In silico Analysis of Iduronate 2 Sulfatase Mutations

in Colombian Patients with Hunter Syndrome (MPSII)

Johanna Galvis1,*, Jannet González2, Daniel Torrente2, Harvy Velasco1,

and George Emilio Barreto2
1
Maestría en Genética Humana, Universidad Nacional de Colombia
djgalvisr@unal.edu.co, juana7@gmail.com
2
Labotarorio de Nutrición y Bioquímica. Pontificia Universidad Javeriana, Colombia
dtorrente@javeriana.edu.co

Abstract. Hunter syndrome or Mucopolysaccharidosis II is an inherited X

linked disease, caused by mutations in iduronate 2 sulfatase (IDS), enzyme
which catalyzes the initial step reaction of heparan and dermatan sulfate degra-
dation. Allelic heterogeneity in MPSII challenges genotype-phenotype correla-
tion. With the aim of understanding the repercussion of mutations on enzyme
structure-function, we performed protein modeling and docking simulations
with wild and mutant forms of hIDS. Mutations were obtained from a molecular
study conducted in Colombian patients. Point mutations affected substrate-
protein interactions. In the case of S71N (attenuated phenotype) further experi-
mentation is required. Novel mutants P160SfsX4, D190Pfs13X and P185GfsX2
have a severely distorted conformation. Detailed analysis of the ligand-protein
interaction is also of great significance in designing molecules for treatment.
This is the first report of molecular docking performed with wild and mutant
forms of iduronate-2-sulfatase as a bioinformatical approach to phenotype-
genotype correlation in patients with Hunter Syndrome in Colombia.

Keywords: Mucopolysaccharidosis II, Sulfoiduronate sulfatase, Bioinformatic

Analysis, Molecular Docking.

1 Introduction

Mucopolysaccharidosis II (MPSII), also known as Hunter syndrome, is a rare, X-

linked disorder caused by deficiency of the lysosomal enzyme iduronate-2-sulfatase
(IDS), which catalyzes the first step in dermatan (DS) and heparan sulfate (HS) de-
gradation. Specifically, IDS removes O-2 linked sulfate from uronic acid, i.e., GlcA
(D-glucuronic acid) in HS, and IdoA (L-iduronic acid) in DS [1]. IDS deficiency
caused by mutations in IDS gene leads to abnormal storage of these glycosaminogli-
cans, leading to pathogenic events which result in multisystemic compromise. MPSII
is chronic and progressive, and occurs in people of all ethnicities, with an estimated
prevalence of ∼1 in 170000 male live births [2]. There are two forms of MPSII:

*
Corresponding author.

L.F. Castillo et al. (eds.), Advances in Computational Biology, 205

Advances in Intelligent Systems and Computing 232,
DOI: 10.1007/978-3-319-01568-2_30, © Springer International Publishing Switzerland 2014
206 J. Galvis et al.

neuropathic, with severe intellectual impairment, and non-neuropathic, with minimal

or absent cognitive involvement.
The gene encoding IDS is located in Xq28 [3]. IDS enzyme belongs to the highly
conserved family of sulfatases [4]. Most frequently gene mutations found in patients
with MPSII are point mutations, with substitution of one aminoacid by another having
different chemical properties. These changes can affect protein stability, processing,
trafficking and even enzyme/substrate interactions. Knowledge about mutations, its
structural implications and patient phenotype could be useful in diagnosis, prognosis
and treatment of MPSII. However, experimental data about structure and function of
IDS mutants are scarce. The crystalized structure of Iduronate 2 sulfatase has not been
obtained, and the study of mutations and its implications on protein structure and
function has been performed in silico by different groups [5-8].
The present study reports both the modeling of native human IDS and mutations
found in a group of Colombian patients with Hunter Syndrome. Finally, molecular
docking is performed with each one of these mutants against the natural substrates
with the aim of understanding the possible mechanisms of the pathogenesis of this
disease.

2 Methodology

2.1 Template Selection, Modeling and Model Assessment

Template search from PDB database was performed by means of Basic Local
Alignment Search Tool for proteins (BLASTp). Tridimensional modeling of hIDS
was performed using protein threading. Modeling using the Molecular Operating
Environment software package [9] indicated that the A chain of human Arylsulfatase
A (1AUK) possessed an arrangement of alpha helical structural elements that could be
superimposed on hIDS. Furthermore, conserved regions and predicted catalytically
active residues were completely covered by 1AUK. Selected force field in MOE was
amber99. Energetic and stereochemical evaluation was performed with Structure
Assessment tool from Swiss-Model workspace server [10]. Overall geometric and
stereochemical qualities were examined by Ramachandran plots generated by
RAMPAGE[11]. Measurement of RMSD (Root median square deviation) was
performed in MOE, to evaluate accuracy of the model.

2.2 Ligand-Protein Docking

Wild type hIDS docking was performed in Autodock Vina software[12] against its
substrates HS and DS. To simulate the posttranslational modification of this residue to
FGly, it was performed on the 3D model atom by atom, with builder tool in Pymol™
v 1.3 for educational use[13] . Using Structure Assessment tool, this modified model
was evaluated to make sure that no steric or energy alterations were generated.
Ligplot+ software package was employed for docking assessment[14], and
visualization of its results was done in Pymol™ v 1.3[13].
 In silico Analysis off Iduronate 2 Sulfatase Mutations in Colombian Patients 207

2.3 IDS Mutants

The source of the differentt IDS mutants evaluated in this work was the unpublished
study Identification of Idurronate 2 Sulfatase Gene Mutations in Colombian Patieents
with Hunter Syndrome[15]. Direct sequencing of the nine exons [16, 17] and ML LPA
(Multiplex ligation-dependeent probe amplification) [18] allowed the identificationn of
seven mutations suitable off bioinformatic simulations. Protein modeling followed the
same steps described abov ve, using wild type IDS model as template. RMSD D of
mutants was determined with
w MOE by superimposition to wild hIDS as referennce.
Subsequently, docking and docking assessment were performed.

3 Results
Human Iduronate 2 sulfattase tridimensional model superposed to its templatee is
shown in figure 1. RMSD D was 0.97Å, indicating high accuracy of the model.
Geometric evaluation show wed that 75.3% of the residues were in the favored regiion,
16.3% were in the allowed d region and 8.3% were in the disfavored region. Thhese
results indicate that the φ and ψ backbone dihedral angles in the hIDS model are
reasonably accurate.

Fig. 1. hIDS model (dark graay) superimposed to its template 1AUK (light gray). Moleccular
Operating Environment v.20100.

Docking results are show wn in Figure 2. Wild type hIDS docking allowed the ideenti-
fication of electrostatic inteeractions between the substrate and the residues at the en-
zyme catalytic pocket.
208 J. Galvis et al.

Fig. 2. Docking simulations. A.

A wt hIDS; B. R468Q showing Tyr300 and Phe105 interaccting
with GlcA/IdoA, and lacking Leu244; C. Q465X lacking Cys84 and other differences in iinte-
racting aminoacids; D. K347 7Q, note hydrogen bonding between O-2sulfate, Asp45 and
Arg247; E. K236N, very simillar to wild type except for lacking Arg297; F. S71N, very sim milar
hydrogen bonding and electrosstatic interactions as seen in K347Q docking; G. del.Q200_E2203,
lacking Leu244; H. R294GfsX X2 (Del. Exon 7), a new mutation which loses part of the cataalyt-
ic core and results in only Phe105 in electrostatic interaction with O-2 sulfate (the other aami-
noacids shown here interact wiith carboxyl group of GlcA/IdoA.
 In silico Analysis off Iduronate 2 Sulfatase Mutations in Colombian Patients 209

Fig. 2. (continued)

In wtIDS significant intteractions involved GlcA O-2sulfate with Cys84, Tyr1165,

and Leu244. Electrostatic interactions
i were also seen between GlcA carboxyl grooup
and Asn106. At the extern nal portion of the pocket, three hydrogen bonds were ob-
served between different GlcA monomer (in the same HS chain), Leu189 and
Asp187. Relating to the diffferent mutants analyzed, results are explained in figure 22.

4 Discussion and Conclusions

C
The present docking simulation showed that HS/DS bound at the wild hIDS iin a
conserved catalytic pockett. Studies related to hIDS tridimensional modeling hhave
reported that putative activve site residues are Asp45, Asn46, Cys84, Arg88, Lys1135,
His138, Asp334, His335, and Lys347, but these groups did not perform med
docking simulations and its arguments were inferred from homology betw ween
sulfatases[8],[19] . In contrast, our docking simulation found not only Cys84, but aalso
210 J. Galvis et al.

Arg297, leu244, Tyr165 and Asn106 as catalytically active, which differs from the
reported. Probably, metal binding required for catalytic activity induces changes in
aminoacids present at the pocket, but this kind of simulations are out of scope of this
study.
Cys84 showed in this in silico analysis an electrostatic interaction with O-2 sulfate,
in agreement with biological function of the enzyme. A hydrogen bond formed at the
edge of the pocket would serve as point of fixation between enzyme and substrate
during the biochemical reaction [20].
Relating to IDS mutants, R468Q (associated with neuropathic phenotype) changes
a positively charged residue, Arginine, to polar uncharged Glutamine. Alteration of
some residues interacting with substrate was seen here with docking (Figure 2). At
experimental level, it was demonstrated by subcellular fractioning that R468Q protein
has poor transport to lysosomes[21]. Kato et al. hypothesized that non conservative
mutation in R468 should affect the electrostatic field for substrate entrance into the
active site cavity resulting in an inactive enzyme [8]. Furthermore, Western blot anal-
ysis published later by the same group showed only primary precursors [19].
Q465X is also a mutation associated with neuropathic phenotype. Docking showed
Cys84 absence in catalytic core. Although downstream residues have no direct partic-
ipation in catalytic site in this model, overall conformational changes are seen. In
K347Q a positively charged aminoacid is substituted by polar uncharged Glutamine.
Lys347 is reported to be located adjacent to the active site, and this non conservative
mutation could severely modify the catalytic core. K236N changes to polar uncharged
Asparagine, and has been only reported in a case from Bulgaria[22].
There were only two patients with non-neuropathic phenotype with the S71N mu-
tation. S71 and N71 in our models are far from catalytic site. Other reported mu-
tants associated with the normal-intelligence phenotype, R48P and W337R, were
founded without a normal maturation process, evidenced by 73–75 kDa precursor in
western blot, but A85T was the only mutant from same phenotype processed to ma-
ture form(55-45kDa). There are no similar studies performed with S71N, so we per-
formed simulations with and without mature form, finding the same results.
S71N docking resulted in hydrogen bonding between Arg297, Asp45 and O2-
sulfate, and electrostatic interactions very similar to wild type IDS. This mutation
requires further investigation for better elucidate its relationship with non-neuropathic
phenotype. Finally, del.Q200_E203 and R294GfsX2 (del. exon 7) are two novel
mutations found in the Colombian patients[15]. Docking analysis showed distorted
interactions within the pocket of R294GfsX2, which lacks the last 254 aminoacids,
thus losing a great part of catalytic core.
Structural analysis indicated that novel frameshift mutants P160SfsX4,
D190Pfs13X and P185GfsX2 lost most of the catalytic domain structure, so it is feas-
ible that these distorted polypeptides will not retain enzymatic activity
Computational analyses are not only useful in contribute to the understanding of dis-
ease mechanisms, but also have considerable relevance in designing therapeutic mole-
cules such as chaperones for genetic diseases that result from misfolded/aggregated
proteins. Different studies suggest that pharmacological chaperone therapy is an
emerging field in lysosomal storage diseases (LSD). In relation to MPSs, Sanfilippo
 In silico Analysis of Iduronate 2 Sulfatase Mutations in Colombian Patients 211

Syndrome type C and Morquio B have chaperones in different stages of investigation

[23-25]. Peripheral IDS mutations such as R468Q, Q465X and S71N could be suitable
of pharmacological chaperones therapy, through the rescue from endoplasmic reticulum
quality control system and correction of protein processing and trafficking.
This is the first report of molecular docking performed with wild and mutant forms
of iduronate 2 sulfatase in Colombia. Even point mutations, not necessarily occurring
at core functional region, may affect substrate-protein interactions.

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