Ile351, Leu355 and Ile461 residues are essential for catalytic activity of bovine cytochrome P450scc (CYP11A1)

Article abstract of DOI:10.1016/j.steroids.2019.01.002

Cytochrome P450scc (CYP11A1) is a mammalian mitochondrial enzyme which catalyzes cholesterol side chain cleavage to form pregnenolone. Along with cholesterol, some other steroids including sterols with a branched side chain like β-sitosterol are the substrates for the enzyme, but the activity towards β-sitosterol is rather low. Modification of the catalytic site conformation could provide more effective β-sitosterol bioconversion by the enzyme. This study was aimed to find out the amino acid residues substitution of which could modify the conformation of the active site providing possible higher enzyme activity towards β-sitosterol. After structural and bioinformatics analysis three amino acid residues I351, L355, I461 were chosen. Molecular dynamics simulations of P450scc evidenced the stability of the wild type, double (I351A/L355A) and triple (I351A/L355A/I461A) mutants. Mutant variants of cDNA encoding P450scc with the single, double and triple mutations were obtained by site-directed mutagenesis. However, the experimental data indicate that the introduced single mutations Ile351A, Leu355A and Ile461A dramatically decrease the target catalytic activity of CYP11A1, and no activity was observed for double and triple mutants obtained. Therefore, isoleucine residues 351 and 461, and leucine residue 355 are important for the cytochrome P450scc functioning towards sterols both with unbranched (cholesterol) and branched (sitosterol) side chains.

Full text of DOI:10.1016/j.steroids.2019.01.002

Accepted Manuscript
Ile351, Leu355 and Ile461 residues are essential for catalytic activity of bovine
cytochrome P450scc (CYP11A1)
Anna V. Glyakina, Nicolai I. Strizhov, Mikhail V. Karpov, Nikita V.
Dovidchenko, Bakhyt T. Matkarimov, Ludmila V. Isaeva, Vera S. Efimova,
Mikhail A. Rubtsov, Ludmila A. Novikova, Marina V. Donova, Oxana V.
Galzitskaya
PII:
S0039-128X(19)30004-2
https://doi.org/10.1016/j.steroids.2019.01.002
STE 8362
DOI:
Reference:
Steroids
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 October 2018
28 December 2018
7 January 2019
Please cite this article as: Glyakina, A.V., Strizhov, N.I., Karpov, M.V., Dovidchenko, N.V., Matkarimov, B.T.,
Isaeva, L.V., Efimova, V.S., Rubtsov, M.A., Novikova, L.A., Donova, M.V., Galzitskaya, O.V., Ile351, Leu355
and Ile461 residues are essential for catalytic activity of bovine cytochrome P450scc (CYP11A1), Steroids (2019),
doi: https://doi.org/10.1016/j.steroids.2019.01.002
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Ile351, Leu355 and Ile461 residues are essential for catalytic activity of
bovine cytochrome P450scc (CYP11A1)
Anna V. Glyakinaa,b, Nicolai I. Strizhovd, Mikhail V. Karpovc,d, Nikita V. Dovidchenkoa, Bakhyt T.
Matkarimove, Ludmila V. Isaevaf, Vera S. Efimovag, Mikhail A. Rubtsovg,h, Ludmila A. Novikovaf,
Marina V. Donovad*, Oxana V. Galzitskayaa*
aInstitute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region 142290,
Russia
bInstitute of Mathematical Problems of Biology, Russian Academy of Sciences, Keldysh Institute of
Applied Mathematics, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia
cInstitute of Biochemistry and Physiology of Microorganisms, Federal Research Center “Pushchino
Center for Biological Research of the Russian Academy of Sciences”, 142290 Pushchino, Moscow
Region, Russia;
dPharmins, Ltd., R&D, 142290 Pushchino, Moscow Region, Russia
eNational Laboratory Astana, Nazarbayev University, Astana, Kazakhstan
fBelozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University,
Leninskie Gory, 1/40, 119234, Moscow, Russia
gDepartment of Molecular Biology, Faculty of Biology, M.V. Lomonosov Moscow State University,
Leninskie Gory, 1/12, 119234, Moscow, Russia
hDepartment of Biochemistry, Institute of Translational Medicine and Biotechnology, I.M. Sechenov
First Moscow State Medical University (Sechenov University), 119991, Moscow, Russia
*Correspondence: Dr. Marina Donova, Institute of Biochemistry and Physiology of Microorganisms,
Federal Research Center “Pushchino Center for Biological Research of the Russian Academy of Sciences”,
142290 Pushchino, Moscow Region, Russia donova@ibpm.pushchino.ru
Dr. Oxana Galzitskaya, Institute of Protein Research, Russian Academy of Sciences, Pushchino,
Moscow Region, 142290 Russia E-mail: ogalzit@vega.protres.ru
1

Abstract
Cytochrome P450scc (CYP11A1) is a mammalian mitochondrial enzyme which catalyzes cholesterol
side chain cleavage to form pregnenolone. Along with cholesterol, some other steroids including
sterols with a branched side chain like β-sitosterol are the substrates for the enzyme, but the activity
towards β-sitosterol is rather low. Modification of the catalytic site conformation could provide more
effective β-sitosterol bioconversion by the enzyme. This study was aimed to find out the amino acid
residues substitution of which could modify the conformation of the active site providing possible
higher enzyme activity towards β-sitosterol. After structural and bioinformatics analysis three amino
acid residues I351, L355, I461 were chosen. Molecular dynamics simulations of P450scc evidenced
the stability of the wild type, double (I351A/L355A) and triple (I351A/L355A/I461A) mutants. Mutant
variants of cDNA encoding P450scc with the single, double and triple mutations were obtained by site-
directed mutagenesis. However, the experimental data indicate that the introduced single mutations
Ile351A, Leu355A and Ile461A dramatically decrease the target catalytic activity of CYP11A1, and no
activity was observed for double and triple mutants obtained. Therefore, isoleucine residues 351 and
461, and leucine residue 355 are important for the cytochrome P450scc functioning towards sterols
both with unbranched (cholesterol) and branched (sitosterol) side chains.
Key words: P450scc; catalytic activity; cholesterol; β-sitosterol; mutations; MD simulations
2

Graphical abstract
Highlights
The considered cytochrome P450 enzymes have low sequence identities, but quite similar three-
dimensional structures.
The cytochrome P450 proteins are depleted in Ala and Gly and enriched with Met, Phe, Ile versus the
proteome values.
Residues Ile351, Leu355 and Ile461 are essential for the functioning of cytochrome P450scc towards
cholesterol and β-sitosterol.
Abbreviations:
P450scc, CYP11A1 – cytochrome P450scc
Adx – Adrenodoxin
AdR – Adrenodoxin reductase
MD simulations – Molecular dynamics simulations
RMSD – Root mean square deviation
3D structure – three-dimensional structure
SDS-PAGE – sodium dodecyl sulfate polyacrylamide gel electrophoresis
3

Introduction
The cytochrome P450 enzymes (P450s or CYPs) are known to form a large superfamily of the
enzymes, which are the external monooxygenases activating molecular oxygen via their heme iron and
catalyze variety of oxidations of the numerous exogenous and endogenous molecules [1]. CYPs are
quite diverse in their functions, types of enzymatic activity and substrate specificity [2]. The oxidation
reactions with involvement of CYPs include transformations of drugs, xenobiotics, synthesis and
transformation of steroids, and other important reactions [3]. These capacities make CYPs essential for
the endogenous metabolism in all living systems.
57 CYPs genes and 58 pseudogenes were identified in humans. Most part of 18 families of
cytochrome P450 genes and 43 subfamilies are related to steroidogenesis, or steroid hydroxylations
(e.g. CYP2, CYP7A, CYP7B, CYP8B, CYP11, CYP17, CYP19, CYP24, CYP27A, CYP27B, CYP39,
CYP46, CYP51)[2]. Mutations in the CYPs genes or deficiencies of the enzymes are responsible for
several human diseases [4–7]. Induction of some CYPs is considered as a risk factor for several types
of cancer since these enzymes can convert procarcinogens to carcinogens [8].
Despite the low homology of the primary structures between CYPs of different families (often
less than 20%), their secondary and tertiary structures are highly conservative thus determining the
general mechanism of the reaction catalysis [9]. A high degree of sequence and structural homology of
the heme-binding site was reported for CYPs [10]. The first three-dimensional (3D) structures were
represented for soluble bacterial CYPs by Poulos et al. [11]. Unlike bacterial CYPs, mammalian ones
are membrane associated proteins. Current data on the 3D structures of P450 enzymes evidence that all
CYPs have similar structural elements: 12 helices (denoted as A–L) and loops [12,13]. In mammalian
CYPs in addition to the common CYP fold helices, other helices were described mainly in the F/G
loop region such as additional helices in CYP11A1 termed A′, G′ and K″ [14]. These helices seem to
provide the enzyme binding to membrane, forming substrate access channel, and play a role in
substrate recognition. All CYPs contain heme (an iron-containing protoporphyrin IX) located between
the helices I and L.
Diversity of the CYPs substrate specificities is explained by the differences in their active-site
structures. The most important segments/amino acid residues of the P450s proteins contacting the
substrate and providing the substrate specificity and regioselective hydroxylation both in bacterial and
mammalian CYPs are the loop between the B and C helices positioned over the heme [15–17], I helix,
located in the active site of all CYPs [16–18] and the loop between the F and G helices [12].
Cytochrome P450scc (CYP11A1) is a mammalian mitochondrial enzyme catalyzing oxidation
cholesterol to pregnenolone (see Fig. 1). This reaction takes place mainly in the male and female
reproductive tissues and the adrenal gland and represents the first, rate-limiting and hormonally
4

regulated step in the synthesis of all steroid hormones in mammals. The CYP11A1 gene is found only
in vertebrates [19].
Figure 1. Chemical reactions catalysed by cytochrome P450scc (CYP11A1) for both substrates
(cholesterol, β-sitosterol) with conversion to product – pregnenolone.
The natural redox partners of CYP11A1 are the [2Fe2S] ferredoxin, adrenodoxin (Adx) and
NADPH-dependent ferredoxin reductase, adrenodoxin reductase (AdR). Adx transfers electrons from
AdR to the heme iron in CYP11A1. The terminal electron acceptor CYP11A1 takes up water-insoluble
cholesterol from the inner mitochondrial membrane and converts it to the less hydrophobic product
pregnenolone, removing the unpolar side chain via the three sequential reactions: production of 22R-
hydroxycholesterol (22HC), 20α,22R-dihydroxycholesterol and further C20-C22 cleavage, which
occurs at a single active site on the CYP11A1 molecule [20].
5

To date, several CYP11A1 models have been designed by computer modeling based on the
experimental 3D structures of cytochromes P450cam, P450-BM3, P450terp, P4502C5 and P4502B4
[21–24]. Storbek et al. [25] presented a model of the active site of P450scc which was created based on
the crystalline structures of CYP102 и CYP2C5. In 2011, the crystalline 3D structure of the bovine
[20] and human CYP11A1 [14] was revealed by X-ray crystallography. Besides, the structures of
CYP11A1 bound with cholesterol, the reaction intermediates and Adx were resolved [14,20]. These
data allowed correct determination of the corresponding protein segments involved in the enzyme
binding to the mitochondrial membrane and interaction with the redox partner. Sequence alignment of
different mammalian P450scc and site-directed mutagenesis in combination with biochemical methods
and analysis of the created 3D-structure models of the protein allowed to obtain essential information
about the organization of the functionally important protein sites, namely, the site responsible for
protein interaction with the inner mitochondrial membrane, the binding site with Adx, the site of
interaction with the substrate, and the catalytic center. These data showed that P450scc has three
additional helices to the common CYP fold termed A′, G′ and K″. Association of protein CYP11A1
with the membrane is mediated by the F-G loop and the A′ helix [23,26]. As identified by
computational analysis [20] and complying with the data obtained earlier using site-directed
mutagenesis and mass spectrometry studies [23,26,27], membrane-interacting residues in P450scc are
Asn19, Trp21, Leu22, Tyr25, Leu216, Leu219, and Phe220.
Cytochrome P450scc interacts electrostatically with its redox partner Adx by means of
positively charged amino acid residues of P450scc with negatively charged ones of Adx [22]. P450scc
amino acid residues important for this interaction were identified: according to the molecular modeling
and the biochemical studies [22], Lys377 and Lys381 (Lys339 and Lys343 for 3mzs) [28] and Lys267,
Lys403, Lys405 and Arg426 (these residues form four key salt bridges with Glu47, Asp76, Asp72 and
Glu73 of Adx) (Lys268, Lys404, Lys406 and Arg427 for 3mzs) play a role in the P450scc interaction
with adrenodoxin [22].
The size of the interacting surfaces and the number of amino acid residues involved in the
formation of the P450 complex with Adx was determined using multiple sequence alignment of the
mitochondrial cytochrome P450 proteins from subphylum Vertebrata [22]. Amino acids conserved for
all mitochondrial P450s were observed in the interface thus suggesting the presence of some common
mechanism for all mitochondrial CYPs. These conserved tertiary structure interactions probably
underlie the specificity of mitochondrial P450s for their shared redox partner Adx.
The enzymatic center of P450 is located inside the protein globule. After binding of the
substrate to the protein surface, it moves to the active site along the hydrophobic channel [29]. The
region of the primary binding of the substrate is located in the N-terminal part of the polypeptide chain
(amino acid residues 8-28) [29,30]. It is suggested that the interaction of Ile98 (Ile99 for 3mzs),
6

located in the B'-helix, with the I-helix opens a hydrophobic channel upon substrate binding to the
P450scc molecule [18].
Positioning of cholesterol in the active site of P450scc is due to its interactions with many
hydrophobic residues of the enzyme. The cholesterol 3β-OH group forms the H-bond with water but
does not directly interact with the protein. According to Mast et al. [20], the active site cavity in
P450scc consists of 39 amino acid residues from 14 different secondary structural elements. Only 12
of these residues are within 4 Å of the substrate. They are Ile351 (β1–4 strand region), Thr291 (I-
helix), Phe203 (F-helix), Met202 (F-helix), Ile85 (B-B' loop), Leu460 (β4-1/4-2 loop), Trp88 (B'-
helix), Gln356 (β1–4 strand region), Thr354 (β1–4 strand region), Val353 (β1–4 strand region), Ser352
(β1–4 strand region) and Phe458 (β1–4 strand). Strushkevich et al. [14] suggested that the amino acid
residues Leu101 (102), Trp87 (88), Phe202 (203), Ile461, Ser352, His39 (40), Tyr61 (62), Asn210
(211), Gln377 are responsible for the preparation of the correct conformation of the substrate for
further catalytic reaction, and the amino acid residues Gly287 and Thr291 are directly involved in the
transfer of the electron to the catalytic center. Thr291 was assumed to play an essential role in the
enzyme interaction with cholesterol, while Arg357 and Ser352 provide its interactions with the
intermediates thus providing hydroxylation and further C-C-cleaving [14]. Structural studies of
P450scc bound with substrates showed that during cascade reactions of cholesterol transformation in
the enzyme active center the position of the steroid core rings remains fixed during changes in the side
chain.
Up to now, little is known about the activity of CYP11A1 towards other steroid substrates. It
was shown that the enzyme transforms 7-dehydrocholesterol (7DHC), the precursor of vitamin D3, to
7-dehydropregnenolone (7DHP), whose photo-transformed 5,7-diene derivatives exhibit an anti-
proliferative effect against melanoma and leukemia cells [19]. The data on the activity of CYP11A1
towards plant sterols, phytosterols, which structurally differ from cholesterol by a branched side chain,
are scarce [19]. It was shown that the enzyme activity with sitosterol is much lower as compared with
cholesterol.
In this study, we have analyzed cholesterol metabolizing CYPs, identified the amino acid
residues essential for the enzyme potent activity towards branched sterols and experimentally
examined their significance for the enzyme activity.
2. Materials and Methods
2.1 Structural and Bioinformatics analysis and modeling
2.1.1 Search for structural homologues of P450scc
7

Twenty-seven structural homologues for P450scc with ability to interact with cholesterol were
selected from the Uniprot database. Of these, 14 structures have resolved 3D structures. Root mean
square deviation (RMSD) and sequence identity of the selected structures were calculated using the
program UCSF Chimera [31]. Flexible/disordered regions in the proteins were identified using the
FoldUnfold [32] and IsUnstruct [33] programs. Affinities for binding to different ligands (cholesterol
and β-sitosterol) of the homologous structures were calculated using the program AutoDock Vina [34].
The amino acid composition of the selected structures was compared with that of the
corresponding proteomes (human, bovine, bacterial). For the bacterial structures the comparison was
carried out with the average bacterial proteomes (averaging was carried out for 18 species of
Mycobacterium tuberculosis: ATCC 25177, F11, KZN 1435, CDC1551A, SUMu001, SUMu002,
SUMu003, SUMu004, SUMu005, SUMu006, SUMu007, SUMu008, SUMu009, SUMu010,
SUMu011, SUMu012, ATCC 25618, Oshkosh).
2.1.2 Molecular dynamics simulations
The objects of the simulation study were wild type proteins P450scc and CYP7A (pdb ids 3mzs
and 3v8d, respectively) and double (I351A/L355A) and triple (I351A/L355A/I461A) mutants of 3mzs
with two sterol substrates, - cholesterol and β-sitosterol. Replacement of the residues and insertion of
the cholesterol and β-sitosterol in the structure was made using the YASARA program [35].
Molecular dynamics simulations were performed using the program PUMA [36,37]. The
system of classical equations of motion for the atoms was resolved in the all-atom force field AMBER-
99 [38]. To maintain a constant temperature, a collisional thermostat [39,40] was used. The mass of
each virtual particle was one atomic mass unit, and the mean collision frequency of the atoms with
virtual particles was 10 ps–1. The simulations were performed at 300 K and 350 K. The equations of
motion were integrated numerically using the velocity version of the Verlet algorithm [41] with a time
step of 1 fs (10-15 s). For water molecules, the TIP3P [42] model was used. The fraction of secondary
structure during the modeling was calculated using the YASARA program [35].
2.2 Experimental procedures
2.2.1 Chemicals, enzymes, oligonucleotides and antibodies
Isopropyl-β-D-thiogalactopyranoside (IPTG), δ-aminolevulinic acid (ALA), diaminobenzidine
tetrahydrochloride hydrate (DAB), 20α-hydroxycholesterol, β-sitosterol, cholesterol oxidase and
horse-radish peroxidase-conjugated anti-rabbit antibodies were supplied by Sigma (USA) and
nitrocellulose filters Hybond-C extra by Amersham (USA). Growth media (LB, TB [43]) were
prepared using reagents from Difco (USA).
8

DNA-modifying enzymes, Pfu DNA polymerase, RNase A and DNA Extraction Kit were
purchased from MBI Fermentas (Lithuania). Oligonucleotides used in the study were synthesized by
Evrogen (Russia). Primary antibodies (IgG-fraction) against cytochrome P450scc were generously
provided by Prof. V. M. Shkumatov (Institute of Physical Chemical Problems, Minsk State University,
Belarus).
2.2.2 Bacterial strain and plasmids
E. coli strain DH5αc (supE44 lac U169 ( 80 lacZ M15) hsdR17 recA1end A1 gyrA96 thi-1
relA1) was supplied by Gibco-BRL, Germany.
Vector pTrc99A/P450scc [28] containing a hybrid trp/lac (trc) promoter and cDNA encoding
the mature bovine cytochrome P450scc was provided by M. R. Waterman (University of Texas,
Dallas, USA). Plasmid pBar_Triple [44] with the cDNAs of mature bovine cytochrome P450scc, AdR
[45] and Adx (4-108) [46] was constructed earlier in our laboratory.
2.2.3 Plasmids construction
The molecular cloning was performed by standard protocols [43]. The pTrc99A/CHL plasmid
containing the tricistronic cassette with the cDNAs encoding the mature forms of cytochrome P450scc,
AdR and Adx1-108 from bovine adrenal cortex was constructed on the base of the vector
pTrc99A/P450scc [28]. The resulting pTrc99A/CHL plasmid has the cDNAs in the following
sequential order: trc promoter, P450scc, AdR and Adx. The construction details of the plasmid will be
described elsewhere soon. The plasmids pBar_Twin[47] and pBar_Triple [44] served as the source of
the cDNAs encoding AdR and Adx1-108. The sequence of the constructed tricistronic cassette in the
pTrc99A/CHL plasmid was confirmed by the DNA sequencing.
Site-directed mutagenesis of P450scc was performed by standard methods with the use of PCR
primers carrying the GCC codon encoding the Ala amino acid residue for substitution of Ile351,
Leu355 and Ile461 in the protein sequence of P450scc. All five resulting mutant DNA sequences
(three single I351A, L355A, I461A, one double I351A/L355A and one triple I351A/L355A/I461A
mutants) were validated by Sanger automatic DNA sequencing. These plasmids including mutated
sequences, generated on the base of the parental plasmid pTrc99A/CHL were designated as
pTrc99A/CHL(Р450-I351A),
pTrc99A/CHL(Р450-L355A),
pTrc99A/CHL(Р450-I461A),
pTrc99A/CHL(Р450-I351A/L355A) and pTrc99A/CHL(Р450-I351A/L355A/I461A).
2.2.4 Protein expression, preparation of bacterial homogenates
9

Expression of the recombinant proteins in E. coli cells was carried out as described earlier[48].
The cell culture was incubated at 28 °C with shaking (160 rpm) in the presence of ALA (0.5 mM) and
ampicillin (100 g/ml) for 22 h. Expression of the proteins encoded by the tricistronic cassette was
induced by adding IPTG (0.5 mM) to the growth medium. E. coli cell homogenates were prepared as
described previously[49]. Debris was removed by centrifugation (4,000 g, 10 min). The supernatant
(homogenate) was used for P450scc activity measurements of the mutant/wild type proteins.
2.2.5 SDS-PAGE and Western immunoblotting
Cell lysates were analyzed by SDS-PAGE[50] followed by Western blotting [51]. Bacterial
cells were pelleted by centrifugation. Pellets were re-suspended in Sample Buffer [49] and disrupted
by boiling (2 min, 100 °C). Samples with 5 µg of total protein in 10 µl were loaded into each lane. The
protein concentration was measured according to Lowry et al. [52] The membranes with transferred
proteins separated by SDS-PAGE were incubated with the primary rabbit polyclonal antibodies against
P450scc (6.0 mg/ml) at 1: 7,500 dilution (v/v) followed by treatment with the conjugate of anti-rabbit
secondary antibodies with horseradish peroxidase at 1:15,000 dilution (v/v) and DAB peroxidase
substrate (0.25 mg/ml). As shown previously, the primary antibodies against bovine P450scc used in
this study demonstrate affinity both to natural mammalian proteins and to the recombinant P450scc
proteins expressed in E. coli [53].
2.2.6 Cholesterol hydroxylase/lyase activity measurements
Catalytic activity of the recombinant P450scc proteins was determined in vitro as described
previously [54]. The reaction mixture contained 1 mg of cell homogenate and 20α-hydroxycholesterol
or β-sitosterol (25 nmol) in 30 mM sodium phosphate buffer (pH 7.2) containing 0.05% Tween-20 in a
total volume of 0.5 ml. The reaction was induced by adding the NADPH-regenerating system and left
to proceed for 30 min at 37 °C. The formed pregnenolone was oxidized to progesterone by adding
cholesterol oxidase (0.5 U). The steroids were extracted with ethyl acetate; the precipitate obtained
after the evaporation of the extracts on a vacuum rotor (SpeedVac Concentrator, Savant, USA) was
dissolved in a sample buffer provided in the reagent kit “IFA PROGESTERONE” (Xema, Russia).
The content of progesterone was determined by means of this immunoassay kit based on
antiprogesterone antibodies according to the manufacturer's protocol.
3. Results and Discussion
3.1 Bioinformatics analysis of selected structures of P450scc
10

Fourteen structures of proteins with resolved 3D structures were selected as described above
(point 2.1.1) for the further analysis. Among them, there are nine structures from human, four from
bacteria, and one from yeast (Table 1). The comparison of the structures with bovine P450scc
(CYP11A1) (pdb id 3mzs) showed low (about 25%) identity, however, the 3D structures of these
proteins were quite similar (RMSD of about 2 Å) (Table 1).
Table 1. Identity and RMSD of the selected proteins with bovine P450scc (pdb id 3mzs), binding
energy with cholesterol, β-sitosterol, living organism
Pdb
id
Native ligand in pdb
Identit RMS
Binding energy,
kcal/mol
Organism
y
D
with
3mzs, 3mzs,
%
with
Cholestero
β-
sitostero
l
l
Å
2q9f
Cholest-5-en-3-yl hydrogen
sulfate
22
2.1
-11.7
-11.6
Homo sapiens
Cholesterol-sulfate
C27H46O4S
4wnu
2hi4
Quinidine
(9S)-6'-methoxycinchonan-9-ol
C20H24N2O2
2-phenyl-4h-benzo[h]chromen-
4-one
24
22
2.1
2.3
-8.4
-6.9
-7.2
-7.9
Homo sapiens
Homo sapiens
7,8-benzoflavone; alpha-
naphthoflavone
C19H12O2
1r9o
3n9y
4uhi
Flurbiprofen
C15H13FO2
Cholesterol
C27H46O
14
73
23
2.3
0.9
2.3
-9.7
-11.7
-11.3
-7.6
-11.5
-11.1
Homo sapiens
Homo sapiens
Homo sapiens
N-[(1R)-1-(3,4'-
difluorobiphenyl-4-yl)-2-(1H-
imidazol-1-yl)ethyl]-4-(5-
phenyl-1,3,4-oxadiazol-2-
yl)benzamide
C32H23F2N5O2
4dvq
Desoxycorticosterone
4-pregnen-21-ol-3,20-dione;
doc; 21-hydroxyprogesterone
C21H30O3
37
24
22
18
1.6
1.9
2.4
2.3
-7.3
-10.5
-13.0
-9.1
-11.4
-10.3
-12.1
-9.0
Homo sapiens
Homo sapiens
Homo sapiens
Mycobacteriu
4d6z Tert-butyl {6-oxo-6-[(pyridin-3-
ylmethyl)amino]hexyl}carbamat
e
C17H27N3O3
3v8d
2wm
(3beta,8alpha,9beta)-3-
hydroxycholest-5-en-7-one
7-ketocholesterol
C27H44O2
-
11

5
m
tuberculosis
Mycobacteriu
m
tuberculosis
Mycobacteriu
m
tuberculosis
Bacillus
subtilis
Saccharomyce
s
2xkr
Tetraethylene glycol
C8H18O5
22
17
2.3
2.4
-7.7
-9.6
-7.3
-9.3
2x5l
-
-
4rm4
4lxj
23
24
2.2
2.2
-
-
Lanosterol
C30H50O
-9.3
-10.9
cerevisiae
The spatial superposition of all selected structures with each other is presented in Fig.2. Dark
red regions correspond to the areas with a smaller RMSD relative to the P450scc structure. The results
show that the topology is preserved in the heme region and in the areas adjacent to the catalytic center.
Figure 2. Spatial superposition of all 15 selected structures with each other. Dark red regions
correspond to the areas with a smaller RMSD relative to the structure of bovine P450scc (pdb id
3mzs).
The spatial superposition of the structure of bovine CYP11A1 (3mzs) on the structure of human
CYP7A1 (3v8d) is presented on Fig. 3. For each amino acid residue, the probability to be ordered or
disordered was calculated for all structures of selected proteins using the IsUnstruct program. The
results for two proteins bovine CYP11A1 (3mzs) and human CYP7A1 (3v8d) are represented in Fig. 3
(B, C). According to this program human CYP7A1 (3v8d) is the most unstructured in comparison with
the selected proteins and has the lowest calculated binding energies with cholesterol and β-sitosterol
(see Table 1). Analysis of the location of flexible regions for the structure of proteins showed that in
general they are located around the exit site of the reaction product. Therefore, it can be assumed that
these regions are important for the release of the reaction product.
12

Figure 3. (A) Spatial superposition of the structure of bovine CYP11A1 (pdb id 3mzs) on the structure
of human CYP7A1 (pdb id 3v8d). Unfolded (unstructured) regions, found by the FoldUnfold [32]
(http://bioinfo.protres.ru/ogu) and IsUnstruct [33] (http://bioinfo.protres.ru/IsUnstruct) programs are
indicated by yellow and red for CYP7A1 and CYP11A1 respectively. (B, C) Probability of each amino
acid residue to be ordered or disordered in structures CYP7A1 and CYP11A1 calculated using the
IsUnstruct program. Disordered regions are colored red.
The docking with cholesterol and β-sitosterol was performed for each of the selected structures (Table
1). In one case (pdb id 4rm4), the affinity value could not be determined, because there was no
significant protein fragment in the structure adjacent to the catalytic pocket. In most cases no
considerable difference in affinity values to the catalytic center between cholesterol and β-sitosterol
was observed. In addition, for most structures, the calculated affinity values are close to those for
bovine P450scc with cholesterol or β-sitosterol (about 12 kcal/mol) (Table 1). When the affinity values
are small, it should be taken into account that the docking was carried out using the original structure
obtained from the Protein Data Bank, and low affinity values do not exclude the possibility that for a
certain protein conformation (for example, after relaxation of the protein with ligand) the values may
vary.
13

The amino acid composition of the selected structures was analyzed and compared with the
corresponding proteome values. This analysis allows us to estimate the difference between the amino
acid compositions of an individual protein and a corresponding proteome. As seen from Fig. 4, the
selected bacterial proteins are enriched with Met, Phe, Ile, Gln, Asn, Glu, Asp, His, Lys and depleted
in Val, Ala, Gly, Thr, Arg versus the average M. tuberculosis proteomes (averaging was carried out for
18 species of Mycobacterium tuberculosis, see Methods section 2.1.1). In the selected human proteins,
in general, there are more Met, Phe, Ile, Leu, His and less Cys, Ala, Gly, Ser, Glu, Gln as compared
with the human proteome. The bovine P450scc protein is enriched with Met, Phe, Ile, Trp, Tyr, Asn,
His, Lys and depleted in Cys, Ala, Gly, Thr, Ser, Gln versus the bovine proteome. It is worth noting
that in all the selected proteins there are less Ala and Gly than in the average proteome values. Human
and bacterial proteins differ in charged amino acid residues Glu, Asp and Lys. Human proteins contain
less of these amino acid residues than the human proteome values, and bacterial proteins contain more
than the average bacterial proteome. The same is observed for Cys and Gln amino acid residues.
14

Figure 4. Comparison of the amino acid composition of the selected structures with the considered
proteomes (human, bovine and mycobacterial).
15

3.2 Molecular dynamics simulations of bovine P450scc
3.2.1 Simulations of wild type P450scc and CYP7A1
Molecular dynamics simulations of structures 3v8d (at T = 350 K) and 3mzs (at T = 300 K)
with the heme and cholesterol have been performed. According to the program IsUnstruct
(http://bioinfo.protres.ru/IsUnstruct), these structures were selected for the following modeling because
one of them (3v8d) has more and the other (3mzs) has less disordered residues (see Fig. 3) in
comparison with other structures.
Fig. 5 represents graphs of changes in the secondary structure during the modeling. Straight
lines on the graphs indicate the initial level of each type of secondary structure before the simulations.
Fig. 5 shows that the increase in the fraction of irregular structure is caused by destruction of α-helices.
In contrast, the β-structure is stabilized. The largest deviations from the initial structure were observed
for 3v8d because its modeling was performed at a higher temperature.
Figure 5. Different changes in secondary structure during simulations of structures 3v8d and 3mzs.
Straight lines on the graphs indicate the initial level of each type of secondary structure before
simulations.
Although a small change in the secondary structure during the simulations was observed, the
root mean square deviation (RMSD) calculated from the C-alpha atoms between the initial structure
and structures obtained after the simulation is within 3 Å (Fig. 6). Thus, these structures remain
relatively stable over the simulation time.
16

Figure 6. Spatial superposition of structures of 3v8d and 3mzs before (gray) and after modeling (blue).
3.2.2 Simulations of P450scc structures with mutated residues I351A, L355A,
I461A
Further, it was necessary to select several mutations in protein 3mzs, leading to an increase in
the size of the binding pocket, so that the sterol (cholesterol or β-sitosterol) molecule could fit there.
To this effect, the number of atom-atom contacts at the distance of 6 Å for each amino acid residue in
protein 3mzs with cholesterol was calculated. Amino acid residues involved somehow in the catalytic
reaction according to Strushkevich et al. [14] have not been considered. By these criteria, the following
mutations were selected: L102A, I351A, L355A and I461A. The affinities of β-sitosterol to various
mutant proteins based on the structure of 3mzs were calculated by using the program for docking
AutoDock Vina [34] (Table 2). According to the affinity of β-sitosterol, the best mutants were the
following: single – I351A and L355A, double – I351A/L355A, and triple – I351A/L355A/I461A. In
Table 2 they are represented in bold. We have found two SNPs only for Leu355
https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?geneId=1583. SNP data confirms our result
that I351, I461, and L355 replacements are lethal to the function of cytochrome P450scc. Two
synonymous L355 substitutions are very interesting. It should be mentioned that leucine is encoded by
6 codons. In our case we have CTG for Leu, for SNP carriers there are CTA and TTG codons. We see
them, but they are only synonymous.
17

Disposition of mutated I351, L355, and I461 residues is shown in Fig. 7. For the following
simulations the structures with double I351A/L355A and triple I351A/L355A/I461A mutations with
cholesterol and β-sitosterol were constructed using the YASARA program [35]. The molecular
dynamics simulations of these mutant constructions were performed at 300 K.
Table 2. Calculated affinities for binding of β-sitosterol to different P450scc mutant proteins
Protein
Binding energy, kcal/mol
-12.5
Native protein with cholesterol
(oxidized form, C22)
Native protein with cholesterol
(normal form)
Native protein with β-sitosterol
-12.2
-12.1
I351A
I351A/I461A
I351A/L355A
I461A
L102A
L102A/I351A
L102A/I461A
L102A/L355A
L355
-11.9
-11.7
-11.9
-11.9
-11.6
-11.4
-11.4
-11.5
-12.0
-11.8
-11.7
-11.3
-11.4
-11.3
-11.2
L355A/I461A
I351A/L355A/I461A
L102A/I351A/I461A
L102A/I351A/L355A
L102A/L355A/I461A
I351A/L355A/I461A/L102A
Figure 7. Spatial structure of bovine CYP11A1 (pdb id 3mzs). Mutated residues I351, L355
and I461 are shown as space-filling models.
18

As seen from Fig. 8, the introduced mutations do not destroy the secondary structure of the
protein, and it remains stable during the simulation. The RMSD between the initial structures and
structures after the simulation is about 3 Å. These mutations also do not dramatically change the size
of the binding pocket.
3mzs
beta-sitosterol
T=300K
3mzs
cholesterol
T=300K
60
40
20
0
60
40
20
0
Helix
Sheet
Turn
Coil
Helix
Sheet
Turn
Coil
0
1000
2000
3000
4000
0
1000
2000
3000
4000
t, ps
t, ps
3mzs (I351AL355A)
cholesterol
3mzs (I351AL355A)
beta-sitosterol
T=300K
60
40
20
0
60
40
20
0
T=300K
Helix
Sheet
Turn
Coil
Helix
Sheet
Turn
Coil
0
1000
2000
3000
4000
0
1000
2000
3000
4000
t, ps
t, ps
19

3mzs (I351AL355AI461A)
cholesterol
3mzs (I351AL355AI461A)
beta-sitosterol
T=300K
60
40
20
0
60
40
20
0
T=300K
Helix
Sheet
Turn
Coil
Helix
Sheet
Turn
Coil
0
1000
2000
3000
4000
0
1000
2000
3000
4000
t, ps
t, ps
Figure 8. Different changes in the secondary structure during simulations of structure 3mzs and its
double I351A/L355A and triple I351A/L355A/I461A mutant proteins with two substrates cholesterol
and β-sitosterol. Straight lines on the graphs indicate the initial level of each type of secondary
structure before simulations.
3.3 Effect of I351A, L355A, I461A mutations on the catalytic reaction rate of
P450scc
Molecular dynamics simulations have demonstrated that the introduced mutations do not
destroy the 3D structure of the cytochrome P450scc in silico. However, it is important to evaluate how
these mutations will affect the catalytic reaction rate of the enzyme.
3.3.1 Mutagenesis of bovine P450scc
The I351A, L355A, I461A mutations were introduced into the cDNA encoding P450scc
protein by site-directed mutagenesis. To this end, mutant variants of cDNA encoding P450scc with
single, double and triple mutations were produced.
The generation of each of the mutant variants of the P450scc cDNA consisted of two steps. The
first step was the production of PCR fragment encoding the cytochrome P450scc with the mutation
using the oligonucleotide guided site-specific mutagenesis method. During the second step the
resulting mutant PCR fragment was used to replace the corresponding fragment of the wild-type
P450scc cDNA in the tricystronic pTrc99A/CHL plasmid.
All five resulting mutant DNA sequences (three single I351A, L355A, I461A, one double
I351A/L355A and one triple I351A/L355A/I461A mutants) were validated with Sanger automatic
DNA sequencing (the numbers of the amino acid residues correspond to their position in the sequence
of the mature form of the protein). Based on the vector pTrc99A/CHL the generated plasmids carrying
mutated cDNA of the cytochrome P450scc were designated as: pTrc99A/CHL(Р450-I351A),
20

pTrc99A/CHL(Р450-L355A), pTrc99A/CHL(Р450-I461A), pTrc99A/CHL(Р450-I351A/L355A) and
pTrc99A/CHL(Р450-I351A/L355A/I461A).
3.3.2 Expression of mutated P450scc proteins in E. coli cells
E. coli DH5αc cells transformed with the recombinant plasmids encoding the mutant and wild-
type cytochrome P450scc were grown as described in 2.2.4 to induce expression of the heterologous
cDNAs. Cell lysates were analyzed with SDS-PAGE, followed by immunoblotting for identification of
the produced Р450scc proteins. As a result of immunodetection with the antibodies against P450scc,
we found that the proteins capable of binding to the specific antibodies have a molecular weight of ~53
kDa for all strains (Fig. 9, lanes 2-7). This molecular weight corresponds to the full-length P450scc.
Moreover, the analysis did not reveal any significant differences in the expression levels of the wild-
type P450scc and the proteins of P450scc with the introduced amino acid substitutions (mutations
I351A, L355A, I461A, I351A/L355 and AI351A /L355A /I461A) (Fig. 9). Thus, the data obtained
clearly indicate that the mutated P450scc proteins are stable and no protein degradation could be
detected.
Figure 9. Western blot analysis of wild-type and mutant forms of P450scc expressed in E. coli cells.
Protein separation of cell lysates (5 µg/lane) was performed by 10% SDS-PAAG. 1 – standard protein
markers (130, 95, 72, 55, 43, 34 and 26 kDa) stained with Coomassie Brilliant Blue; 2 - 7 - lysates of
E. coli cells transformed with pTrc99A/CHL, pTrc99A/CHL(Р450-I351A), pTrc99A/CHL(Р450-
L355A),
pTrc99A/CHL(Р450-I461A),
pTrc99A/CHL(Р450-I351A/L355A),
pTrc99A/CHL(Р450I351A/L355A/I461A), respectively; 8 – lysate of control plasmid-less E. coli
cells. The membrane (2-8 lanes) was probed with antibodies against P450scc.
3.3.2 Side-chain cleavage activity of P450scc mutant proteins
21

Sterol side-chain cleavage activity of the mutant and wild-type cytochrome P450scc proteins was
analyzed as the rate of the side-chain cleavage from substrates: 20α-hydroxycholesterol and β-
sitosterol. Substrate conversion rate to pregnenolone was recorded using the ELISA method. The
results of the activity measurements (minus the background value obtained for the cell homogenate of
control E. coli DH5αc strain) are shown in Table 3. Mean values (minus K) from 4 to 6 measurements
using 20α-hydroxycholesterol and from 2 measurements conducted with β-sitosterol are represented.
K, the activity measured in the homogenate of control untransformed (without P450scc) cells was 15.9
± 6.3 (with 20α-hydroxycholesterol) and 0.57 ± 0.07 (with β-sitosterol) (pmol of pregnenolone/mg
homogenate×h, respectively. As follows from the presented results, the introduction of any of the
mutations leads to a sharp decrease or a complete loss of protein activity.
Table 3. Catalytic activity of wild-type and single mutant P450scc proteins (pmol of pregnenolone/mg
homogenate×hour)*
Protein
P450scc
I351A
20α-hydroxycholesterol
540.00±9.40
β-sitosterol
129.00±29.50
1.24±0.36
21.40±7.70
0
11.45±8.03
L355A
I461A
383.00±7.92
12.09±4.50
*The data presented mean the values of activity measurement minus the background value obtained for
the cell homogenate of control E. coli DH5αc strain.
The maximum recorded activity for a protein with a single amino acid replacement was found
for P450scc mutant L355A, which is lower by ~ 1.4 times with 20α-hydroxycholesterol and ~ 6.5
times with β-sitosterol than the activity of native P450scc. I351A and I461A substitutions in the
cytochrome P450scc molecule resulted in a dramatic decrease in activity with both substrates: 20α-
hydroxycholesterol (more than 50 times) and with β-sitosterol (to almost/complete loss of activity).
Moreover, the introduction of another additional mutation totally inactivates P450scc protein. All the
double (I351A/L355A) and triple (I351A/L355A/I461A) mutants that we used were completely
inactive.
4. Conclusions
We examined 14 structural homologues of P450scc CYP11A1 capable of interacting with
cholesterol. These structures have low homology in the primary structure (about 25%), but are very
similar in the 3D structure (RMSD is about 2 Å). It turned out that for these structures, only one amino
22

acid residue from those involved in the preparation of the correct conformation of cholesterol is
conservative (Ile461). However, the amino acid residues that are involved in electron transfer are
strictly conservative (Gly287 and Thr291). For these 14 proteins the heme binding site and the areas in
the vicinity of the catalytic center are structurally very similar. The results of docking of cholesterol
and β-sitosterol suggest that for half of the structures (7 of 14) the substrate molecules have the free
energy similar to that of P450scc in the catalytic pocket. Molecular dynamics simulations of P450scc
(pdb 3mzs) and its double (I351A/L355A) and triple (I351A/L355A/I461A) mutants with cholesterol
and β-sitosterol suggest that these structures are stable during 3 ns simulations. However, the
experimental data have proved that the introduced single mutations, either I351A, L355A and I461A
dramatically decrease the catalytic activity of P450scc with both substrates: cholesterol and β-
sitosterol. Thus, we can conclude that the catalytic activity of the enzyme is affected by the
replacement of amino acid residues not directly interacting with the substrate and proved to be
essential for the function
The results contribute to the better understanding of structure-function relationship of the
unique and key enzyme of steroidogenesis – cytochrome P450scc.
Acknowledgments: We are grateful to T.B. Kuvshinkina for assistance in preparation of the
manuscript. This work was supported by the Russian Science Foundation (No. 18-14-00321) for OVG,
AVG and NVD. MD, MK and NS are greatly acknowledged the Russian Science Foundation (Grant
No. 18-14-00361) for the support of their work; LN, LI, VE and MR are greatly acknowledged the
Russian Foundation for Basic Research (Grant No. 16-54-00139). BM is greatly acknowledged the
Nazarbayev University ORAU. The work was supported by the Ministry of Science and Higher
Education of the Russian Federation (agreement No. 075-11-2018-096 / 14.588.21.0008) within the
framework of implementing the Federal Target Program (for MVD, MVK and NIS).
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Highlights
The considered cytochrome P450 enzymes have low sequence identities, but quite similar three-
dimensional structures.
The cytochrome P450 proteins are depleted in Ala and Gly and enriched with Met, Phe, Ile versus the
proteome values.
Residues Ile351, Leu355 and Ile461 are essential for the functioning of cytochrome P450scc towards
cholesterol and β-sitosterol.
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