Supplementary information:
Silicatein-mediated polycondensation of orthosilicic acid: Modeling of catalytic mechanism
involving ring formation
H. C. Schröder§ - M. Wiens - U. Schloßmacher - D. Brandt - W. E.G. Müller§
Institute for Physiological Chemistry, University Medical Center of Johannes Gutenberg University Mainz,
Duesbergweg 6, D-55128 Mainz, Germany
The phylogenetic tree shown in Fig. S1 was constructed after alignment of the deduced protein sequences for
various
silicateins
and
cathepsins.
Gapped
BLAST
search
was
performed
using:
http://swissmodel.expasy.org/workspace/index.php?func=tools_targetidentification. The alignment of the
deduced amino acid sequence of silicatein α from the demosponge Suberites domuncula against the Cys25→Ser
mutant of human cathepsin S is shown in Fig. S2. The amino acid residue that is changed in the cathepsin S
sequence is indicated. The identity is 51.6%.
The Ramachandran plot of the silicatein model is shown Fig. S3. The results revealed that only glycine residues
are located outside of the allowed regions (Table S1).
The Modeler parameters and the LibDock parameters are given in Tables S2 and S3.
The results of the ligand docking analysis with the silicatein substrate molecules, Si(OH)4 and TEOS, are
summarized in Table S4.
Fig. S1. Phylogenetic analysis of silicatein within the cathepsin family. The deduced proteins were aligned and
the phylogenetic tree was constructed. The hitherto known three hexactinellid sequences were included;
silicatein from Crateromorpha meyeri silicatein (SILCA_CRATEROMORPHA; AM920776) and from
Monorhaphis chuni (SILCAa_MONORHAPHIS; FN394978) and the silicatein-like protein Aulosaccus sp.
(SILCA_AULOSACCUS; ACU86976.1). The bulk of silicatein sequences has been identified in demosponges.
First, the silicateins-α sequences from Suberites domuncula (SILCAa_SUBERITES; CAC03737.1), Tethya
aurantium (SILCAa_TETHYA; AAC23951.1), Geodia cydonium (SILCAa_GEODIA; CAM57981.1) and
Acanthodendrilla sp. Vietnam (SILCAa_ACANTHODENDRILLA; ACH92669.1), as well as from Lubomirskia
baicalensis (SILCAa2_LUBOMIRSKIA; AJ968945) and from Ephydatia fluviatilis (SILCA_EPHYDATIA;
BAE54434.1). Second, the silicatein-β sequences from Suberites domuncula (SILCAb_SUBERITES;
CAH04635.1), Tethya aurantium (SILCAb_TETHYA; AF098670_1) and Acanthodendrilla sp. Vietnam
(SILCAb_ACANTHODENDRILLA; FJ013043.1). Third, silicateins that had been identified in marine sponges
from which only one isoform had been obtained; silicatein from Petrosia ficiformis (SILCA_PETROSIA;
AAO23671.1) and from Halichondria okadai (SILCA_HALICHONDRIA; BAB86343.1). As reflected in the
rooted tree, these silicateins derived from the cathepsins among which in this tree the following sequences have
been included; cathepsin-like protein 2 Crateromorpha meyeri (CATL2_CRATEROMORPHA; CAP17585.1),
cathepsin-like protein 1 [Crateromorpha meyeri] (CATL1_CRATEROMORPHA; CAP17584.1), mRNA for
cathepsin L (catl gene) Aphrocallistes vastus (CATL_APHROCALLISTES; AJ968951), cathepsin B Suberites
domuncula
(CATLB_SUBERITES;
CAH04630.1),
cathepsin
X/O
Suberites
domuncula
(CATLX/O_SUBERITES; |CAH04633.1), cathepsin L Suberites domuncula (CATLL_SUBERITES;
CAH04632.), cathepsin H [Suberites domuncula (CATLH_SUBERITES; CAH04631.1), human cathepsin L1
(CATLL_HUMAN; NP_666023.1) as well as human cathepsin S (CATLS_HUMAN; AAB22005.1). The
resulting tree was rooted with the sequence from the papain-like cysteine peptidase XBCP3 Arabidopsis thaliana
(PAPAIN_ARABIDOPSIS; AF388175_1).
SD
1GLO:A_PDBID_CHAIN_SEQUENCE
Consensus
SD
1GLO:A_PDBID_CHAIN_SEQUENCE
Consensus
SD
1GLO:A_PDBID_CHAIN_SEQUENCE
Consensus
SD
1GLO:A_PDBID_CHAIN_SEQUENCE
Consensus
SD
1GLO:A_PDBID_CHAIN_SEQUENCE
Consensus
1
50
(1) DYPEAVDWRTKGAVTAVKDQGDCGASYAFSAMGALEGANALAKGNAVSLS
(1) -LPDSVDWREKGCVTEVKYQGSCGASWAFSAVGALEAQLKLKTGKLVSLS
(1)
PDAVDWR KG VT VK QG CGASWAFSAMGALEA
L G VSLS
51
100
(51) EQNIIDCSIP-YGNHGCHGGNMYDAFLYVIANEGVDQDSAYPFVGKQSSC
(50) AQNLVDCSTEKYGNKGCNGGFMTTAFQYIIDNKGIDSDASYPYKAMDLKC
(51) QNIIDCS
YGN GC GG M AF YII N GID DAAYPF A
C
101
150
(100) NYNSKYKGTSMSGMVSIKSGSESDLQAAVSNVGPVSVAIDGANSAFRFYY
(100) QYDSKYRAATCSKYTELPYGREDVLKEAVANKGPVSVGVDARHPSFFLYR
(101) NY SKYKA S S
I G E L AVAN GPVSVAIDA
AF Y
151
200
(150) SGVYDSSRCSSSSLNHAMVVTGYGSYNGKKYWLAKNSWGTNWGNSGYVMM
(150) SGVYYEPSCTQN-VNHGVLVVGYGDLNGKEYWLVKNSWGHNFGEEGYIRM
(151) SGVY
CS
LNHAMLV GYG NGK YWL KNSWG NFG GYI M
201
219
(200) ARNKYNQCGIATDASYPTL
(199) ARNKGNHCGIASFPSYPEI
(201) ARNK N CGIAS SYP I
Fig. S2. Alignment of the deduced amino acid sequence of silicatein α from Suberites domuncula (accession
number CAC03737.1) against the Cys25→Ser mutant of human cathepsin S (PDB ID: 1GLO). Identical amino
acids are in red letters and highlighted in yellow, and similar amino acids are highlighted in green. The amino
acid residue that was changed in the cathepsin S sequence is indicated in blue (serine; large S).
Fig. S3. Ramachandran (φ, ψ) plot of the silicatein model. Glycine residues are depicted as open triangles, other
amino acids as open circles. The allowed regions αL, αR, and β are indicated. Blue line: high score; red line: low
score.
Table S1. Evaluation of the silicatein model.
Statistics for non-glycine, non-proline residues:
Number of terminal residues
2
Number of non terminal residues
185
Number of residues in allowed region
180
(97.3%)
Number of residues in marginal region
5
(2.7%)
Number of residues in disallowed region
0
(0.0%)
Number of glycine residues
Number of proline residues
Statistics for proline and glycine:
25
5
Table S2. Modeler parameters.
Cut overhangs
Disulfide bridges
Cis-prolines
Additional restraints
Copy ligands
Copy chains
Reference template
Reference template copy regions
Optimize side-chains
Number of models
Start model index
Optimization level
Refine loops
Refine loops number of models
Refine loops optimization level
Refine loops use DOPE method
False
True
5
1
High
False
1
Medium
True
Table S3. LibDock parameters.
Input site sphere
Number of hot spots
Docking tolerance
Docking preferences
Max hits to save
Max number of hits
Minimum LibDock score
Final score cutoff
Max BFGS steps
Rigid optimization
Keep hydrogens
Max conformation hits
Max start conformations
Steric fraction
Final cluster radius
Apolar SASA cutoff
Polar SASA cutoff
-7.903, 37.125, 17.084, 14
100
0.25
High quality
100
100
100
0.5
50
False
False
30
1000
0.10
0.5
15.0
5.0
Surface grid steps
Conformation method
Maximum conformations
Discard existing conformations
Energy threshold
DS report summary
Separate conformations
Minimization algorithm
RMSD cutoff
Minimization sphere of flexible atoms
Flexible residues
Minimization forcefield
Minimization iterations
Minimization dielectric constant
Minimization distant-dependent dielectrics
Minimization energy tolerance
Minimization gradient tolerance
Minimization nonbond cutoff
Verbose
sp2-sp2 rotation
Grid scoring
18
FAST
255
True
20.0
False
False
Do not minimize
1.0
CHARMm
1000
1.0
True
0.0
0.001
13.0
0
True
True
Table S4. Number of conformers, polar and apolar HotSpots, and the LibDock score for the silicatein substrate
molecules Si(OH)4 and TEOS.
Ligand
Conformers
Polar
Si(OH)4
TEOS
1
123
94
57
HotSpots
Apolar
LibDock score
73
45
51,0041
34,0053