)
B
WT L-hFABP
20
control
10 M PFHxS
50 M PFHxS
100 M PFHxS
15
M74G
15
control
10 M PFHxS
50 M PFHxS
100 M PFHxS
15
control
10 M PFHxS
50 M PFHxS
100 M PFHxS
10
5
0
CD (mdeg)
10
CD (mdeg)
CD (mdeg)
10
S39G
20
5
0
5
0
-5
-5
-5
-10
-10
200
210
220
230
240
250
200
wavelength (nm)
control
10 M PFHxS
50 M PFHxS
100 M PFHxS
15
CD (mdeg)
10
CD (mdeg)
220
230
240
250
5
0
R122G
control
10 M PFHxS
50 M PFHxS
100 M PFHxS
15
10
5
0
-5
-5
-10
7
-10
200
210
220
230
wavelength (nm)
240
250
200
210
220
230
wavelength (nm)
200
210
220
230
wavelength (nm)
wavelength (nm)
N111D
20
210
240
250
240
250
8
C
15
10
CD (mdeg)
0
control
10 MPFNA
50 MPFNA
100 MPFNA
200 MPFNA
15
10
CD (mdeg)
5
control
10 MPFNA
50 MPFNA
100 MPFNA
200 MPFNA
20
control
10 MPFNA
50 M PFNA
100 MPFNA
200 MPFNA
10
CD (mdeg)
M74G
S39G
WT hL-FABP
15
5
0
-5
5
0
-5
-5
-10
-10
-10
200
210
220
230
240
200
250
220
230
R122G
N111D
control
10 MPFNA
50 MPFNA
100 MPFNA
200 MPFNA
15
240
250
10
5
0
-5
5
0
-5
-10
-10
200
210
220
230
wavelength (nm)
240
250
200
210
220
230
wavelength (nm)
Fig. S1
9
200
210
220
230
wavelength (nm)
control
10 MPFNA
50 MPFNA
100 MPFNA
200 MPFNA
15
CD (mdeg)
10
CD (mdeg)
210
wavelength (nm)
wavelength (nm)
240
250
240
250
A
(b)
WT hL-FABP
500000
WT hL-FABP
20
400000
1/(1-F/Fmax)
Fluorescence Intensity (a.u.)
(a)
300000
200000
15
10
5
100000
0
420
440
460
480
500
520
0
540
0
5
10
[ANS]/F/Fmax
wavelength (nm)
15
B
Relative Fluorescence (%)
Relative Fluorescence (%)
120
S39G
100
80
60
40
20
0
M74G
100
80
60
40
20
0
-2
-1
0
1
2
3
-2
-1
0
1
2
3
log [PFOA] (M)
log [PFOA] (M)
C
S39G
WT hL-FABP
80
60
40
20
0
-2
-1
0
1
log [PFNA] (M)
2
100
M74G
Relative Fluorescence (%)
Relative Fluorescence (%)
Relative Fluorescence (%)
100
80
60
40
20
0
-2
-1
0
1
log [PFNA] (M)
10
2
100
80
60
40
20
0
-2
-1
0
1
log [PFNA] (M)
2
Fig. S2
11
A
Time (min)
Time (min)
0
10
20
30
0
40
40
0
-0.20
-0.40
kcal mol-1 of injectant
0.0
-0.3
-0.6
-0.9
WT hL-FABP
0.0
0
-1.8
-2.4
1.0 1.5 2.0 2.5
Molar Ratio
Time (min)
10
20
30
40
S39G
0.0
0
0.00
20
30
40
-0.20
-0.40
-1.2
0.5
10
0.00
kcal mol-1 of injectant
kcal mol-1 of injectant
Time (min)
30
cal/sec
cal/sec
cal/sec
-0.10
-0.20
-1.0
-2.0
-3.0
0.5
1.0 1.5 2.0 2.5
Molar Ratio
Time (min)
10
20
30
40
M74G
0.0 0.5 1.0 1.5 2.0 2.5
Molar Ratio
0.00
cal/sec
cal/sec
20
0.00
0.00
-0.10
-0.05
-0.20
-0.10
kcal mol-1 of injectant
kcal mol-1 of injectant
10
-0.40
-0.60
-0.80
0.0
N111D
0.5
1.0 1.5 2.0
Molar Ratio
0.00
-0.10
-0.20
R122G
-0.30
2.5
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Molar Ratio
B
Time (min)
0
10
20
40
0
40
0
-0.60
-0.90
-2.0
-4.0
-6.0
WT hL-FABP
0.0
0
0.5
1.0 1.5 2.0
Molar Ratio
Time (min)
10
20
30
-2.5
-5.0
S39G
-7.5
0
2.5
1
2
3
Molar Ratio
4
Time (min)
40
0
0.00
cal/sec
0.00
-0.30
-0.60
10
20
30
0.00
-0.30
kcal mol-1 of injectant
kcal mol-1 of injectant
30
10
1220
30
40
cal/sec
-0.60
-0.90
cal/sec
20
-0.40
kcal mol-1 of injectant
-0.30
-0.30
10
0.00
cal/sec
cal/sec
0.00
-0.15
Time (min)
Time (min)
30
-1.20
-0.80
-1.0
-2.0
-3.0
M74G
0.0
0.5
1.0 1.5 2.0
Molar Ratio
2.5
13
C
Time (min)
10
20
40
0
cal/sec
0.00
cal/sec
Time (min)
Time (min)
30
-0.30
-0.60
10
20
30
40
0.00
0.00
-0.20
-0.50
cal/sec
0
-0.40
-2.0
-4.0
WT hL-FABP
-2.0
-4.0
S39G
0.0 0.5 1.0 1.5 2.0 2.5
Molar Ratio
Time (min)
0
10
20
30
40
0.0 0.5 1.0 1.5 2.0 2.5
Molar Ratio
Time (min)
0
10
20
30
40
0.00
cal/sec
cal/sec
0.00
-0.15
-0.30
-0.5
-1.0
-1.5
N111D
-2.0
0
2
4
6
Molar Ratio
8
kcal mol-1 of injectant
kcal mol-1 of injectant
-0.45
10
0.0
0.5
20
30
40
-1.00
-1.50
0.0
-0.40
-0.80
-1.20
0.0
-3.0
-6.0
R122G
0.0
0.5
1.0
Molar Ratio
Fig. S3
14
kcal mol-1 of injectant
0.0
kcal mol-1 of injectant
kcal mol-1 of injectant
-0.60
0
0.0
-2.0
-4.0
M74G
1.0
1.5
Molar Ratio
2.0
2.5
Fig. S4
15
Time (min)
0
10
20
30
40
kcal mol-1 of injectant
cal/sec
0.00
-0.02
-0.04
-0.06
-0.10
-0.15
-0.20
WT hL-FABP
-0.25
0
1
2
3
Molar Ratio
Fig. S5
16