Computational Biology and Chemistry 96 (2022) 107620

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Computational Biology and Chemistry

journal homepage: www.elsevier.com/locate/cbac

In silico analysis and characterization of medicinal mushroom cystathionine

beta-synthase as an angiotensin converting enzyme (ACE)
inhibitory protein
Neng-Yao Goh a, Muhammad Fazril Mohamad Razif a, Yeannie Hui-Yeng Yap b, Chyan
Leong Ng c, *, Shin-Yee Fung a, *
a
Medicinal Mushroom Research Group (MMRG), Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
b
Department of Oral Biology and Biomedical Sciences, MAHSA University, Selangor, Malaysia
c
Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

A R T I C L E I N F O A B S T R A C T

Keywords: Angiotensin-converting enzyme (ACE) regulates blood pressure and has been implicated in several conditions
Cystathionine beta-synthase including lung injury, fibrosis and Alzheimer’s disease. Medicinal mushroom Ganordema lucidum (Reishi) cys­
Angiotensin-converting enzyme tathionine beta-synthase (GlCBS) was previously reported to possess ACE inhibitory activities. However, the
Ganordema lucidum
inhibitory mechanism of CBS protein remains unreported. Therefore, this study integrates in silico sequencing,
Tiger milk mushroom
structural and functional based-analysis, protein modelling, molecular docking and binding affinity calculation
Lignosus rhinocerus
Protein-protein docking to elucidate the inhibitory mechanism of GlCBS and Lignosus rhinocerus (Tiger milk mushroom) CBS protein
Chloride blocking (LrCBS) towards ACE. In silico analysis indicates that CBSs from both mushrooms share high similarities in terms
Protein inhibitor of physical properties, structural properties and domain distribution. Protein-protein docking analysis revealed
that both GlCBS and LrCBS potentially modulate the C-terminal domain of ACE (C-ACE) activity via regulation of
chloride activation and/or prevention of substrate entry. GICBS and LrCBS were also shown to interact with ACE
at the same region that presumably inhibits the function of ACE.

1. Introduction
Angiotensin-converting enzyme (ACE), also known as peptidyl-
dipeptidase A, CD143, kininase II or EC 3.4.15.1, is part of the renin-
angiotensin-aldosterone system (RAAS) (Skeggs, 1993; Ruiz-Dueñas
et al., 2020). Two distinct isoforms, namely somatic ACE (sACE) and
testicular ACE (tACE) are found in human (Erdös, 1990). sACE is
composed of 1306 residues; it contains N-terminal (N-ACE) and C-ter­
minal (C-ACE) domains that share similar folding pattern, each con­
taining a functional catalytic site that contributes to the ability of ACE to
hydrolyze a variety of peptides such as bradykinin, angiotensin I (AngI),
substance P, amyloid peptides, tetrapeptide N-acetyl-Ser-Asp-Lys-Pro,
neurotensin and enkephalins (Bernstein et al., 2011; Cozier et al.,
2020b). tACE found in testis, is a 701-residue protein identical to the
C-terminal domain of sACE. The high-resolution crystal structure of
tACE is widely used in the drug discovery of C-ACE specific inhibitors
(Acharya et al., 2003).
ACE is a substrate- and zinc-dependent peptidase, activated by the
presence of chloride ions (Wei et al., 1991). The crystal structure of tACE
shows the existence of two chloride ions embedded in each of the
chloride binding pockets. The chloride ion furthest away from the zinc
ion is known to interact with residues Arg186, Trp485, Arg489 and
water molecules at the CL1 binding pocket (CL1C-domain). The second
chloride ion, closer to the zinc ion, was shown to interact with Arg522
and Tyr224 at a region called the CL2 binding pocket (CL2C-domain) (Guy
et al., 2003). Nonetheless, the residues in CL1C-domain was found to not
be fully conserved in the N-terminal-domain ACE (N-ACE) (Corradi
et al., 2006). Hence, only one chloride ion is present in the N-ACE
chloride 2 binding pocket (CL2N-domain) coordinated by Arg500 and
Tyr202. C-ACE requires significantly higher chloride concentration for
optimal catalytic activity compared to N-ACE (Wei et al., 1991),
attributed to the importance of chloride ion in the stabilization of the
ACE active site (Rushworth et al., 2009; Yates et al., 2014).
Chemically synthesized ACE inhibitors i.e. (captopril and trando­
lapril) have been successfully applied clinically to reduce mortality in
patients with high blood pressure and heart failure (De Leo et al., 2009).

* Corresponding authors.
E-mail addresses: clng@ukm.edu.my (C.L. Ng), syfung@um.edu.my (S.-Y. Fung).

https://doi.org/10.1016/j.compbiolchem.2021.107620
Received 16 August 2021; Received in revised form 20 December 2021; Accepted 20 December 2021
Available online 23 December 2021
1476-9271/© 2021 Elsevier Ltd. All rights reserved.
N.-Y. Goh et al. Computational Biology and Chemistry 96 (2022) 107620

Other than blood pressure control, the regulation of ACE and its peptide
substrates by ACE inhibitors show promising results against conditions
such as proteinuria, lung injury, Alzheimer’s disease, oncological dis­
eases and sepsis (Pacurari et al., 2014; Bernstein et al., 2012; Yn Liu
et al., 2019; S. Liu et al., 2019; Regulska et al., 2019; Kutyrina et al.,
1994). Recently, some ACE inhibitors are predicted with potential to be
repurpose as an angiotensin-converting enzyme 2 (ACE2) inhibitor
against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
infection (Choi et al., 2020); attributed to the homology between ACE
and ACE2 with 42% sequence identity and 75% similarity (Lubbe et al.,
2020).
Medicinal mushrooms (MM) are macroscopic fungi that are used to
alleviate and/or prevent diseases (Abdel-Azeem et al., 2019). MM have
been studied for their naturally occurring ACE inhibitors due to the
incidence of side effects, such as cough and skin rashes, caused by
synthetic drugs, (Choi et al., 2001; Hyoung Lee et al., 2004; Gao et al.,
2012). Reishi mushroom or Ganordema lucidum is a widely recognized
medicinal basidiomycete used in traditional remedy throughout China
and Southeast Asia (Liu et al., 2015; Wachtel-Galor et al., 2011).
Mohamad Ansor et al. (2013) reported the presence of ACE inhibitory
proteins in the proteome of G. lucidum, one of it being cystathionine
beta-synthase (CBS) (Mohamad Ansor et al., 2013). Within the same
fungi family, tiger milk mushroom or Lignosus rhinocerus is similarly
endorsed for its medicinal properties and is commonly consumed raw or
drank as a decoction for the treatment of cough, asthma and fever by
indigenous communities (Abdullah et al., 2013). L. rhinocerus was pre­
viously shown to exhibit immunomodulation, anti-inflammatory,
anti-proliferative, antioxidative and anti-glycation properties (Lee
et al., 2018; Johnathan et al., 2016; Lai et al., 2014; Pushparajah et al.,
2016; Yap et al., 2018). Of interest, it was also annotated in both L.
rhinocerus genomic and transcriptomic data the presence of CBS-like
proteins (Yap et al., 2015b; Yap et al., 2014; Yap et al., 2015b).
CBS (EC 4.2.1.22) is a key enzyme in the reverse transsulfuration
pathway that catalyzes the formation of cystathionine from homocys­
teine and serine along with the biogenesis of hydrogen sulfide (H2S)
(Chen et al., 2004). Depending on the organism, CBS adopts different
magnitudes of domain distribution and oligomeric variation (Fig. 1).
Based on previous studies, five types of domain distribution (termed
Class 1–5) and 3 major oligomers have been identified. Class 1, con­
sisting of one single type II family pyridoxal-5′ -phosphate

Fig. 1. Structural features of various CBS. (A) Diagrammatical representation of catalytic domains predicted by SMART for GlCBS (position 11–329) and LrCBS
(position 11–330). (B) Graphical representation of CBS domain architectures and oligomeric information in different organisms. Class 1, a single catalytic domain
found in Bacillus anthracis (BaCBS), Lactiplantibacillus plantarum (LpCBS), Helicobacter pylori (HpCBS), L. rhinocerus (LrCBs) and G. Lucidum (GlCBS); Class 2, two
tandem catalytic domains reported in C. elegans (CeCBS); Class 3, the combination of a catalytic domain and Bateman module reported in S. cerevisiae (ScCBS) and
T. gondii (TgCBS). Class 4, modulation of 3 domains of heme, catalytic and Bateman Module, reported in Apis mellifera (AmCBS). Class 5, reported in human, contains
the Bateman module with AdoMet binding site. The degree of oligomerization is represented by the number of circles. Single asterisk represents solved crystal
structure. Double asterisk represents predicted structure through modelling protocol.

2
N.-Y. Goh et al.
(PLP)-dependent enzyme, has a catalytic domain that functions as a PLP
co-factor binding site via the presence of a Schiff base aided by a
conserved lysine (Evande et al., 2004). Class 1 CBS exhibits
self-assembly properties into an active dimer and are usually found in
bacteria (Matoba et al., 2017). Class 2 CBS are monomer in nature,
found in the nematode Caenorhabditis elegans (CeCBS) and consists of
two tandem catalytic domains with only one PLP binding site (Vozdek
et al., 2012).
Class 3 CBS are characterized by the presence of a catalytic domain
followed by a C-terminal Bateman module consisting of two consecutive
cystathionine β-synthase motifs (CBS1, CBS2) that are associated with its
regulatory function and tetramer formation (Tu et al., 2018;
Oliveriusová et al., 2002). Class 4, typically found in insects such as
honeybee and fruit fly (Giménez et al., 2017; Su et al., 2013), is a CBS
dimer comprised of three separate domains with a middle conserved
catalytic domain flanked by an N-terminal heme-binding domain and a
C-terminal Bateman module. Finally, Class 5, the most complex of them
all, are found in mammals (Su et al., 2013). It is a tetrameric CBS with
one functioning heme-binding domain, a catalytic domain and a Bate­
man module.
Current studies are greatly inclined towards protein hydrolysate or
short inhibitory peptides (Boschin et al., 2014). Therefore, general un­
derstanding on the inhibitory mechanism of large bulky proteins, such
as CBS, towards ACE is lacking. This is because large bulky proteins were
previously speculated to be weak ACE inhibitors due to the presence of
ACE α1 (position 40–71), α2 (position 74–107), and α3 (position
109–120) helices which hinder the entry of bulky polypeptides to the
substrate-binding site (Fang et al., 2019). Nonetheless, identification of
significant ACE inhibition by several mushroom ACE inhibitory proteins
with high molecular weight (>30 kDa) prompts the need to further
understand the inhibitory mechanism of those proteins (Mohamad
Ansor et al., 2013; Ibadallah et al., 2015; Geng et al., 2015).
Our study demonstrates the in silico identification, characterization,
structural modelling of GlCBS and LrCBS. The molecular docking anal­
ysis of GlCBS, LrCBS and ACE are employed to elucidate the potential
interaction and inhibitory mechanism of CBSs towards ACE. The iden­
tification of CBS-interacting residues can lead to the discovery of a new
druggable site on ACE for the design of peptide binding epitopes derived
from CBS protein for ACE inhibition.
2. Material and methods
2.1. Genomic data mining of potential ACE inhibitory CBS-like protein in
Lignosus rhinocerus
The protein sequences of GlCBS (NCBI ID: AUN37950.1) was
retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov/
protein/). The GlCBS sequence was used as the query for sequence
similarity (“homology”) search with L. rhinocerus genomic-
transcriptomic in-house database (Yap et al., 2015a) using locally
installed blast-2.4.0 + software (BLASTP cut-off e-value ≤1e-4) to
identify LrCBS (Suppl. Table S1).
2.2. Determination of physical parameters
The physiochemical characteristics of GlCBS and LrCBS enzymes
were analyzed via the ExPASy-ProtParam tool (http://web.expasy.org/
protparam) to explore its characteristics such as molecular weight,
theoretical pI value, instability index, aliphatic index, and grand average
of hydrophobicity (GRAVY).
2.3. Sequence alignment and domain
GlCBS and LrCBS sequences were subjected to BLASTP search under
non-redundant database. Five sequences with the highest sequence
identity to the CBS protein from NCBI protein blast were aligned with
3
Computational Biology and Chemistry 96 (2022) 107620
Expresso multiple sequences (Armougom et al., 2006) on the T-COFFEE
Server (http://tcoffee.crg.cat/) (Notredame et al., 2000) and were dis­
played via ESPript 3.0 (https://espript.ibcp.fr/ESPript/ESPript/) (Rob­
ert and Gouet, 2014). The domain architecture was investigated by
SMART genomic mode (http://smart.embl-heidelberg.de/) (Letunic
et al., 2020).
2.4. Secondary structure analysis
PSIPRED 4.0 (Jones, 1999) and DISOPRED3 (Jones and Cozzetto,
2015) in PSIPRED server (http://bioinf.cs.ucl.ac.uk/psipred/) (Buchan
and Jones, 2019) were used for secondary structure and disorder region
prediction (Suppl. Fig. S1).
2.5. Protein modelling of CBS proteins
The protein structure of both CBS proteins remains unresolved.
Therefore, composite protein modelling approaches using (1) contact
assisted protein structure prediction (C-I-TASSER) (https://zhanglab.
ccmb.med.umich.edu/C-I-TASSER/) (Zhang et al., 2018); (2)
transform-restrained Rosetta (TrRosetta) and (3) Comparative model­
ling based Rosetta (RosettaCM) (https://robetta.bakerlab.org/) (Kim
et al., 2004) were employed to obtain the structural model of GlCBS and
LrCBS proteins. Default modelling protocol was employed in
C-I-TASSER and Robetta pipeline and the model with the highest C-score
and confident score were selected, respectively (Suppl. Fig. S2). Roset­
taCM supports oligomeric modelling where GlCBS and LrCBS structural
models were obtained as a dimer while C-I-TASSER and TrRobetta
outputs the model as a single subunit. The selected models are provided
in (Suppl. Fig. S3) and were further evaluated in Sections 2.6 and 2.7 to
obtain GlCBS and LrCBS models with the highest reliability. Upon
release of AlphaFold2 algorithm (Thornton et al., 2021), ab initio
structural prediction of both GlCBS and LrCBS were carried out using
AlphaFold2 at ColabFold server (https://colab.research.google.com/
github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb).
2.6. Model structural analysis and superimposition
The generated models underwent DALI PDB search (http://ekhidna
2.biocenter.helsinki.fi/dali/), to detect structurally similar models.
Structural comparisons were performed via superimposition using
PyMol (Schrödinger, www.pymol.org) to compare structural conserva­
tion. LrCBS and GlCBS models generated as a single subunit, i.e., C-I-
TASSER and TrRosetta, were further evaluated to detect potential
clashing of dimeric interface prior to further dimerization of the models
(Suppl. Fig. S4). Structural comparison of RosettaCM and Colabfold
models were also conducted (Suppl. Fig. S5).
2.7. Model stereochemical evaluation
The structure models of LrCBSRosettaCM, GlCBSRosettaCM and GlCBSC-I-
TASSER
were subjected for validation using three independent servers:
SAVES, SWISS-Model and ProSa to aid final model selections. Swiss-
Model structural assessment tool was employed (https://swissmodel.
expasy.org/assess) to obtain QMEAN Z-score and Ramachandran plots
using MolProbity version 4.4 (Suppl. Fig. S6 & S7), SAVES 6.0 server
(https://saves.mbi.ucla.edu/) for ERRAT (Suppl. Fig. S8) (Colovos and
Yeates, 1993) and Verify3D (Suppl. Fig. S9) (Lüthy et al., 1992) analysis
and ProSa (Suppl. Fig. S10 & S11) (Wiederstein and Sippl, 2007)
(https://prosa.services.came.sbg.ac.at/prosa.php).
2.8. Model Refinement and dimerization model construction of GlCBS
and LrCBS
Dimerized model of GlCBSC-I-TASSER was generated using Galax­
yHomomer (Baek et al., 2017) (Suppl. Fig. S12). LrCBSRosettaCM and
N.-Y. Goh et al.
GlCBSRosettaCM models were refined with GalaxyRefineComplex (Heo
et al., 2016) with the option of symmetric refinement for homo-oligomer
(http://galaxy.seoklab.org/refinecomplex). The results were provided
as 10 refined models with five lowest-energy models from two different
refinement protocols: (1) distance restraints; (2) both distance and po­
sition restraints (Heo et al., 2016). GlCBSRosettaCM and GlCBSC-I-TASSER
model quality were further evaluated to choose the best model for
GlCBS. LrCBS and GlCBSRosettaCM models with the most improved ste­
reochemical quality were selected as shown in Table 3. The dimer
interface of the final selected structural model of GlCBS and LrCBSRo­
settaCM
were further investigated and validated by PISA (Krissinel and
Henrick, 2007) to examine the buried interface area between CBS pro­
tomers, the solvation free-energy gain (ΔiG), P-value of ΔiG (statistical
probability to validate the protein interfaces as biological importance),
amount of hydrogen bonds and salt bridges.
2.9. Protein-protein docking and interaction evaluation
The full crystal structure of human somatic angiotensin-converting
enzyme (sACE) was not available. Therefore, human testicular ACE,
representing the C-domain of sACE (C-ACE, PDB Id: 1O8A and N domain
of sACE (N-ACE, PDB Id: 2C6F) crystal structure of protein in native state
were obtained separately from the Research Collaboratory for Structural
Bioinformatics (RCSB) Protein Data Bank (http://www.rcsb.org) for
subsequent docking study. Before docking analysis, polar hydrogen was
added along with the removal of irrelevant water and ligand molecules
using Chimera 1.15 (Pettersen et al., 2004) software (University of
California, San Francisco, CA, USA), whereas the zinc and chloride ions
were retained. The GlCBSRosettaCM and LrCBSRosettaCM protein models
were docked individually onto the C- and N-ACE receptors using (1)
Cluspro 2.0 (Suppl. Fig. S13) (Kozakov et al., 2017) (https://cluspro.org
/home.php), (2) HADDOCK 2.4 (Suppl. Table S2) (van Zundert et al.,
2016) (https://wenmr.science.uu.nl/HADDOCK2.4/) and (3) Hdock
(Suppl. Fig. S14) (Yan et al., 2020b) under default setting. The required
active and passive residues by HADDOCK pipeline are predicted by
CPORT (Suppl. Table S3), which incorporate six different interface tools
into a consensus predictor (de Vries and Bonvin, 2011). The binding
poses of the top docking complexes were selected and compared. The
interacting residues of CBS-ACE were evaluated by Ligprot+ Dimplot
function (Laskowski and Swindells, 2011) to generate schematic dia­
grams of protein-protein interactions (Suppl. Fig. S16). Potential
C-domain specific inhibition of CBS was studied using molecular blind
docking of substrate Hip-His-Leu (HHL) (.sdf file obtained from Pub­
Chem) into the selected CBS-C-ACE complexes with CB-Dock server, a
cavity guided protein-ligand docking server coupled with Autodock
Vina docking algorithm(Yn Liu et al., 2019; S. Liu et al., 2019). The
binding affinity and dissociation constant of the plausible solution were
predicted with contact-based method, PRODIGY (PROtein binDIng en­
erGY prediction) using non-interface surface intermolecular contacts of
the CBS-ACE and C-ACE-HHL interactions (Xue et al., 2016).
3. Results and discussion
3.1. Genomic data mining of potential ACE inhibitory CBS-likened protein
in Lignosus rhinocerus
Blastp homology search of in-house L. rhinocerus genomic-
transcriptomic database revealed a protein sequence (Seq. ID
GME6470_g) that shares 82% identity and 89% similarity with GlCBS
protein, term LrCBS (Suppl. Table S1).
3.2. Determination of physical parameters
Protparam analysis (Table 1) showed that LrCBS and GlCBS possess
similar physical parameters and amino acid constituents (Fig. 2).
4
Computational Biology and Chemistry 96 (2022) 107620
Table 1
Protparam physicochemical characterization of LrCBS and GlCBS.
Parameter LrCBS GlCBS
Sequence length (A.A) 399 396
MW (Da) 42049.94 42141.79
Theoretical pI: 5.81 5.81
Total number of negatively charged residues (Asp + 46 49
Glu)
Total number of positively charged residues (Arg + Lys) 41 44
The instability index (II) 36.87 37.82
Aliphatic index 97.54 92.63
Grand average of hydropathicity (GRAVY) -0.084 -0.230
Note: Protein with a stability index less than 40 is stable; Positive aliphatic index
indicates the increase of thermostability of globular proteins and is defined as
the relative volume occupied by aliphatic side chains (Ala, Val, Ile, and Leu).
Negative GRAVY indicates hydrophilic proteins.
3.3. Sequence alignment, domain, disordered region and secondary
structure analysis
LrCBS shares high sequence identity (%) with mushroom CBS i.e.,
G. lucidum (82.20), Dichomitus squalens (86.75), Polyporus brumalis
(85.96), Polyporus arcularius HHB13444 (85.96), Lentinus tigrinus
(85.21), Trametes coccinea (85.60) (Fig. 3). Both LrCBS and GlCBS se­
quences were similarly predicted to contain 12 extended strands and 19
α helices (Suppl. Fig. S1) with the presence of intrinsically disordered
regions (IDRs) at position (1− 3) and (366− 399) for LrCBS as well as
position (1–4, 389 and 396) for GlCBS (Suppl. Fig. S1).
3.3.1. LrCBS and GlCBS are class I CBS
SMART domain search revealed that both LrCBS and GlCBS were
similarly annotated with the presence of a single type II family pyri­
doxal-5′ -phosphate (PLP)-dependent catalytic domain, which contains
the PLP-cofactor binding sites (Fig. 1). The C-terminal Bateman module
present in the C-terminal of Toxoplasma gondii (TgCBS) (position
366–419) was non-existent in both mushroom CBSs (Fig. 3). Previous
studies speculated that the ACE inhibitory activity of GlCBS was
potentially derived from the zinc-binding capability demonstrated by
the bateman module on CBS C-terminal domain by interfering with the
zinc ion active centre of ACE (Mohamad Ansor et al., 2013). Unlike other
CBS proteins in higher-order organisms with greater complexity i.e.,
containing N-terminal heme binding and/or C-terminal Bateman mod­
ule, simplicity of the domains can be observed in both LrCBS and GlCBS
along with other closely similar mushrooms CBSs (Fig. 1). Hence, ACE
inhibitory mechanism of GlCBS is not Bateman module dependent.
Based on sequence and domain analysis, the native structure of both
mushroom CBS showed greater similarity with that of Class 1 CBS and
may exist predominantly in the dimeric form. The potential ACE
inhibitory mechanisms of GlCBS and LrCBS proteins are investigated in
Section 3.6.
Also, the lysine residue (position 50) that makes a Schiff base with
PLP (pyridoxal 5’-phosphate) (indicated by an asterisk in Fig. 3), crucial
for the catalytic activity of CBS are conserved in both L. rhinocerus and
G. lucidum CBS. The important conserved catalytic residue Lys50 sug­
gests that both LrCBS and GlCBS proteins are likely functionally active as
a key regulator in the reverse transsulfuration pathway. This is in
agreement with a study reported by Tian et al. (2019) whereby inhibi­
tion of GlCBS resulted in the loss of ~60% of endogenous H2S biosyn­
thesis. GlCBS transcription was reported to influence H2S biogenesis
which regulates the synthesis of bioactive secondary metabolites such as
ganoderic acid. The conserved nature of LrCBS and GlCBS domain ar­
chitecture and catalytic residue infers the possibility of similar H2S
regulation in secondary metabolite biosynthesis present in L. rhinocerus.
N.-Y. Goh et al.
Fig. 2. Amino acid composition profile of LrCBS and GlCBS.
3.4. Structural and stereochemical quality-based protein model selection
Homology modelling servers HHpred and Swiss-modelling generated
incomplete protein models due to the lack of template similarity at the
IDR, which is known to be highly variable (Not shown). Therefore,
protein modelling servers C-I-TASSER, TrRosetta and RosettaCM with
inclusion of ab initio protocol were employed to obtain the full protein
structure for GlCBS and LrCBS. DaliPDB search showed the obtained
LrCBS models bear significant structural resemblance with TgCBS while
GlCBS models share significant similarity with either ScCBS or TgCBS,
all with significant Z-scores > 50 (Z-score >2 represents significance)
(Table 2).
Unlike RosettaCM, both C-I-TASSER and TrRosetta pipeline do not
support oligomeric modelling. Further oligomerization modelling of the
protein models were required to be performed via GalaxyHomomer.
Nonetheless, additional structural superimposition of dimeric models
for GlCBSTrRosetta, LrCBSTrRosetta and LrCBSC-I-TASSER with TgCBS (PDB
ID: 6xyl) revealed clashing dimerization interface at the C-terminal re­
gion with IDR characteristics (Suppl. Fig. S4). Hence, only the GlCBSC-I-
TASSER
model without any notable structural clashing in the dimerization
interface during structural comparison was selected for further dimer­
ization and refinement. Structural comparison revealed two protein
model candidates for GlCBS i.e. GlCBSC-I-TASSER and GlCBSRosettaCM;
Conversely, only one for LrCBS that is LrCBSRosettaCM.
The inaccuracy of a protein 3D model can arise from incorrect
template alignment or prediction mistakes. Those inaccuracies can be
detected based on high conformational energies or quality assessment
scores (Bhattacharya et al., 2007). GlCBSC-I-TASSER, GlCBSRosettaCM and
LrCBSRosettaCM models were evaluated with five structure validation
tools including Molprobity, QMEAN, ProSa, Verify-3D and ERRAT using
3 independent servers (Table 3). Further comparisons of model qualities
were carried out between the dimerized GlCBSC-I-TASSER model and
GlCBSRosettaCM for the final GlCBS model selection. SWISS-Model struc­
ture assessment server employs MolProbity version 4.4 to generate a
Ramachandran (RC) plot and clashscore analysis. MolProbity is an
all-atom measurement of structural correctness based on steric clashes,
rotamer outliers, and Ramachandran-plot while Molprobity score is a
log-weighted combination of those functions that reflect crystallography
resolution (Davis et al., 2004). ERRAT is mainly used to verify protein
via the study of non-bonded atom-atom interactions (Colovos and
Yeates, 1993). On the other hand, Verify-3D functions to determine the
compatibility of a 3D atomic model with its amino acid sequence (Lüthy
5
Computational Biology and Chemistry 96 (2022) 107620
et al., 1992). QMEAN Z-score or QMEAN4 is a linear combination of
statistical potential terms, and a value less than four indicates a reliable
model. ProSa evaluates experimental structures by comparing its
knowledge-based score to random structures with the same sequence
(Wiederstein and Sippl, 2007).
Both LrCBSRosettaCM and GlCBSRosettaCM model were unable to satisfy
the Verify-3D (%) cutoff point of 80%, with only 79.95% and 79.29% of
the amino acids scoring > 0.2 in the 3D/1D profile, respectively
(Table 3). This indicates poor compatibility of the amino acid sequences
with its 3D model. Hence, further refinement was performed with Gal­
axyRefineComplex. Refinement of LrCBSRosettaCM model improved the
Molprobity score of 2.98–1.58, RC favoured (%) of 96.47–98.24%
(>98%) (Elsliger and Wilson, 2012), clashscore of 195.74–11.76 with a
tolerable RC outlier (%) of 0.63%. QMEAN value of 0.19 (close to zero)
indicates a model of high quality. Additionally, Verify-3D analysis with
an improvement from 79.95% to 81.70% of the amino acids scoring
> 0.2 in the 3D/1D profile and an ERRAT score of 95.24 (greater than 5)
indicates a good quality model. Besides, ProSA Z-score of − 10.01 shows
the Z-value of the protein was similar to native protein of equivalent
sizes (Suppl. Fig. S10). The reliability of the model was also shown by
the ProSA energy plot with no obvious problematic regions with a
positive value in the ProSA energy plot (Suppl. Fig. S11).
The two GlCBS models, GlCBSC-I-TASSER and GlCBSRobettaCM, were
further evaluated. GlCBSRobettaCM refined model revealed significant
improvement in Molprobity score of 3.01–1.58, RC Favored (%)
96.07–98.24%, clash score from 189.32 to 11.76 with acceptable RC
outliers of 0.63(%). QMEAN Z-score of 0.70 to − 0.40 also indicate the
closeness of the model with experimental structures (Feig, 2016).
Moreover, model quality improvement was also observed in the
Verify-3D analysis, from 79.29% to 81.57% of the amino acids scoring
> 0.2 in the 3D/1D profile. ProSA Z-score improvements from − 9.01 to
− 9.06 indicated that its energy value was similar to those found for
native protein of equivalent sizes (Suppl. Fig. S10) with no obvious
problematic regions with positive value in the ProSA energy plot (Suppl.
Fig. S11). Although there is a decrease in the ERRAT value, from 94.57
to 92.65, the value is still higher than the generally accepted range of
> 50 for a high-quality model (Laskowski et al., 1993).
GlCBSC-I-TASSER dimerized model was evaluated with Molprobity
score (2.05), RC favoured (94.16%), RC outlier (1.27%), clash score
(14.13), QMEAN Z-score (− 1.98), ERRAT score (87.61), Verify-3D score
(82.20%) and ProSA Z-score (− 8.62), all of which signifies an acceptable
model with good quality. Although GlCBSC-I-TASSER and GlCBSRobettaCM
N.-Y. Goh et al. Computational Biology and Chemistry 96 (2022) 107620

Fig. 3. Comparison of amino acid sequences of LrCBS and GlCBS with other CBS proteins. Multiple sequence alignments were visualized by ESPript 3.0 (Robert and
Gouet, 2014) subsequent to alignment of protein sequences using Expresso T-Coffee (Notredame et al., 2000). Secondary structure features of RosettaCM generated
LrCBS model and DALI homolog, Toxoplasma gondii CBS, (TgCBS, PDB ID: 6XYL) are shown above and below the alignments, respectively. α-helices and β-strands are
represented as helices and arrows, respectively. β-turns are marked with TT while η symbol refers to a 310-helix. Fully conserved areas are shaded in red. Conserved
sequences of greater than 70% similarity are indicated by a box. Dichomitus squalens (TBU30616.1), Polyporus brumalis (RDX54216.1), Polyporus arcularius HHB13444
(TFK86983.1), Lentinus tigrinus ALCF2SS1–6 (RPD64447.1), Trametes coccinea (OSD05344.1), Ganoderma lucidum (AUN37950.1) and Toxoplasma gondii (6xyl).
Asterisk depicts the conserved Lysine residue responsible for PLP cofactor binding. Single red dot represents conserved amino acid residues involved in hydrogen
bonding or salt bridging by TgCBS dimer interface, respectively. Black triangle represents recurring interacting residues in both C-ACE-GlCBS and C-ACE-LrCBS
interactions predicted by Cluspro and HADDOCK. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article.)

Table 2
DALI structural homolog with highest Z-score with the predicted CBS models.
Protein type Modelling pipelines Oligomeric state Z-score PDB ID & chain Protein name %ID Species

LrCBS C-I-TASSER – 52.8 6xyl-A Cystathionine beta-synthase 53 Toxoplasma gondii

TRrosetta – 51.6 6xyl-A Cystathionine beta-synthase 53 Toxoplasma gondii
Rosetta CM Dimer 50.2 6xyl-A Cystathionine beta-synthase 53 Toxoplasma gondii
GlCBS C-I-TASSER – 54.0 6c2h-A Cystathionine beta-synthase 49 Saccharomyces cerevisiae
TrRosetta – 51.8 6xyl-A Cystathionine beta-synthase 54 Toxoplasma gondii
Rosetta CM Dimer 50.2 6xyl-A Cystathionine beta-synthase 54 Toxoplasma gondii

Note: %ID = Percentage identity.

models were observed to possess similar structural resemblance, ho­
mologous to the TgCBS structure, nonetheless, the GlCBSRobettaCM model
analyzed had better stereochemical qualities compared to GlCBSC-I-
TASSER
across Molprobity, QMEAN, ERRAT and ProSa scores. Quality
evaluation of the GlCBS models revealed that the GlCBSRosettaCM model
was superior overall, with better stereochemical, and geometrical
qualities compared to the GlCBSC-I-TASSER model. Hence, the GlCBSRo­
settaCM
and LrCBSRosettaCM were selected for the following docking study
(Fig. 4A).
Upon the release of the recent Alphafold2 modelling algorithm
(Pereira et al., 2021)., ab initio model of GlCBS and LrCBS were
generated using Colabfold server. Structural comparison between the
RosettaCM and AlphaFold2 models showed high similarity at the core
structure of CBS with low RMSD value (<1.34 Å) (Suppl. Fig S5). The
AlphaFold2 predicted models showed low structural confidence at the
CBS C-terminal (IDR), shown in the predicted aligned error (PAE) plot
and is possibly the reason for the observed conformation differences
when compared with RosettaCM models.
DALI server and all of the quality assessment analysis from SAVES,
Swiss-Model structural assessment and ProSa servers revealed that both

6
N.-Y. Goh et al. Computational Biology and Chemistry 96 (2022) 107620

Table 3
Stereochemical validation of pre/post refinement CBS proteins models.
Protein Modelling server SWISS-Model structure assessment server SAVES ProSA Z-
score
MolProbity RC Favored (%) RC Outliers (%) Clash QMEAN ERRAT Verify- 3D (%)
Score score

LrCBS RosettaCM 2.98 96.47 0.25 195.74 0.77 97.03 79.95 -9.86
RosettaCM (refined) 1.58 98.24 0.63 11.76 0.19 95.24 81.70 -10.01
GlCBS C-I-TASSER (dimerized and 2.05 94.16 1.27 14.13 -1.98 87.61 82.20 -8.62
refined)
RosettaCM 3.01 96.07 0.38 189.32 0.70 94.57 79.29 -9.01
RosettaCM (refined) 1.58 98.24 0.63 11.76 -0.40 92.65 81.57 -9.06

Note = RC: Ramachandran.

Fig. 4. 3D structural model. (A) Two protomers

of LrCBS (left) and GlCBS (right) obtained from
RosettaCM. Chain A and B are coloured in blue
and cyan, respectively. N- and C-terminals are
represented with symbol N and C, respectively.
Structural superimposition of (B) LrCBSRo­
settaCM
-TgCBS; (C) GlCBSRosettaCM-TgCBS; (D)
LrCBSRosettaCM-GlCBSRosettaCM Chain. Cyan
arrow indicates the longer helices (position
174–184) observed in LrCBSRosettaCM model.
Black circle represents the region with notable
structural divergence between the LrCBS and
GlCBSRosettaCM model. Chain A and B of TgCBS
are coloured in pink and red, respectively. (E)
Interdimeric residues in TgCBS (Red & pink,
chain A and B) showing the formation of non-
covalent bonding between Asp15-Arg28 and
Asp15-Arg31, represented with stick models.
Equivalent residues in LrCBSRosettaCM and
GlCBSRosettaCM are shown and coloured as blue
and orange respectively. (For interpretation of
the references to colour in this figure legend,
the reader is referred to the web version of this
article.)

LrCBSRosettaCM and GlCBSRosettaCM model refined by GalaxyR­

efineComplex protocol were more appropriate for protein modelling of
dimeric CBS structures.

7
N.-Y. Goh et al.
3.5. Structural comparison and dimer interface analysis
3.5.1. LrCBS and GlCBS dimer interface analysis and structural
comparison with TgCBS
Structural comparisons between LrCBSRosettaCM and GlCBSRosettaCM
model revealed that the catalytic domain structure was highly conserved
in comparison to TgCBS (Fig. 4B & 4C). One major insertion at position
174–184 of LrCBSRosettaCM was observed, contributing to the formation
of longer helices (Fig. 2). The structural variants were observed to not
impose any structural obstructions that might affect the dimeric inter­
face and oligomeric state of the protein (Fig. 4B). In addition, there are
some minor differences due to small insertions or deletions in the
mushroom CBS relative to TgCBS. Nonetheless, secondary elements
were predominantly unaltered concerning the equivalent region in the
TgCBS (Fig. 4B & 4C).
The inter-dimeric formation of TgCBS at the catalytic core was
supported by the formation of a hydrogen bond and salt bridge between
Asp15-Arg28, Asp15-Arg31 (Fig. 4E). Similar conservation of those
residues in LrCBS and GlCBS suggests that the interdimeric interactions
in the dimer interface of LrCBS and GlCBS were retained (indicated by
red dot in Fig. 3). However, further structural comparisons revealed the
presence of Asp9-Arg22 interaction in the equivalent position of LrCBS
and GlCBS, but Asp9-Arg25 interaction was not observed (Fig. 4E).
Nonetheless, the oligomeric state of the predicted LrCBSRosettaCM and
GlCBSRosettaCM models was further validated via PISA dimer interface
analysis (Table 4) where dimer formation for LrCBS and GlCBS models
are favoured, gaining solvation energy during interface formation with
Δi G values of − 39.4 and − 41.7 kcal/mol, along with interface area of
3324.4 and 3811.7 Å2, respectively. A total of 40 and 44 hydrogen
bonds as well as 4 and 12 salt bridges, formed during the dimeric
interface formation of LrCBS and GlCBS, respectively. Moreover, the
obtained P-value > 0.5 also indicates that the interface was interaction-
specific and biologically important. PISA analysis supports the notion of
homodimer formation between CBS protomers in LrCBS and GlCBS,
respectively.
3.5.2. Structural comparison of LrCBS with GlCBS
Further structural comparisons between the LrCBSRosettaCM and
GlCBSRosettaCM models indicated some variations can be observed in the
coiled regions as well as the IDR in the C-terminal region, but the pre­
dicted secondary elements in the main catalytic domain were mostly the
same (Fig. 4D). The high structural resemblance of the main catalytic
domain implies great possible conservation of protein functionalities
and interactions.
3.6. Protein-protein docking and interactions between CBS proteins and
ACE
Protein-protein docking algorithms can achieve successful predictive
rates in many complexes, as shown in the Critical Assessment of Pre­
dicted Interactions (CAPRI). Protein docking servers that possess great
reliability, based on recent CAPRI community assessment experiments,
are Cluspro 2.0, HADDOCK 2.4 and Hdock (Lensink et al., 2020). All
three were used to elucidate the ACE inhibitory mechanism of GlCBS
and LrCBS with the C- and N-domain of ACE. The outputs of Cluspro
involved ten models defined by centres of highly populated clusters of
Table 4
PISA analysis of CBS protein dimeric interface.
Dimer Interface area ΔiG (kcal/ ΔiG P- No. of H- No. of Salt
(Å2) mol) value bond bridge
LrCBS 3324.4 -39.4 0.018 40 4 CL1C-domain showed greater possibility as the most plausible inter­
GlCBS 3811.7 -41.7 0.021 44 12
Note: Å2 = interface area; ΔiG = solvation free energy gains upon formation of
the interface.
8
Computational Biology and Chemistry 96 (2022) 107620
low-energy docked structures. Model cluster size was emphasized
instead of docking energy for the selection of probable cluster solution
(Kozakov et al., 2017). Similarly, Hdock will output 10 docking solu­
tions as the most plausible binding positions based on its docking scoring
and algorithm. These are applicable for docking studies where the
reference template possesses a sequence identity of greater than 30%
(Yan et al., 2020b). Besides, HADDOCK docking complexes are indicated
by its Z-score which provides an indication of the amount of standard
deviation from the average cluster. Cluster with a positive Z-score are
usually unfavourable and not selected for further investigation (van
Zundert et al., 2016).
3.6.1. Docking studies uncover two potential binding site on C-ACE
Our preliminary docking study revealed a possible interaction be­
tween GlCBS with entrance of CL1/CL2 binding pocket (CL1/2 C-domain)
of C-ACE (not shown). Therefore, a subsequent docking study between
C-ACE and CBSs was conducted with removal of the chloride ion in both
CL1/2 C-domain to affirm the ability of the CBS protein to block and
prevent entrance of Cl- ion into the C-ACE. All of the selected binding
positions (Fig. 5), binding energies and affinities (Table 5) and inter-
molecular interacting residues (Suppl. Table S4) are provided. Cluspro
server unveiled that GlCBS interacts with C-ACE at CL1C-domain with
binding affinity (BA) and dissociation constant (Kd) of − 14.2 kcal/ mol
and 3.7E-11 mol/L, respectively. Comparable binding positions for
LrCBS was also observed at cluster 6 with BA and Kd of − 13.7 kcal/mol
and 8.9E-11 mol/L, respectively.
Intriguingly, similar binding interaction with the CL1C-domain (Fig. 5A
& 5B) can be observed in the HADDOCK top clusters for both GlCBS (BA:
− 12.2 kcal/mol; Kd: 1.1E-0.9 mol/L) and LrCBS (BA: 13.1 kcal/mol; Kd:
2.6E-10 kcal/mol), where stronger binding affinity was observed for
HADDOCK C-ACE-LrCBS complexes compared to GlCBS. Although the
detailed mechanism is still unknown, it was postulated that conforma­
tion changes near the surface of CL1 pocket assist the acceptance of
chloride ion into the pocket (Fig. 6) and both CBS proteins are found to
similarly interact and conceal the supposed entrance of CL1C-domain (Guy
et al., 2003; Rushworth et al., 2009).
HADDOCK complexes of LrCBS and GlCBS with CL1C-domain pre­
dicted several conserved inter-molecular interactions with the formation
of hydrogen bonding between C-ACE Lys174 and Asp235 in both CBSs.
There were also similar electrostatic interactions between Glu176 and
Ile230 (GlCBS) and Ile229 (LrCBS), Gln262 with Gly243, Pro245
(GlCBS) and Gly242, Pro244 (LrCBS) (Fig. 7A) found in both CBSs. The
partake of recurrent residues Lys174, Glu176, Gln262, His263 in C-ACE
with the CBS proteins via Cluspro and HADDOCK signifies the impor­
tance of those residues in the inhibitory mechanism of CBS proteins.
Top Hdock docking solution for C-ACE-GlCBS showed a different
binding location compared to both Cluspro and HADDOCK (Fig. 5A),
where binding occurs at the supposed entrance of the CL2C-domain with
BA − 17.3 kcal/mol and Kd 2.0E-13 mol/L. Nonetheless, similar LrCBS-
CACE docking interaction (Fig. 5B) was observed at solution 3 with BA
− 12.9 kcal/mol and Kd 3.5E-10 mol/L. Both CBS proteins were
revealed to similarly interact with CL2C-domain surrounding residues of
Gln231, Asp232, Glu234, Arg235, Gln238, Gln239, Pro242, Gln579,
Pro585, Asn586, Ser590, Leu593, Arg604 (Fig. 7B). Similar hydrogen
bonding formation between C-ACE and both CBS protein were observed
in Asp 232-Tyr166. The conserved negatively charged Glu174 in CBS
protein could also be an important residue in the PPI between C-ACE
CL2 binding site and CBS, as it actively engages in salt bridge formation
with a positively charged arginine on C-ACE of Arg604 and Arg235 in
LrCBS and GlCBS docking complexes, respectively. Two conserved res­
idues, Tyr166 and Glu174, in CBS proteins could be the crucial residues
assisting in blocking the entrance of the CL2C-domain.
acting site compared to CL2C-domain with mutual agreement of binding
location predicted by Cluspro and HADDOCK top clusters. However,
based on the binding affinities between the three docking algorithms,
N.-Y. Goh et al. Computational Biology and Chemistry 96 (2022) 107620

Fig. 5. Protein-protein docking solutions predicted by Cluspro, HADDOCK and Hdock. (A) Cluspro and HADDOCK top clusters predicted GlCBS interaction with the
C-ACE CL1 chloride binding site (CL1C-domain); Hdock predicted GlCBS binding at the entrance of C-ACE CL2 chloride binding site (CL2C-domain). (B) Cluspro and
HADDOCK predicted similar mechanism of LrCBS interaction with the CL1C-domain; while Hdock predicted similar GlCBS binding at the entrance of CL2C-domain
binding site. (C) Cluspro, Haddock and Hdock top docking complexes all showed GlCBS binding to the entrance of N-ACE CL1 binding pocket (CL1N-domain). (D) Top
docking complexes of Cluspro, Haddock and Hdock revealed LrCBS binds similarly to CL1N-domain. Magenta-coloured atoms represent the supposed removed chloride
ions in C-ACE and are only shown to show the location of the binding site, the chloride atom in N-ACE is coloured in green. ACE are coloured in orange. Chain A and
chain B of CBS proteins are coloured in blue and cyan, respectively. The possible entry way of chloride 1 and 2 into its respective binding pocket in C-ACE (circled in
black and orange, respectively) could be blocked upon CBS binding. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)

Hdock predicted C-ACE-GlCBS interacting interface has the highest
binding affinity at the CL2C-domain. The observed discrepancy possibly
arises due to the template-dependent nature of Hdock that sets itself
apart from the template-free docking algorithm of HADDOCK and Clu­
spro. Hdock algorithm with the inclusion of biological information may
be the source of these differently predicted docking outputs (Yan et al.,
2020b).
Nevertheless, two potential binding sites on C-ACE were uncovered
by the C-ACE-GlCBS protein-protein interaction studies. (1) Cluspro and
HADDOCK predicted binding onto the surrounding residues of CL1C-
domain
; (2) Hdock proposed interaction with the entrance of CL2C-domain.
Despite both Cluspro and Hdock top docking solution showing different
interacting sites, Arg144 on GlCBS was observed to actively partake in
hydrogen bond formation in both cases with Gln183 and Gln483 of C-
ACE (Fig. 7C), respectively. This indicates the potential importance of
Arg144 in C-ACE-GlCBS interactions.
The two predicted potential binding sites on C-ACE are potentially
linked with the attenuation of chloride activation activity in C-ACE. ACE
was determined to be chloride-dependent and substrate-specific, espe­
cially in the catalysis of Angiotensin I (Ehlers and Kirsch, 1988). Certain
substrates (eg. AngI) require the initial binding of a chloride ion to ACE
prior to Angiotensin II (AngII) formation (Shapiro et al., 1983). Chloride

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N.-Y. Goh et al. Computational Biology and Chemistry 96 (2022) 107620

Table 5
Comparison of top docking cluster or solutions for GlCBS against LrCBS with similar binding conformation in C-ACE and N-ACE interaction.
Receptors Protein ligand Cluspro HADDOCK Hdock

C. no. LES BA Kd C. no. H. S. Z-score BA Kd S. no. D. S. BA Kd

C-ACE GlCBS 1 -895.4 -14.2 3.7E-11 11 25.3 + /- 6.0 -2.5 -12.2 1.1E-09 1 -286.69 -17.3 2.0E-13
LrCBS 6 -797.3 -13.7 8.9E-11 1 -21.7 + /- 5.9 -2.6 -13.1 2.6E-10 3 -221.69 -12.9 3.5E-10
N-ACE GlCBS 1 -856.9 -14.0 5.2E-11 1 45.2 + /- 9.9 -2.2 -15.7 3.0E-12 1 -291.56 -14.4 2.7E-11
LrCBS 1 -762.6 -12.6 5.9E-10 3 -4.3 + /- 12.6 -1.3 -13.4 1.5E-10 1 -259.15 -14.7 1.6E-11

Note: BA = Binding affinity/ΔG (kcal/mol), Kd = dissociation constant (mol/L) C. no. = Cluster number, D. S. = Docking score, H. S. = HADDOCK score LES= Lowest
energy score, S. no. = Solution number. The docking solution with the highest reliability in Cluspro, HADDOCK and Hdock are decided by cluster number/size
(Kozakov et al., 2017), Z-score (van Zundert et al., 2016) and docking score (Yan et al., 2020b). BA and Kd were calculated by PRODIGY.

Fig. 6. Important regions and residues in C terminal domain of ACE (C-ACE). (A) (tACE crystal structure, 1O8A, coloured in orange). Two chloride ions can be found
in the C-ACE (coloured in green). The binding site of GlCBS on entrance of CL1C-domain. The pocket with chloride ion bound which furthest away from the zinc ion
(coloured in grey) is termed CL1 binding pocket binding pocket. The chloride ion closest to zinc ion is termed CL2 binding pocket. Six flexible hinge regions involved
in hinge bending activity of C-ACE were coloured in red (Watermeyer et al., 2006), Glu403-Lys118 salt bridging that controls the movement of chloride ion into the
CL2 pocket (Masuyer et al., 2014) is represented as blue sticks. (B) Frontal view of the proposed entrance of CL1 binding site, showing the residues affecting chloride
sensitivity of C-ACE. (C) Overview of GlCBS-C-ACE interacting surface that may be involve in blocking the entrance of CL2 into C-ACE. (D) Entrance of C-ACE CL2
binding pocket is circled. Critical salt bridging residues that control the entry of chloride into CL2 binding pocket colored in blue. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)

was studied to modulate the affinity of ACE inhibitors i.e. (enalaprilat,
lisinopril and captopril) towards ACE domains (Wei et al., 1992). A
mutagenesis study by Rushworth et al. (2009) showed that both
CL1C-domain and CL2C-domain modulate the chloride dependence of
C-ACE, hence it is relevant to catalysis. It was also proposed by Tzakos
et al. (2003) that CL1 is a synergistic anion for high-affinity substrate
binding. The absence of chloride ion in the CL1C-domain would result in
an unfavorable shift of residue Lys511, required for the stabilization of
the substrate C-terminal carboxylate in C-ACE. Likewise, the absence of
chloride ion in the CL2C-domain was found to greatly affect catalytic site
stability via chloride coordination of Arg522 (Yates et al., 2014). The
interacting interface between C-ACE-CBS interface potentially results in
the blockage of chloride ion entry into either one of the chloride binding
sites on C-ACE, thus, inhibiting C-ACE catalytic activity. Therefore,
based on our docking analysis, it is likely that the inhibitory mechanism
of mushroom CBS proteins is dependent on the modulation of chloride
activation in C-ACE via prevention of chloride ion entry into either one
of the CL1/CL2C-domain.
The acceptance of substrate entry into ACE is characterized by mouth
opening and closing aided by a hinge-bending motion (Cozier et al.,
2020b). ACE complex is known to undergo various open, semi-open and
closed conformations in the presence of AngII (Vy et al., 2020). An
inhibitory mechanism was proposed for the mixed-non-competitive in­
hibitor hexapeptide (Thr-Met-Glu-Pro-Gly-Lys-Pro), in which the pep­
tide is able to stabilize the closed and semi-open states of ACE while
preventing the open state, resulting in a dead-end complex that holds

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N.-Y. Goh et al. Computational Biology and Chemistry 96 (2022) 107620

Fig. 7. Consolidated protein-protein interaction studies of C-ACE residues engaging in non-covalent interactions with GlCBS/LrCBS. (A) Four surrounding residues
on C-ACE CL1 binding pocket (CL1C-domain) partake in interactions with several residues on GlCBS/LrCBS, predicted by Cluspro and Haddock, in the selected protein-
protein complexes. (B) Surrounding residues on C-ACE CL2 binding pocket (CL2C-domain) interacts with both GlCBS and LrCBS predicted by Hdock. Chain A and B on
CBS proteins are represented by (1) and (2), respectively. (C) Arg144 on GlCBS was found to be actively engaging in C-ACE-GlCBS intermolecular interactions among
the top complexes predicted by Cluspro and Hdock.

AngII in the active site. Watermeyer et al. (2006) reported that hinging
of C-ACE was closely related to the flexibility of residues at positions
98–125, 296–297, 400–409, 434–439, 535–537 and 569–578 (Fig. 6A).
In line with this, the Hdock PPIs analysis of GlCBS against CL2C-domain
identified several key hinging residues (i.e., Arg100, Asn105, Gln106,
Gln108, Pro575, Glu576 and Gln579) that potentially could restrain the
open-close transition of ACE and prevent substrate entry and indirectly
(Suppl. Table S4). Disruption of the Glu403-Lys118 salt bridge was re­
ported to promote entry of chloride ion into the CL2 pocket. Hence,
interactions with C-ACE hinge bending residues could potentially
sequester the hinge bending motion and help to stabilize the
Glu403-Lys118 interaction that blocks chloride ion entry into the
CL2C-domain. This eventually reduces the chloride affinity of CL2C-domain
(Yates et al., 2014).
Our docking analysis revealed that the inhibitory action of GlCBS
and LrCBS most likely occur via (1) the regulation of chloride activation
by blocking either CL1C-domain or CL2C-domain, or (2) formation of CBS-
ACE complex that prevents the entry of substrate into the active site

11
N.-Y. Goh et al.
via an allosteric interaction.
3.6.2. Inhibition activity of LrCBS and GlCBS are likely C-domain specific
Astoundingly, Cluspro, HADDOCK and Hdock top docking solutions
(Fig. 5C) unequivocally predicted GlCBS to interact with the surround­
ing residues of N-ACE CL1 binding pocket (CL1N-domain) with binding
energies − 14.0 kcal/mol, − 15.7 kcal/mol and − 14.4 kcal/mol and
dissociation constant 5.2E-11 mol/L, 3.0E-12 mol/L and 2.7E-11 mol/L,
respectively (Table 5). Similarly, Cluspro, HADDOCK, and Hdock top
docking solutions (Fig. 5D) showed LrCBS able to interact with
convergent binding site of CL1N-domain with binding energy of
− 12.6 kcal/mol, − 13.4 kcal/mol and − 14.7 kcal/mol and dissocia­
tion constant of 5.9E-10 mol/L, 1.5E-10 mol/L and 1.6E-11 mol/L,
respectively.
Cluspro and HADDOCK N-ACE-CBS top docking solutions also
revealed several conserved residues on the PPi of both LrCBS and GlCBS,
such as Thr4, Tyr166 and Glu174, that contribute to the stabilization of
protein complexes, mostly via electrostatic interactions (Fig. 8A). The
overlap in convergent binding site across all top docking complexes
revealed that Leu603 and Asn606 on N-ACE appear to be critical resi­
dues that contribute to the interaction of LrCBS and GlCBS with N-ACE,
engaging in various non-covalent bonding interactions (Fig. 8B).
All docking algorithms for GlCBS and LrCBS reported a similar
binding mechanism to the surrounding residues of CL1N-domain. None­
theless, these interactions may not result in significant inhibition of N-
ACE catalytic activity. Unlike C-ACE, only one chloride ion can be found
in the crystal structure of N-ACE at the CL2N-domain, due to the residue
difference between His164 of N-ACE and the equivalent Arg186 residues
of C-ACE, that restrict the entry of chlorine into the CL1N-domain (Natesh
et al., 2003). Therefore, the binding and blocking of CL1N-domain will
likely not affect chloride activation mechanism on N-ACE. Moreover,
N-ACE was reported to exhibit lower chloride requirement and can
remain catalytically active even without the presence of chloride ions
(Masuyer et al., 2014; Jaspard et al., 1993), possibly attributed to the
presence of only one chloride ion at the CL2N-domain (Fang et al., 2019).
Hence, any interactions towards the CL1N-domain of N-ACE-CBS are less
Fig. 8. Graphical representation of important residues on ACE-CBs interactions. (A) Conserved residues on LrCBS and GlCBS are denoted in blue boxes with recurrent
interactions with surrounding residues of N-ACE CL1 binding site predicted by Cluspro and HADDOCK. (B) Conserved residues on N-ACE denoted in blue boxes
engaging in non-covalent interactions with GlCBS and LrCBS top docking complexes predicted by Cluspro, HADDOCK and Hdock. Chain A and B on CBS proteins are
represented by (1) and (2), respectively.
12
Computational Biology and Chemistry 96 (2022) 107620
likely to result in an inhibitory effect on N-ACE catalytic activity.
3.6.3. C-ACE inhibition of GlCBS are likely conserved in LrCBS and
potentially in a non-competitive manner
LrCBS demonstrated binding at the entrance of CL1 and CL2 binding
pocket of C-ACE with good resemblance against GlCBS top docking so­
lution, in Cluspro and HADDOCK, and Hdock, respectively (Section
3.6.1). Several equivalently conserved residues of GlCBS engage in inter-
molecular interactions with CL1C-domain such as Asp235-Lys174, Ile230-
Glu176 and Gly243 & Pro245-Gln262, respectively, shown in
HADDOCK and Cluspro top docking complexes, are found in LrCBS with
similar interplay against CL1C-domain (i.e. Asp235-Lys174, Ile229-
Glu176, Gly242 & Pro244-Gln262, respectively). Interestingly, residues
Gly243 and Pro245 in GlCBS are not present in TgCBS and are only
present in some mushroom CBS proteins including LrCBS and Lentinus
tigrinus (LtCBS) (Fig. 3). Cluspro and HADDOCK revealed some over­
lapping residues present in both GlCBS and LrCBS that interact with
CL1C-domain; these are also only conserved in some mushroom CBSs.
The presence of overlapping convergent binding sites on PPI can
potentially signify the intrinsic characteristics of protein-protein in­
teractions (DeLano, 2002; Cukuroglu et al., 2014). Hence, Lys174,
Glu176 and Gln262 located at the surrounding entrance of the
CL1C-domain could potentially serve as a druggable site for selective in­
hibition of C-ACE due to its repetitive formation and intermolecular
interactions with both CBS proteins as predicted by Haddock. Stronger
binding affinity was observed for C-ACE-LrCBS compared to
C-ACE-GlCBS in HADDOCK top docking solutions. Hence, this suggests
that LrCBS may exert similar or potentially stronger ACE inhibitory
properties than reported for GlCBS.
There are three main active site pockets in an ACE molecule,
including the S1 pocket (i.e., Ala 354, Glu 384, and Tyr 523), S2′ pocket
(i.e., Gln 281, His 353, Lys 511, His 513, and Tyr 520), and S1′ pocket
(Glu 162) (Suppl. Fig. S15A) (Ma et al., 2019). Further molecular
docking simulation was employed to study the potential effect of
absence of chloride ion in the CL1/CL2 binding pocket of C-ACE. Mo­
lecular docking simulation revealed C-ACE substrate, Hip-His-Leu
N.-Y. Goh et al.
(HHL), established various interactions with the S1 (Ala 354, Glu 384,
Tyr 523) and S2 pocket (Gln 281, His 353, Lys 511, His 513, Tyr 520) of
ACE when both chloride ions are present (Suppl. Fig. S17A). Loss of HHL
interaction with the S1 pocket residues were observed in Cluspro,
Haddock and Hdock predicted GlCBS-C-ACE complexes (Suppl.
Fig. S17B), with decreased binding affinity in Haddock and Hdock
(Suppl. Table S5). On the other hand, most interactions with the S1 and
S2 pockets are retained in HHL-LrCBS-C-ACE complexes predicted by
Cluspro, Haddock and Hdock (Suppl. Fig. S17C), but with decreasing
binding affinity compared with uncomplexed C-ACE (Suppl. Table S5).
This further demonstrates that CBS binding to C-ACE may inhibit C-ACE
activity. However, this hypothesis requires further in vitro in­
vestigations to examine the hydrolysis ratio of Z-Phe-His-Leu (ZPHL)
and HHL, in order to determine its domain selective activity (Danilov
et al., 2008).
At present, C-ACE specific drugs such as phosphinic peptide
RXPA380 inhibits C-ACE competitively by occupying the S2’, S2, S1’,
and S1 subsites of the C-ACE substrate binding site (Corradi et al., 2007).
Past studies comparing RXPA380 and N-domain specific RXP407 pep­
tides also revealed that the domain selectivity of those peptides are
mainly governed by interactions to the S2 subsite (Phe 391, Glu 403)
(Kröger et al., 2009). Discovery of drugs with C-domain inhibitory
specificity are crucial for blood pressure regulation due to its ability to
bypass unfavourable side effects such as cough and angioedema, which
arise from N-ACE inhibition (Burger et al., 2014). Based on
protein-protein docking prediction, C-domain selective inhibitory
mechanism of GlCBS and LrCBS are different compared to RXPA380.
This is likely because (i) CBS was predicted to interact with potential
allosteric sites of C-ACE i.e., CL1/2 C-domain, but not substrate binding
site, and (ii) the bulky structure of CBS proteins may pose a challenge for
CBS to enter ACE substrate binding site, unlike the small peptide of
RXPA380.
The high molecular weight (>30Kda) fraction of L. rhinocerus scle­
rotium cold water extract was previously shown to possess anti-
inflammatory and anticancer properties (H.-Y.Y. Yap et al., 2018; H.Y.
Y. Yap et al., 2018; Lee et al., 2014). It was also reported to mediate the
activity of cell surface Gαq-coupled protein receptor (GPCR), contrib­
uting to airway relaxation. High molecular weight proteins, such as
LrCBS, with excellent predicted solubility and stability (Table 1) are an
excellent candidate for future drug discovery due to its potential func­
tion as an ACE inhibitor, alleviating conditions related to the over­
expression of the ACE/AngII axis. However, further studies on the actual
13
Computational Biology and Chemistry 96 (2022) 107620
expression of LrCBS protein in Lignosus rhinocerus is required.
Protein-protein interaction studies on mushroom CBS with two ACE
domains have revealed preliminary clues on its probable ACE inhibitory
mechanism, as demonstrated by GlCBS in vitro [Graphical summary of
all predicted mushroom CBS binding sites on ACE are provided in
Fig. 9]. Several conserved residues in LrCBS and GlCBS were found to
actively interact with residues in ACE, that could set up the foundation
for the future discovery of novel peptide binding epitope based-drug
discovery. Nevertheless, the potential inhibitory activity of LrCBS re­
quires further validation in vitro, along with confirmation of the po­
tential chlorine-activation and domain-specific inhibitory mechanisms
in both CBS proteins.
4. Conclusion
Genomic data mining of L. rhinocerus revealed a CBS protein (LrCBS)
with prominent structural similarity (>80%) with ACE inhibitory pro­
tein GlCBS. Domain analysis revealed the conservation of the catalytic
domain in both LrCBS and GlCBS with high structural resemblance.
Protein-protein docking analysis revealed an ACE inhibitory mecha­
nism, whereby both GlCBS and LrCBS potentially bind to the entrance of
the CL1/CL2 binding pocket, thereby moderating C-ACE activity. Resi­
dues Lys174, Glu176 and Gln262, and Asp232 on tACE (C-ACE) are
potentially important residues that stabilize the C-ACE-CBS interface on
CL1C-domain and CL2C-domain, respectively. All top binding solutions for
both N-ACE-CBS studies predicted by Cluspro, HADDOCK and Hdock
revealed convergent binding at the CL1 binding pocket of N-ACE with
recurring residues Leu603 and Asn606 on N-ACE. However, the
blockage of CL1N-domain is proposed to have minimum effect on the
catalytic activity of N-ACE as the binding site serves no purpose in
chloride activation activity. Our results highlight the potential of G.
lucidum and L. rhinocerus as a natural source of ACE inhibitory proteins,
potentially in a C-domain specific manner. However, further in vitro
investigations are required to affirm the presence of such ACE inhibitory
activities in LrCBS and GlCBS. The identification of recurring interacting
residues between ACE and CBS proteins can potentially be exploited for
further studies on C domain-selective ACE inhibitors in order to develop
prospective therapeutics.
CRediT authorship contribution statement
Neng-Yao Goh: Conceptualization, Data curation, Formal analysis,
Fig. 9. Graphical representation of sACE as an
integral-membrane protein with two active sites
of N- and C- domain. C-domain contains two
chloride binding pockets of chloride 1 (CL1C-
domain
) and 2 (CL2C-domain), while N-domain
contains only 1 chloride ion at the CL2N-domain
binding pocket. Docking analysis revealed three
possible binding modes. I: Binding of CBS at
CL1N-domain binding. II: Binding of CagBS at
CL1C-domain binding pocket, leading to blockage
of CL1 entrance. III: Binding of CBS at CL2C-
domain
binding pocket, leading to blockage of
CL2 entrance. Prevention of either CL1/CL2 C-
domain
binding leads to destabilization of the
active site and prevents high-affinity substrate
binding to the S1, S2 and S1’ pocket. Chloride
ion is coloured in green. Zinc ion is represented
as a grey circle. (For interpretation of the ref­
erences to colour in this figure legend, the
reader is referred to the web version of this
article.)
N.-Y. Goh et al.
Investigation, Methodology, Resources, Visualization, Validation,
Writing – original draft, Writing – review & editing. Shin-Yee Fung:
Conceptualization, Supervision, Resources, Project administration,
Writing – review & editing. Muhammad F M Razif: Conceptualization,
Funding acquisition, Project administration, Resources, Supervision,
Writing – review & editing. Chyan Leong Ng: Supervision, Resources,
Project administration, Writing – review & editing. Hui-Yeng Y Yap:
Supervision, Resources, Project administration, Writing – review &
editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by Faculty Research Grant, University of
Malaya, Malaysia [Grant numbers: GPF003A-2020 and GPF003B-2020].
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.compbiolchem.2021.107620.
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