Supplementary Information
Structure-Guided Directed Evolution of Highly Selective P450-based Magnetic Resonance Imaging
Sensors for Dopamine and Serotonin
Eric M. Brustad1, Victor S. Lelyveld2, Christopher D. Snow1, Nathan Crook1, Sang Taek Jung1, Francisco
M. Martinez5, Timothy J. Scholl5, Alan Jasanoff2-4* and Frances H. Arnold1*
1Division
of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA
2Department
of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
3Department
of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA,
USA
4Department
of Nuclear Science and Engineering, Massachusetts Institute of Technology, Cambridge,
MA, USA
5Department
of Physics and Astronomy, University of Western Ontario, London, ON, Canada
Supplementary Fig. 1. High-throughput optical screening and directed evolution for Improved
neurotransmitter binding in BM3h. A customized optical screening method was developed to evolve
ligand binding within BM3h1. Fatty acids are so-called ‘type I’ substrates, inducing a hypsochromic shift in
the P450’s characteristic Soret absorbance band associated with catalytically important changes in the
heme electronic environment2; 3. The bathochromic shift observed upon DA binding (from λmax = 419 nm
to 425 nm) is consistent with ‘type II’ ligand binding, indicative of DA coordination to the heme iron 2.
Binding isotherms can be generated by monitoring absorbance changes as a function of ligand
concentration. For panels a - c, we show data from BM3h-2G9C6 as a representative example of P450
ligand binding measurements described in the Methods. a) Characteristic P450-BM3h Soret band with
MAX at ~ 419 nM (solid line). In the presence of saturating biogenic amine neurotransmitters such as
serotonin (5HT), this absorbance is red shifted to ~425 nm (dashed line). b) Difference absorbance
spectra are acquired by subtracting ligand bound and unbound Soret spectra. Maximum absorbance
changes at 415 nm and 435 nm (triangles) were used to generate binding isotherms (panel c) at each
ligand concentration. c) Typical binding isotherm. d) Directed evolution protocol: DNA libraries were
constructed by active site-saturation mutagenesis using degenerate NNK oligonucleotides.
Libraries
were transformed into competent BL21-(DE3) E. coli. Individual BM3h variants colonies were picked,
expressed in 96 well blocks, and harvested cell lysates were distributed into 3 microtitre 96 well plates. As
shown in the middle of this panel, BM3h expression was readily observed by the characteristic red color
(red circles) consistent with heme bound proteins. Each library was co-screened against DA, 5HT and NE
to allow positive/negative for improved affinity and selectivity. Promising variants were analyzed for ligand
dependent MRI changes. This process was repeated for multiple cycles to yield final evolved BM3h
variants.
a
Compound
R1
R2
R3
KD (M)
dopamine (DA)
OH
OH
NH2
3.3  0.6
tyramine
OH
H
NH2
7.0  1.2
m - hydroxyphenethylamine
H
OH
NH2
18.0  1.7
phenethylamine
H
H
NH2
17.9  0.8
4-ethylcatechol
OH
OH
H
no binding
b
Compound
R1
R2
KD (M)
serotonin (5HT)
OH
H
0.7  0.1
tryptamine
H
H
0.6  0.1
N - acetylserotonin
OH
Ac
No binding
O - methylserotonin
OCH3
H
0.4  0.1
melatonin
OCH3
Ac
No binding
R3
L-tryptophan
H
No binding
5-hydroxytryptophan
OH
-
No binding
5-hydroxyindoleacetic acid
-
-
No binding
5-hydroxyindoleacetic acid
Supplementary Fig. 2. Structure-activity relationship of BM3h neurotransmitter binding. Type II
ligand binding in P450s is indicative of direct ligand coordination to the heme iron. To assess the nature
of this binding, a variety of commercially available dopamine and serotonin homologues, modified at the
exocyclic hydroxyl groups and aliphatic amine moieties, were screened for binding to variants BM3h-B7
(a) and BM3h-2G9C6 (b). Modification of the exocyclic hydroxyls has a minimal effect on ligand binding
whereas deletion or acetylation of the neurotransmitter amine functionality abrogates ligand binding. 5HT
precursors such as tryptophan and hydroxytryptophan show no binding to BM3h-2G9C6 as measured by
absorbance. In addition, serotonin metabolites such as 5-hydroindoleacetic acid and melatonin do not
bind BM3h-2G9C6.
a.
b.
c.
d.
e.
f.
Supplementary Fig. 3. Electron density of BM3h-bound DA and 5HT. Maximum likelihood weighted
electron density maps of protein-bound neurotransmitter and the heme cofactor. (a) BM3h-2G9C6 +
serotonin (b) BM3h-2G9 + serotonin (c) BM3h-9D7 + dopamine (d) BM3h-8C8 + dopamine (e) BM3h-8C8
+ serotonin and (f) BM3h-B7 + dopamine. All panels were contoured at  = 1.5.
Supplementary Fig. 4. Global protein structure in evolved BM3h variants. Neurotransmitter binding
in P450-BM3 does not contribute to the closed conformation. Panels a – c and g – i show alignments of
BM3h variants to the open wild type BM3h conformation (green) taken from PDB:2IJ2 4. Panels d – f and j
– l show residue-by-residue C RMSD values for the corresponding alignment above.
The closed conformation in the presence of cognate fatty acid ligand is shown in (a), taken from
PDB:1JPZ5) and is characterized by three regions that show significant substrate-induced conformational
changes: the N-terminal beta subdomain (region 1), the tip of the active site capping F- and G-helices
(region 2) and the hinge region at the bottom of the G-helix (region 3). The corresponding regions are
also marked in the residue-by-residue RMSD graph (d). Panels (b/e) and (d/f) show minor deviations from
open wild type for BM3h-8C8 in the absence (b/e) or presence (c/f) of dopamine. Small movements in
region 1 are observed in both structures, regardless of ligand binding. Serotonin bound BM3h-2G9 (g/j)
closely resembles the open wild type P450 structure with little movement observed in all three regions.
BM3h-2G9C6 in the absence (h/k) or presence (i/l) of serotonin shows movement in region 1 and 2, but
no change in the hinge region (3). Observed backbone movements are likely crystal packing artifacts. For
example, in the BM3h-2G9C6 structure in the absence of ligand (h), alternate conformations can be
observed between both chains in the asymmetric unit (colored brown and gold in panel h) suggesting that
these movements are likely influenced by interactions within the protein crystal. The absence of ligand
dependent conformational changes suggests that global protein backbone changes are not required for or
induced by neurotransmitter binding in evolved fMRI sensors.
a.
b.
Supplementary Fig. 5. Results from screening double NNK libraries in parent scaffold BM3h-B7. a)
Apparent KD values for 5HT-selective hits observed during screening. KD values (given in M) are colorcode from greatest affinity (green) to least affinity (red). Apparent K D titrations for these experiments were
performed in clarified cell lysates. Typically these values are at least 3-fold lower than KD titrations
performed on purified BM3h variants. b) Soret optical shift observed for BM3h variant 3DB10 in the
absence of ligand (dashed line) and presence of ligand (dotted line). For comparison, the Soret band for
wild type BM3h is also shown (solid line) with a MAX at ~ 419 nm. BM3h-3DB10 is slightly red-shifted
even in the absence of ligand. Electronic or structural changes at the heme center that cause this shift
may also lead to the decrease in r1 observed for this mutant.
Supplementary Fig. 6. Heterogeneous active site density in unbound BM3h structures. a) For
reference, a histogram of relaxivity (r1) for several ligand-free variants described in this work is shown. b)
Normalized Soret absorbance spectra for all BM3h variants in the absence of ligand (solid line) is
consistent with a low spin heme center (max = 419 nm). For comparison, high spin wild type BM3h,
bound to saturating lauric acid (a model fatty acid substrate) to induce spin shift is shown (dotted line)
with a max ~ 390 nm. Based on these data, changes in the ferric iron spin state are probably not
involved in relaxivity differences.
Supplementary Fig. 6. Heterogeneous active site density in unbound BM3h structures: continued.
Averaged kicked omit maps were generated to display axial heme density in ligand-free BM3h crystal
structures (heme = 1.5 and axial = 1.0). Averaged kick omit maps were generated using the calculate
maps tool of the PHENIX crystallographic refinement software package 6. Panels c – g show heme bound
omit-density for BM3h variants in order of increasing r1: BM3h-8C8 (c), wild type BM3h (d; taken from
pdb:2IJ24), BM3h-2G9 (e), BM3h-9D7 (f), and BM3h-2G9C6 (g). Top and bottom panels for each
structure refer to chains A and B of asymmetric unit, respectively. Amorphous regions of proximal-heme
density among existing BM3h structures have been attributed to mixed occupancy water molecules. We
are unable to rule out the possibility that increased density in high-r1 variants is the result of promiscuous
binding to components of the crystallization mother liquor. However, the most likely contaminant, acetate
(a crystallization co-salt), fails to account for the total observed electron density nor does acetate yield
changes in the P450 Soret spectrum upon titration. Nevertheless, the observed trend of increasing
electron density around the heme iron may reflect the improved relaxivity observed in variants such as
BM3h-2G9C6.
Supplementary Fig. 7. B factor analysis of BM3h variants in the absence of ligand. a - c) Active
sites for BM3h variants BM3h-8C8 (a), wild type (b, taken from pdb:2IJ2)4 and BM3h-2G9C6 (c). In the
high relaxivity mutant, BM3h-2G9C6, several amino acid side chains within the active site, including the
mutation at L437Q, are observed to exist in multiple conformations within the crystal structure. (chain A is
shown in green, chain B is shown in orange). d – f) The same active site structures from above are
shown color-coded for B factor. A color bar is shown under panel e. For the higher relaxivity mutant,
BM3h-2G9C6, increased mobility is observed for the regions shown. g) Plots of α-carbon B factor versus
relaxivity (r1) are shown for select active site mutations as well as the proximal heme ligand, cys400, for
reference. B factors were taken from ligand free crystal structures solved during this work (BM3h-8C8,
BM3h-2G9, BM3h-9D7 and BM3h-2G9C6) as well as ligand free wild type protein (pdb: 2IJ2)4. To
compare B factors between structures, we normalized C B-factor with the average heme B-factor to
account for differences in resolution. Others have applied similar normalization strategies to compare
different protein structures7 and normalizations to other BM3h residues provided similar results. Plotting
residue-by-residue B-factors against ligand-free r1 showed three regions with marked correlation between
these two properties (panels g and h). h) The residue-by-residue squared correlations (R2) of α-carbon B
factor and relaxivity, such as those shown in panel g, are shown for the entire protein sequence with a
sliding average over 5 residues (solid line). In addition, the slope of the linear regression of B factor
versus relaxivity used to generate R2 is also shown (dotted line). Three regions, highlighted, appear to
demonstrate an observed correlation (R2) between increasing local B factor and the improvements in
relaxivity observed in evolved BM3h variants. These regions include the active site I-helix including
residues 263 – 273 (green), the active site loop comprised of residues 319 – 329 (blue), as well as the
active site loop comprised of residues 425 – 440 (red), which includes the mutation L437Q (highlighted in
panel c) in the highest relaxivity variant, BM3h-2G9C6. For comparison, one region that demonstrates
large deviations in B factor with poor correlation relative to relaxivity changes includes the loop linking the
F and G helices (residues ~ 180 – 200), which is known to be highly mobile and often poorly structured in
various BM3h structures. As shown in panels i and j, these regions (color coded to match panel h) are
located within the active site of the protein, near the heme center, and flank 2 of the 3 largest solvent
channels (shown by arrows) leading to the heme.
Supplementary Fig. 8. NMRD analysis of 5HT- and DA-binding BM3h mutants. Purified BM3h protein
T1 molar relaxivity measured in a field cycling relaxometer at 25°C as a function of applied magnetic field
strength (solid circles), shown here as proton Larmor frequency, and best-fit Solomon and Bloembergen
dipolar coupling models of a single bound water (q = 1) in the first coordination sphere, with the following
fitted parameters (inset): effective proton-ion distance r (units in Å), first coordination sphere bound water
residence time τM (units in µs), and frequency-independent electronic relaxation time τS (units in ns). The
fitting procedure assumed a uniform population of low spin ferric ions (total electron spin, S = 1/2), a
Stokes-Einstein radius of 3.5 nm (corresponding to a rotational correlation time, τR = 39 ns), a modeled
diamagnetic contribution based on apotransferrin8, and that the scalar contribution to the overall
relaxation process was negligible.
Supplementary Table 1. Data collection and refinement statistics for BM3h-8C8 crystals
pdb accession #
BM3h-8C8
(no ligand)
4DTY
BM3h-8C8:DA
complex
4DTZ
BM3h-8C8:5HT
complex
4DTW
Data collection
Space group
Wavelength
P 1 21 1
1.033
P 1 21 1
1.033
P 1 21 1
1.033
58.75, 145.66, 63.53
97.10
58.70, 146.37, 63.98
97.59
58.61, 146.25, 64.06
97.51
38.5 - 1.45 (1.45 - 1.53)
*
5.6(55.5)
10.3(2.1)
93.8(93.0)
3.4(3.3)
38.9 - 1.55 (1.55 - 1.63)
*
5.1(44.8)
11.8(2.3)
95.7(98.1)
2.6(2.6)
38.9 - 1.8 (1.8 – 1.9) *
a, b, c (Å)
a
b
g
()
Resolution (Å)
Rmerge
I / σI
Completeness (%)
Redundancy
7.8(58.0)
9.0(1.9)
92.5(95.0)
2.4(2.4)
Refinement
Resolution (Å)
36.4 - 1.45
37.4 - 1.55
38.9 - 1.8
No. reflections
165582
140219
86403
Rwork / Rfree
0.18 / 0.22
0.18/0.22
0.19/0.25
No. atoms
Protein
7370
7343
7308
Ligand/ion
86
108
112
Water
1036
1098
1059
B-factors
Protein
16.9
15.9
17.7
Ligand/ion
9.87
9.6
12.3
Water
27.8
28.0
29.6
R.m.s. deviations
Bond lengths (Å)
0.027
0.027
0.023
2.21
2.22
1.97
Bond angles ()
*All data sets were collected from single crystals. Highest-resolution shell is shown in parentheses.
Supplementary Table 2. Data collection and refinement statistics for BM3h-B7 crystals
pdb accession #
BM3h-B7:DA
complex
4DU2
Data collection
Space group
wavelength
P 1 21 1
1.033
a, b, c (Å)
a
b
g
()
Resolution (Å)
Rmerge
I / σI
Completeness (%)
Redundancy
58.73, 147.23, 63.22
98.11
39.0 - 1.9 (1.9 – 2.0) *
5.1(44.0)
11.8(1.9)
94.1(93.0)
2.6(2.4)
Refinement
Resolution (Å)
39.0 - 1.9
No. reflections
74344
Rwork / Rfree
0.20/0.25
No. atoms
Protein
7040
Ligand/ion
108
Water
436
B-factors
Protein
24.2
Ligand/ion
17.5
Water
31.1
R.m.s. deviations
Bond lengths (Å)
0.023
1.89
Bond angles ()
*All data sets were collected from single crystals. Highest-resolution shell is shown in parentheses.
Supplementary Table 3. Data collection and refinement statistics for BM3h-9D7 crystals
pdb accession #
BM3h-9D7
(no ligand)
4DUA
BM3h-9D7:DA
complex
4DUB
Data collection
Space group
wavelength
P 1 21 1
0.979
P 1 21 1
1.000
58.83, 152.30, 61.78
94.58
58.91, 153.58, 61.10
95.10
39.2 - 2.0 (2.0 - 2.1) *
8.6(60.7)
10.1(2.2)
97.5(97.7)
3.4(3.5)
39.1 - 1.7 (1.7 - 1.8) *
4.8(66.3)
13.7(2.0)
97.7(97.9)
3.5(3.5)
a, b, c (Å)
a
b
g
()
Resolution (Å)
Rmerge
I / σI
Completeness (%)
Redundancy
Refinement
Resolution (Å)
38.4 - 2.0
38.6 - 1.7
No. reflections
67311
109611
Rwork / Rfree
0.16 / 0.21
0.16/0.20
No. atoms
Protein
7280
7328
Ligand/ion
95
108
Water
516
731
B-factors
Protein
24.9
22.5
Ligand/ion
22.2
14.8
Water
32.0
30.5
R.m.s. deviations
Bond lengths (Å)
0.023
0.028
1.90
2.09
Bond angles ()
*All data sets were collected from single crystals. Highest-resolution shell is shown in parentheses.
Supplementary Table 4. Data collection and refinement statistics for BM3h-2G9C6 crystals
pdb accession #
BM3h-2G9C6
(no ligand)
4DUD
BM3h-2G9C6:5HT
complex
4DUE
Data collection
Space group
wavelength
P 1 21 1
1.000
P 1 21 1
1.033
58.73, 153.30, 60.98
94.68
29.8 – 1.85 (1.85 – 1.95)
*
7.4(59.3)
9.7(2.0)
98.6(99.1)
3.5(3.5)
58.61, 148.42, 64.29
98.88
38.4 - 1.7 (1.7 – 1.79) *
a, b, c (Å)
b
)
Resolution (Å)
Rmerge
I / σI
Completeness (%)
Redundancy
4.9(65.1)
15.1(1.8)
97.3(98.3)
3.4(3.5)
Refinement
Resolution (Å)
29.8 – 1.85
38.4 - 1.7
No. reflections
84485
108321
Rwork / Rfree
0.17 / 0.21
0.17 / 0.21
No. atoms
Protein
7497
7287
Ligand/ion
86
112
Water
562
836
B-factors
Protein
21.3
21.1
Ligand/ion
12.3
16.3
Water
28.2
31.6
R.m.s. deviations
Bond lengths (Å)
0.025
0.027
1.95
2.16
Bond angles ()
*All data sets were collected from single crystals. Highest-resolution shell is shown in parentheses.
Supplementary Table 5. Data collection and refinement statistics for BM3h-2G9 crystals
pdb accession #
BM3h-2G9
(no ligand)
4DUC
BM3h-2G9:5HT
serotonin complex
4DUF
Data collection
Space group
wavelength
P 1 21 1
1.000
P 1 21 1
0.979
a, b, c (Å)
b
)
Resolution (Å)
Rmerge
I / σI
Completeness (%)
Redundancy
58.65, 153.75, 60.98
94.82
29.2 – 1.9 (1.9 – 2.0) *
8.9(42.5)
6.3(2.0)
95.5(78.9)
3.4(3.0)
79.69, 150.49, 87.71
90.32
29.6 - 1.8 (1.8 – 1. 9) *
6.3(51.3)
11.5(2.9)
98.1(96.0)
3.5(3.3)
Refinement
Resolution (Å)
29.2 – 1.9
29.4 - 1.8
No. reflections
73543
177347
Rwork / Rfree
0.18 / 0.23
0.16 / 0.20
No. atoms
Protein
7270
14906
Ligand/ion
86
224
Water
568
1101
B-factors
Protein
19.7
16.0
Ligand/ion
12.7
15.1
Water
20.1
24.1
R.m.s. deviations
Bond lengths (Å)
0.022
0.026
1.91
2.07
Bond angles ()
*All data sets were collected from single crystals. Highest-resolution shell is shown in parentheses.
Supplementary Table 6.
Ramachandran statistics.
Structure
Crystallization
Crystallization
condition a,b
conditions,
MOLREP
template
molecular
Rfree
Template
c
8C8 (no ligand)
8C8 +
dopamine
8C8 +
serotonin
B7 + dopamine
9D7 (no ligand)
9D7 +
dopamine
2G9 (no ligand)
2G9 +
serotonin
2G9C6 (no
ligand)
2G9C6 +
serotonin
0.1 M Tris, pH 8.5
0.2 M MgCl2,
22 % PEG 3350
0.1 M Tris, pH 8.2
0.2 M MgCl2,
20 % PEG 3350
0.1 M Tris, pH 8.5
0.2 M MgCl2,
18 % PEG 3350
0.1 M Tris, pH 8.2
0.2 M MgCl2,
19 % PEG 3350
0.1 M Na cacodylate,
pH 5.5,
0.14 M Ca(Ac)2,
14 % PEG 8000
0.1 M Na cacodylate,
pH 5.5,
0.14 M Ca(Ac)2,
14 % PEG 8000
0.1 M Na cacodylate,
pH 5.5,
0.14 M Ca(Ac)2,
14 % PEG 8000
0.1 M Na cacodylate,
pH 5.5,
0.14 M Ca(Ac)2,
14 % PEG 8000
0.1 M Na cacodylate,
pH 5.5,
0.14 M Ca(Ac)2,
14 % PEG 8000
0.1 M Na cacodylate,
pH 5.5,
0.14 MgCl2,
13 % PEG 3350
replacement
models
and
%
Ramachandran
outliers (total)
0.1 % ( 1 )
%
Ramachandran
favored
97.8 %
0.0 % ( 0 )
97.9 %
8C8
+
dopamine
2IJ2 (WT)
8C8
+
dopamine
New Rfree
8C8
+
dopamine
8C8
+
dopamine
9D7
+
dopamine
8C8
+
dopamine
8C8
+
dopamine
9D7
+
dopamine
0.2 % ( 2 )
97.9 %
0.2 % ( 2 )
96.6 %
0.1 % ( 1 )
97.5 %
2IJ2 (WT)
New Rfree
0.0 % ( 0 )
97.7 %
2IJ2 (WT)
New Rfree
0.1 % ( 1 )
97.8 %
2IJ2 (WT)
New Rfree
0.0 % ( 0 )
97.3 %
2IJ2 (WT)
New Rfree
0.0 % ( 0 )
97.8 %
2G9C6
(no
ligand)
2G9C6
(no
ligand)
0.0 % ( 0 )
97.3 %
crystallization conditions involved the addition of 1 L protein at 15 mg/mL + 1 L mother liquor listed
in the table.
aAll
bCrystal
drops were microseeded with protein crystals grown in the absence of seeding. Seeding crystals
usually grew in conditions containing slightly higher PEG concentrations (+ 4 to 6 %) with same salt and
buffer concentrations.
cIn
structures where molecular replacements models were generated from other protein models from this
work (i.e. not from wild type, WT), RFree data sets were carried over to minimize over fitting of the data.
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6.
Adams, P. D., et al. (2010). PHENIX: a comprehensive Python-based system for macromolecular
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Smith, D. K., Radivojac, P., Obradovic, Z., Dunker, A. K. & Zhu, G. (2003). Improved amino acid
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8.
Bertini, I., Fragai, M., Luchinat, C. & Parigi, G. (2000). 1H NMRD profiles of diamagnetic proteins:
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