Cation-π interactions contribute to substrate recognition in γ-butyrobetaine hydroxylase catalysis

Article abstract of DOI:10.1002/chem.201503761

γ-Butyrobetaine hydroxylase (BBOX) is a non-heme Fe^II- and 2-oxoglutarate-dependent oxygenase that catalyzes the stereoselective hydroxylation of an unactivated C-H bond of γ-butyrobetaine (γBB) in the final step of carnitine biosynthesis. BBOX contains an aromatic cage for the recognition of the positively charged trimethylammonium group of the γBB substrate. Enzyme binding and kinetic analyses on substrate analogues with P and As substituting for N in the trimethylammonium group show that the analogues are good BBOX substrates, which follow the efficiency trend N⁺>P⁺>As⁺. The results reveal that an uncharged carbon analogue of γBB is not a BBOX substrate, thus highlighting the importance of the energetically favorable cation-π interactions in productive substrate recognition. What's in the BBOX? Enzyme kinetics and substrate binding studies reveal that γ-butyrobetaine hydroxylase (BBOX)-catalyzed stereoselective hydroxylation of γ-butyrobetaine involves energetically favorable cation-π interactions.

Full text of DOI:10.1002/chem.201503761

DOI: 10.1002/chem.201503761
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&
Biocatalysis |Hot Paper|
Cation–p Interactions Contribute to Substrate Recognition in
g-Butyrobetaine Hydroxylase Catalysis
Jos J. A. G. Kamps,[a] Amjad Khan,[b] Hwanho Choi,[b] Robert K. Lesniak,[b] Jürgen Brem,[b]
Anna M. Rydzik,[b] Michael A. McDonough,[b] Christopher J. Schofield,[b]
[b]
[a]
´
Timothy D. W. Claridge, and Jasmin Mecinovic*
Chem. Eur. J. 2016, 22, 1270 – 1276
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Abstract: g-Butyrobetaine hydroxylase (BBOX) is a non-
heme FeII- and 2-oxoglutarate-dependent oxygenase that
catalyzes the stereoselective hydroxylation of an unactivated
CÀH bond of g-butyrobetaine (gBB) in the final step of carni-
tine biosynthesis. BBOX contains an aromatic cage for the
recognition of the positively charged trimethylammonium
group of the gBB substrate. Enzyme binding and kinetic
analyses on substrate analogues with P and As substituting
for N in the trimethylammonium group show that the ana-
logues are good BBOX substrates, which follow the efficien-
cy trend N+ >P+ >As+. The results reveal that an un-
charged carbon analogue of gBB is not a BBOX substrate,
thus highlighting the importance of the energetically favor-
able cation–p interactions in productive substrate recogni-
tion.
Introduction
catalyzes the stereoselective hydroxylation of g-butyrobetaine
(gBB, 1) to form l-carnitine (l-CAR) in eukaryotes and some
prokaryotes (Figure 1a).[3–6] l-Carnitine is required for the trans-
port of fatty acids into the mitochondrial matrix, where they
are converted into acetyl-CoA.[7] Structural analyses on human
BBOX (hBBOX) reveal that the active site FeII is chelated by
a 2His-1Asp triad and that the 2OG cosubstrate binds in a simi-
lar mode to other 2OG oxygenases (Figure 1b).[8,9] The BBOX
active site contains an apparent “aromatic cage,” which binds
the gBB substrate’s trimethylammonium group, and two aspar-
agine residues that hydrogen bond with the gBB carboxylate
(Figure 1b).[8,9] The chiral environment of the enzyme’s active
site enables hBBOX to catalyze the oxidative desymmetrization
of achiral N,N-dialkyl piperidine-4-carboxylates.[10] hBBOX also
catalyzes an unusual Stevens-type rearrangement of Mildro-
nate (also known as Meldonium), a gBB competitive inhibitor
that is clinically used in the treatment of myocardial infarction
in order to inhibit fatty acid metabolism.[11,12] Recent studies re-
vealed that subtle differences in the active sites of human and
Pseudomonas sp. AK1 BBOX (hereafter psBBOX) can result in al-
tered substrate-analogue selectivities.[13]
2-Oxoglutarate (2OG) and FeII-dependent oxygenases play im-
portant roles in human physiology, including in hypoxia sens-
ing, DNA repair, chromatin modification and fatty acid metabo-
lism.[1,2] g-Butyrobetaine hydroxylase (BBOX), a 2OG oxygenase,
Functionally and structurally diverse proteins contain aro-
matic cages (or aromatic boxes) as recognition modules for
substrate binding. Aromatic cages typically comprise the side-
chains of 2–4 aromatic residues (Trp, Tyr, Phe), and are ob-
served on both exposed and buried sites.[14–16] Work by Dough-
erty and coworkers has demonstrated that aromatic cages can
recognize positively charged quaternary ammonium species
via favorable cation–p interactions.[17–19]
Figure 1. BBOX-catalyzed hydroxylation: a) hBBOX-/psBBOX-catalyzed hy-
droxylation of gBB 1; b) view of the hBBOX active site (yellow sticks, upper
residue numbers, PDB ID: 3O2G) and the psBBOX model (blue sticks, lower
residue numbers) with gBB (white sticks), N-oxalylglycine (NOG, a 2OG ana-
logue, cyan sticks) and ZnII substituting for FeII (grey sphere).[13]
Cation–p interactions are involved in associations of Cys-
loop receptors and G-protein-coupled receptors with neuro-
transmitters, including acetylcholine, serotonin, dopamine, epi-
nephrine, and histamine.[20] Studies on chromatin interactions
involved in the regulation of gene expression reveal that
“reader domain” proteins that recognize trimethyllysine-con-
taining histone tails interact through cation–p interactions.[14,21]
Thus, along with hydrophobic effects, hydrogen bonding, ion
pairing, and van der Waals interactions, cation–p interactions
are crucial noncovalent forces in protein–protein and protein–
ligand associations.[22]
[a] J. J. A. G. Kamps,+ Dr. J. Mecinovic
´
Institute for Molecules and Materials, Radboud University Nijmegen
Heyendaalseweg 135, 6525 AJ Nijmegen (The Netherlands)
E-mail: j.mecinovic@science.ru.nl
[b] A. Khan,+ Dr. H. Choi, R. K. Lesniak, Dr. J. Brem, Dr. A. M. Rydzik,
Dr. M. A. McDonough, Prof. C. J. Schofield, Prof. T. D. W. Claridge
Chemistry Research Laboratory, Department of Chemistry
University of Oxford, 12 Mansfield Road, Oxford OX1 3TA (UK)
[+] These authors contributed equally to this work.
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under http://dx.doi.org/10.1002/chem.201503761.
The BBOX aromatic cage contains the electron-rich aromatic
residues Phe184, Phe188 and Tyr201; Tyr368 is also located in
close proximity, with its side-chain OH group likely positioned
to form an H-bond with Asp211 (Figure 1b).[13] The location of
the gBB trimethylammonium group inside the aromatic cage
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medium, provided the original work is properly cited.
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suggests that the association between gBB and BBOX could be
substantially mediated by energetically favorable cation–p in-
teractions. In support of this proposition, previous work with
recombinant psBBOX employing a radioactive-based assay
with the C-analogue 4 of gBB (1), provided evidence that it is
a poor substrate.[23] Herein, we report on the use of NMR and
MS assays to investigate gBB analogues with P, As, and C sub-
stituting for N (2–4, respectively; Scheme 1) as psBBOX sub-
alogue 4, would provide insights into the role of cation–p in-
teractions in BBOX catalysis (Scheme 1). Like gBB (1), the phos-
phorus (2) and arsenic (3) derivatives possess a “fixed” positive
charge with respect to their trimethyl group, but are slightly
larger than 1, whereas the carbon analogue (4) has nearly the
same size and shape, but lacks a positive charge. We hypothe-
sized that direct comparison of binding and psBBOX-catalyzed
hydroxylation of the positively charged gBB 1 and neutral 4
+
would inform on the interactions between the NMe3 group
and the aromatic cage of psBBOX. Thus, if 4 is a much poorer
psBBOX ligand and substrate than 1, the requirement of
cation–p interactions in psBBOX catalysis would be implied. In
contrast, if 4 is a better ligand and substrate for psBBOX, that
would suggest that hydrophobic interactions dominate the
psBBOX–gBB association.
Results and Discussion
The phosphorus (2) and arsenic (3) analogues of gBB (1) were
synthesized in concise three-step sequences from 4-bromobu-
tyric acid (Scheme 1 and Figure S1 in the Supporting Informa-
tion). 5,5-Dimethylhexanoic acid (4) was synthesized from 4-
bromobutanoic acid and a twofold excess of tert-butylmagne-
sium chloride (Scheme 1 and Figure S1).
Scheme 1. Syntheses of gBB analogues 2–4. DMAP=4-dimethylaminopyri-
dine.
We then used LC-MS analyses to test for psBBOX-catalyzed
hydroxylation of the three gBB analogues. In the presence of
psBBOX (1 mm), 1 was efficiently hydroxylated (complete con-
version in 5 min; Figure 2a and Figure S2 in the Supporting In-
formation). Phosphorus analogue 2 was also a good substrate,
with about 70% conversion (Figure 2b); arsenic analogue 3
was less well hydroxylated (approximately 45%), but clear evi-
strates; the results clearly support the proposal that cation–p
interactions are crucial in the recognition of gBB by psBBOX.
We envisaged that replacement of the trimethylammonium
group of gBB by three closely analogous functionalities, that is,
positively charged trimethylphosphonium and trimethylarsoni-
um “Group V analogues” 2 and 3, and the neutral tert-butyl an-
Figure 2. Mass spectrometry data for psBBOX-catalyzed hydroxylations: a) natural substrate g-BB 1; b) phosphorus analogue 2; c) arsenic analogue 3; d) neu-
tral carbon analogue 4. Top panel=starting substrate; bottom panel=psBBOX-catalyzed reaction [a–c) 1 mm psBBOX, 5 min; d) 10 mm psBBOX, 3 h].
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dence for hydroxylation was obtained (Figure 2c). Importantly,
the neutral analogue 4 was not hydroxylated by psBBOX
within our limits of detection; use of an increased amount of
psBBOX (10 mm) and prolonged incubation (3 h) did not result
in the observation of hydroxylated product by LC-MS (Fig-
ure 2d). In the absence of psBBOX, no hydroxylation of 2 or 3
took place (see the Supporting Information, Figure S3). Time-
course NMR studies were consistent with the order of efficien-
cy as observed by LC-MS (1>2>3) and revealed that in each
case psBBOX-catalyzed hydroxylation of 1–3 is tightly coupled
to oxidation of 2OG to succinate (Figure 3a–c). Consistent with
the LC-MS results, the NMR assays revealed that 4 was not hy-
droxylated, even in the presence of 10 mm psBBOX (Figure 3d).
Controls with 2 and 3 showed a lack of hydroxylation without
psBBOX (see the Supporting Information, Figures S4 and S5).
Overall, these results reveal the importance of a positively
charged XMe3 substrate group for psBBOX catalysis.
occurs at C3 (see the Supporting Information, Figures S6–S11).
These assignments were confirmed by stereoselective synthesis
of the (3R)- and (3S)-hydroxylated phosphorus and arsenic de-
rivatives 5–8 (Scheme 2 and Figure S12 in the Supporting In-
1
formation). The intensity of H NMR peaks that correspond to
the hydroxylated products increased upon the addition of au-
thentic (3R)-hydroxylated phosphorus- (5) and arsenic-contain-
ing (6) products into the respective reaction mixtures, confirm-
ing that psBBOX-catalyzed hydroxylation of the analogues
occurs at C3 (see the Supporting Information, Figures S13 and
S14). Together, these results imply that the psBBOX-catalyzed
hydroxylation of 2 and 3 results in the C3-hydroxylated prod-
ucts 5 and 6, likely with (3R) stereochemistry (Scheme 3).
LC-MS analyses testing (3R)- and (3S)-hydroxylated phospho-
rus derivatives 5 and 7 as substrates showed that the latter is
a poor psBBOX substrate, giving a small amount of the 3-keto
product in the presence of 20 mm psBBOX (as assigned on the
basis of a À2 Da mass shift), whereas the (3R)-5 enantiomer
appears not to be converted within limits of detection for this
COSY- and HSQC-based 2D NMR spectroscopy of the prod-
ucts implied that psBBOX-catalyzed hydroxylation of 2 and 3
Figure 3. 1H NMR monitoring of the hydroxylations by psBBOX in the presence of 2OG as a cosubstrate and FeII as a cofactor: a) 1; b) 2; c) 3; d) 4 (blank=re-
action mixture in the absence of psBBOX). The substrate/product ratio is measured for each spectrum as a function of reaction time.
Scheme 2. Stereoselective syntheses of (3R)- and (3S)-hydroxylated phosphorus- and arsenic-containing derivatives 5–8.
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oxygen atom in the hydroxylated products from 2 and 3 deriv-
ing (at least predominantly) from atmospheric oxygen and not
from water, as also found in oxygen-labeling studies on most
other 2OG oxygenases.[24]
To quantify the relative efficiencies of the substrate ana-
logues 2 and 3, we carried out kinetic analyses using LC-MS.
The results revealed that the natural NMe3+-containing sub-
strate 1 is a superior substrate to the PMe3+- and AsMe3+-con-
taining analogues 2 and 3, by approximately 2- and 3-fold, re-
spectively, as measured by kcat/Km values (Table 1 and Fig-
Table 1. Kinetic parameters for conversion of gBB 1 and the phosphorus
(2) and arsenic (3) analogues into the corresponding hydroxylated prod-
ucts by psBBOX.[a]
1
2
3
Vmax [mmsÀ1
kcat [sÀ1
Km [mm]
]
2.81Æ0.31
7.02Æ0.77
1.00Æ0.21
1.65Æ0.16
4.13Æ0.40
1.20Æ0.20
1.04Æ0.13
2.61Æ0.31
1.37Æ0.28
]
[a] 400 nm psBBOX was used with varying concentrations of substrates
from 50 mm to 1.5 mm.
ure S24 in the Supporting Information). The differences in kcat
and Km values are relatively small for 1, 2, and 3, implying the
importance of a positively charged XMe3 group. There is
a trend in decreasing kcat from 1 to 3, suggesting that the
longer CÀP and CÀAs bond lengths (1.9 Š and 2.0 Š, respec-
tively) relative to the CÀN bond length (1.5 Š) may cause sub-
strates 2 and 3 to adopt a non-optimal binding mode with re-
spect to the FeIV=O intermediate, as observed with substrate
analogue studies on other 2OG oxygenases.[1] The Km values
for all three positively charged substrates were within error
(1.00–1.37 mm).
Although the kinetic studies and time-course analyses imply
that binding in the aromatic cage is important, the small differ-
ences in the kcat and Km values and the complexity of 2OG oxy-
genase catalysis motivated us to carry out NMR titration stud-
ies to obtain KD values for the binding of 1–3 to the psBBOX·
ZnII·2OG complex (using ZnII as an unreactive FeII substitute);
the KD values for analogues 1, 2, and 3 were 5 mm, 7 mm, and
17 mm, respectively (see the Supporting Information, Fig-
ures S25–S27). The NMR experiments also implied that 4 does
not bind in the active site of psBBOX within detection limits
(see the Supporting Information, Figure S28). Similar trends
have been observed in inhibition studies on the serine pro-
tease factor Xa, which contains an aromatic cage in its “S4”
substrate residue binding pocket. That is, a ligand possessing
a quaternary ammonium moiety inhibits factor Xa to a similar
extent as one with an analogous phosphonium group, where-
as the neutral carba analogue is substantially (60-fold) less
potent.[25,26]
Scheme 3. psBBOX-catalyzed stereoselective hydroxylation of positively
charged phosphorus (2) and arsenic (3) analogues of gBB, and their (3R)-
and (3S)-hydroxylated derivatives. Black dashed arrows and grey dashed
arrows indicate poor and very poor conversions, respectively.
assay (see the Supporting Information, Figure S15). We did not
detect ketone formation with the hydroxylated arsenic deriva-
tives (3R)-6 or (3S)-8 by LC-MS (see the Supporting Informa-
tion, Figure S16). However, NMR assays indicated that all four
hydroxylated derivatives 5–8 are very poor psBBOX substrates
with low levels of conversion being observed in the presence
of 10 mm psBBOX, likely to the C3 ketone, which we could not
fully characterize due to the low levels of conversion
(Scheme 3 and Figures S17–S21 in the Supporting Information).
The observation of a product with the same chemical shift for
+
the PMe3 group from incubation of 5 and 7 supports the pro-
posed formation of the 3-ketone (as with hydroxylation of the
enantiomeric arsenic derivatives 6 and 8; Figure S17–S21). As
for the carnitine enantiomers,[13] the (3S) enantiomers 7 and 8
were substantially better substrates than the (3R) enantiomers
5 and 6, which afforded barely detectable products (36% vs.
7% for phosphorus derivatives 7 and 5; 22% vs. 3% for arsenic
derivatives 8 and 6). These results are consistent with previous
results of hBBOX- and psBBOX-catalyzed hydroxylation of d-
and l-carnitine.[13]
Under (near) anaerobic conditions (Ar atmosphere), only
traces (<5%) of hydroxylated products were observed for 2
and 3 (see the Supporting Information, Figure S22). Reactions
of 2 and 3 in H218O (90% 18O, Tris buffer, pH 7.5) afforded hy-
droxylated products with the same molecular mass as products
from standard reaction in Tris buffer/H216O (see the Supporting
Information, Figure S23). These results are consistent with the
To investigate the binding modes of 2 and 3 to psBBOX, we
used an X-ray crystal structure of hBBOX in complex with ZnII,
NOG, and gBB [Protein Data Bank (PDB) ID: 3O2G] to make
a homology model of psBBOX. All three positively charged
substrates (1, 2, 3) are predicted to bind in a similar manner in
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tential for utilization in radioactive tracing inside cells. Phos-
phorus and arsenic analogues of naturally occurring molecules
that contain the quaternary ammonium groups might become
useful probes for other genuinely important biomolecular pro-
cesses that are driven by strong cation–p interactions. Given
the central role of carnitine in eukaryotic fatty acid metabo-
lism, our results also highlight biomedicinally important
cation–p interactions.
Experimental Section
BBOX production and purification
Recombinant psBBOX was produced according to a previously de-
scribed procedure.[13] In brief, cells were cultured in 2TY media sup-
plemented with 50 mgmLÀ1 ampicillin until mid-log phase growth
was achieved (OD600 0.7). Production of the recombinant proteins
was then induced by addition of 0.2 mm IPTG and the cells were
cultured for further 16 h at 158C. Cells were harvested by centrifu-
gation (8 min, 8 g), then resuspended in lysis buffer (50 mm Tris
pH 7.5/500 mm NaCl) supplemented with 0.2% Tween 20, DNAse,
Lysosyme, and EDTA-free protease-inhibitors.
Figure 4. Modeled structure of psBBOX (blue) complexed with ZnII (red), N-
oxalylglycine (cyan), and substrates 1 (white), 2 (yellow), and 3 (magenta).
the psBBOX active site, consistent with the observation of C3
+
hydroxylation, and with the XMe3 group positioned in the ar-
omatic cage (Figure 4). Due to strong negative inductive effect
+
of a large XMe3 group, the C4 position (adjacent to the
+
XMe3 group) is activated, but also the most sterically hin-
The cell lysates were loaded onto a 5 mL HisTrap HP column (GE
Healthcare Life Sciences, Little Chalfont, UK), with 50 mm Tris
pH 7.5/500 mm NaCl, containing 20 mm imidazole, then eluted
with an imidazole gradient (up to 500 mm imidazole). Fractions
containing the purified psBBOX protein were concentrated by cen-
trifugal ultrafiltration (50 kDa cutoff membrane). The protein solu-
tion was then injected onto a Superdex S200 column (300 mL) and
eluted with 20 mm Tris pH 7.5/200 mm NaCl supplemented with
10 mm EDTA. Fractions containing purified psBBOX were concen-
trated by centrifugal ultrafiltration (50 kDa cutoff filter) and buffer
exchanged by using a PD-10 column to a Chelex 100-treated
metal-free buffer (50 mm Tris pH 7.5/200 mm NaCl). The purity of
the resulting fractions was ascertained to be >90% by SDS-PAGE
analysis. Concentrations of the purified proteins were determined
by using a ND-1000 NanoDrop spectrophotometer.
dered. The C2 position, although the most activated, is posi-
tioned away from the FeIV=O intermediate. Thus, of all three
potential sites, psBBOX-catalyzed hydroxylation occurs at the
C3 site of 1–3.
We calculated the CHELPG atomic charges (see Experimental
Section) of the X atom of the XMe3 group and the attached
three carbon and nine hydrogen atoms for the docked confor-
mations for 1–3 and the minimized energy conformation for 4
(see the Supporting Information, Table S1). The average partial
+
charges of the nine hydrogen atoms of XMe3 show a very
slight incremental trend of +0.1506 (for 3), +0.1596 (for 2),
and +0.1652 (for 1). The calculated partial charge of hydrogen
atoms in the neutral tert-butyl group of 4 is +0.0884. These re-
sults are in agreement with trends in binding affinities as ob-
served by NMR spectroscopy, that is, the more positively
Enzyme kinetics experiments
+
charged H atoms of the XMe3 substrates result in stronger
Kinetics experiments were conducted at 296 K in Tris buffer
(20 mm) and NaCl (200 mm) at pH 7.5. To a premixed solution of
psBBOX (400 nm), FeSO4 (50 mm), 2OG (1.5 mm), and ascorbate
(5 mm) was added the substrate in a range of different concentra-
tions. After 1 min, an aliquot (20 mL) of the reaction mixture was
quenched with MeCN (80 mL). Subsequently the sample was ana-
lyzed by LC-MS. Each experiment was performed in duplicate.
cation–p interactions with the psBBOX aromatic cage.
Conclusions
In conclusion, our substrate analogue studies employing both
turnover and binding assays with purified recombinant
enzyme clearly support the proposal that recognition and
BBOX-catalyzed hydroxylation of gBB involve energetically fa-
vorable cation–p interactions between the positively charged
trimethylammonium group of gBB and the aromatic cage of
psBBOX. The observation that the neutral carbon analogue of
gBB does not bind to psBBOX and does not undergo psBBOX-
catalyzed hydroxylation in our assays further supports this
view. Furthermore, the results reveal that the positively
charged trimethylphosphonium and trimethylarsonium ana-
logues of gBB are good substrate mimics and have a potential
to act as small-molecule probes for functional studies of carni-
tine biosynthesis. Thus, for example, the enzymatic conversion
of the phosphorus analogue could be probed by 31P NMR
spectroscopy, and the 32P-labeled substrate might have a po-
NMR experiments
All NMR experiments were performed on a Bruker Avance III
700 MHz spectrometer equipped with a TCI inverse cryoprobe and
on
a Bruker Avance II 500 MHz spectrometer equipped with
a 5 mm 13C(1H) dual cryoprobe at 298 K and data analyzed using
Bruker Topspin 3.2. All spectra were processed with a Lorentzian
line broadening of 0.3 Hz. The solutions were buffered in 50 mm
Tris-D11·HCl pH 7.5, in 90:10 H2O/D2O. Bruker MATCH 3 mm diame-
ter and 5 mm NMR tubes, with total sample volumes of 160 mL
and 500 mL, respectively, were used. For the psBBOX-catalyzed sub-
strate-turnover experiments, the assay mixture was incubated in an
Eppendorf tube and whenever necessary the reaction was
quenched (stopped) with the addition of 1m HCl (5 mL) and the
spectrum was recorded for analysis. To measure the ligand binding
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constant by psBBOX titration, separate samples were prepared.
The PROJECT-CPMG pulse sequence (908x–[t–1808y–t–908y–t–
1808y–t]n–acquisition), as described by Aguilar et al.,[27] was used
to remove the broad resonances of the protein. The relaxation
edited (CPMG) 1H NMR experiments were recorded with a total
filter time of 32 ms. Protein titration data were fitted using Origin-
Pro 9.0 (Origin lab, Northampton, MA, USA) to calculate the ligand
binding constant (KD). Water suppression was achieved by presatu-
ration.
Keywords: cation–pi interactions · CÀH oxidation · enzyme
catalysis · molecular recognition · oxygenases
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Computational methods
For psBBOX protein-ligand docking simulation, the X-ray crystal
structure of hBBOX in complex with NOG and gBB was employed
(PDB entry: 3O2G) to make a homology structure of psBBOX using
Modeller 9v4.[28] Careful attention was paid to the assignment of
protonation states for Asp, Glu, His, and Lys residues. We calculat-
ed partial charge distribution by using quantum mechanical calcu-
lation at PBE1PBE/LanL2DZ level of theory.[29] Their partial charge is
assigned with charges from electrostatic potentials using a grid-
based method (CHELPG).[30] After reassigning CHELPG partial charg-
es to X–gBB using the above quantum mechanical calculation,
docking simulations of the X–gBB substrate with psBBOX were car-
ried using the empirical AutoDock[31] scoring function improved by
implementation of a new solvation model. The modified scoring
function has the following form [Eq. (1)]:
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ꢀ
ꢁ
ꢀ
ꢁ
XX
XX
Aij Bij
Cij Dij
aq
bind
DG
¼ WvdW
12 À
þ Whbond
EðtÞ 12 À
10
rij
ri6j
rij
r
ij
i¼1 j>1
i¼1 j>1
XX
q1q2
À Á
À
þWelec
þ WtorNtor þ DGsol
e rij rij
i¼1 j>1
(
!
)
i¼j
i¼j
atoms
ri2j
2s2
ri2j
2s2
X
X
X
¼
Si Omi ax
Vje
þ Pi
Vje
i
j
j
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ð1Þ
where WvdW, Whbond, Welec, Wtor, and Wsol are the weighting factors
for the van der Waals, hydrogen-bond and electrostatic interac-
tions, the torsional term, and the desolvation energy of the inhibi-
tors, respectively. rij represents the interatomic distance and Aij, Bij,
Cij, and Dij are related to the depths of the potential energy well
and the equilibrium separations between the two atoms. The hy-
drogen bond term has an additional weighting factor, E(t), repre-
senting the angle-dependent directionality. A cubic equation ap-
proach was applied to obtain the dielectric constant required to
compute the interatomic electrostatic interactions between
psBBOX and X–gBB. In the entropic term, Ntor is the number of sp3
bonds in the ligand. In the desolvation term, Si, Pi, and Vi are the
solvation parameter, self-solvation parameter, and fragmental
volume of atom i, respectively, whereas Occmi ax is the maximum
atomic occupancy.[32,33]
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ˇ
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Acknowledgements
We thank the Netherlands Organisation for Scientific Research,
the Wellcome Trust, Biotechnology and Biological Sciences Re-
search Council, Cancer Research UK, the European Union for fi-
nancial support. A.K. thanks Abdul Wali Khan University,
Mardan, Pakistan for financial support.
Received: September 18, 2015
Published online on December 14, 2015
Chem. Eur. J. 2016, 22, 1270 – 1276
www.chemeurj.org
1276 ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim