CMD: A Database to Store the Bonding States of Cysteine Motifs with Secondary Structures

Abstract

Computational approaches to the disulphide bonding state and its connectivity pattern prediction are based on various descriptors. One descriptor is the amino acid sequence motifs flanking the cysteine residue motifs. Despite the existence of disulphide bonding information in many databases and applications, there is no complete reference and motif query available at the moment. Cysteine motif database (CMD) is the first online resource that stores all cysteine residues, their flanking motifs with their secondary structure, and propensity values assignment derived from the laboratory data. We extracted more than 3 million cysteine motifs from PDB and UniProt data, annotated with secondary structure assignment, propensity value assignment, and frequency of occurrence and coefficiency of their bonding status. Removal of redundancies generated 15875 unique flanking motifs that are always bonded and 41577 unique patterns that are always nonbonded. Queries are based on the protein ID, FASTA sequence, sequence motif, and secondary structure individually or in batch format using the provided APIs that allow remote users to query our database via third party software and/or high throughput screening/querying. The CMD offers extensive information about the bonded, free cysteine residues, and their motifs that allows in-depth characterization of the sequence motif composition.

Full text

Hindawi Publishing Corporation
Advances in Bioinformatics
Volume 2012, Article ID 849830, 5pages
doi:10.1155/2012/849830
Research Article
CMD: A Database to Store the Bonding States of Cysteine Motifs
with Secondary Structures
Hamed Bostan,1Naomie Salim,2Zeti Azura Hussein,3
Peter Klappa,4and Mohd Shahir Shamsir1
1Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia, 81310 Johor Bahru, Johor, Malaysia
2Faculty of Computer Science and Information Systems, Universiti Teknologi Malaysia, 81310 Johor Bahru, Johor, Malaysia
3School of Bioscience and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia,
43600 Bangi, Selangor, Malaysia
4School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK
Correspondence should be addressed to Mohd Shahir Shamsir, shahir@utm.my
Received 30 July 2012; Accepted 6 September 2012
Academic Editor: Huixiao Hong
Copyright © 2012 Hamed Bostan et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Computational approaches to the disulphide bonding state and its connectivity pattern prediction are based on various descriptors.
One descriptor is the amino acid sequence motifs flanking the cysteine residue motifs. Despite the existence of disulphide bonding
information in many databases and applications, there is no complete reference and motif query available at the moment. Cysteine
motif database (CMD) is the first online resource that stores all cysteine residues, their flanking motifs with their secondary
structure, and propensity values assignment derived from the laboratory data. We extracted more than 3 million cysteine motifs
from PDB and UniProt data, annotated with secondary structure assignment, propensity value assignment, and frequency of
occurrence and coefficiency of their bonding status. Removal of redundancies generated 15875 unique flanking motifs that are
always bonded and 41577 unique patterns that are always nonbonded. Queries are based on the protein ID, FASTA sequence,
sequence motif, and secondary structure individually or in batch format using the provided APIs that allow remote users to query
our database via third party software and/or high throughput screening/querying. The CMD offers extensive information about
the bonded, free cysteine residues, and their motifs that allows in-depth characterization of the sequence motif composition.
1. Background
Disulphide bonds are formed by oxidation of two cysteine
residues in a protein and are significant to a protein’s
conformational stability as they confer greater thermal and
chemical stability as well as stabilizing structural interme-
diates to ensure the correct folding pathway. However, the
connectivity of the disulphide bonds in protein sequences
can only be determined experimentally. Given this dif-
ficulty, the ability to evaluate or predict the disulphide
bonding state and connectivity from the sequence would
prove to be highly valuable in engineering proteins for
biotechnological and medical applications. Computational
approaches towards disulphide connectivity prediction have
been based on various descriptors. One of these descriptors
is the sequence motifs generated by combining the flanking
residues on the either side of the the cysteine residue [1,
2]. These immediate residues flanking the cysteine have
been shown to influence the cysteine’s redox potential and
the cysteine’s steric accessibility [3]. These sequence motifs
have been fed into various prediction methods [4]suchas
machine learning approaches (i.e., statistical methods, neural
networks (NNs) [5], and support vector machine (SVM) [6–
8]suchasDiaNNA[3], DISULFIND [9], DCON [10], and
CysView [11]. Currently, all the cysteine motifs are extracted
by parsing data from protein databases and feeding them into
the prediction tools. Motivated by the absence of a database
and usefulness of the cysteine flanking motifs in predicting
the cysteine bonding state and connectivity prediction, we
have developed cysteine motif database (CMD) as a tool to
mine and store these motifs. The creation of CMD allows the
motif extraction and facilitates the study of their secondary

2Advances in Bioinformatics
structures, bonding and connectivity propensities. In this
paper, we present CMD as a publicly available tool that
complements existing prediction tools.
2. Construction and Content
2.1. Content. The CMD data was compiled from Pro-
tein Data Bank (PDB) (http://www.rcsb.org) and UniProt
(http://www.uniprot.org). For each databank, two different
datasets were created; a complete protein dataset and a
second 100% nonhomologous unique sequence dataset
(100% similar sequences were omitted). We have featured
CMD with both datasets for each PDB and UniProt, allowing
researchers to utilize the database in its entirety (73656
structures for PDB and 531462 structures for UniProt) or to
include only unique sequences (33874 for PDB and 140723
for UniProt). Using these datasets, we extracted 878,000
cysteine motifs based on 1st, 2nd, 3rd, 4th, and 5th flanking
residues of the cysteine as these immediate residues are
within proximity to exert influence on the cysteine (Tab l e 1).
The assignment of the bonding state of cysteine residues
and their bonding partners is based on the SSBOND and
DISULPHIDE BOND tags in each PDB and UniProt files.
The motifs were clustered according to the occurrence of the
bonding state, that is, always bonded, always nonbonded,
and both bonded and nonbonded (nonbonded state with
another cysteine or to other atoms such as metals). Each of
the bonded cysteine is also mapped to each inter and intra-
chain disulphide bond cysteine partner.
The motifs were categorized between inter and intrado-
main with the secondary structure assignments for each
motif sequence (if available) determined using secondary
structure reference files retrieved from PDB.
2.2. Construction. The data contained in CMD is stored
in Microsoft SQL server 2005 data storage architecture.
Cysteine motif pattern tables are indexed based on Protein
ID, motifs, chain number, and secondary structure to
enhance the efficiency of the querying performance. Table-
based partitioning was used to increase the flexibility and
performance on Motif data tables. In these tables, over
three million motifs are stored which can be queried and
processed. All preprocessing, data extraction, and injection
for motif sequences and their secondary structure were
carried out in Net 4.0 platform using C# programming
language. The web interface of CMD is based on ASP.
Net extension integrated with Ajax technology to provide a
strong, simple, and user friendly environment for end users.
The web application is hosted on an Internet Information
Services (IIS) HTTP server version 7.5.7600.16385. CMD
will be updated automatically with latest data from PDB and
Uniprot.
In addition, several APIs available in CMD enable
developers to query our database remotely and embed
the results in their own system independently. A complete
list of available APIs together with the method of inline
implementation is available in the FAQ section of the CMD
website.
Tab l e 1
PDB
(All)
PDB
(NH)
UniProt
(All)
UniProt
(NH)
Proteins 73656 33874 531462 140723
Patterns 535544 230213 2509611 966374
Bonded motifs 148505 64246 189238 113365
Nonbonded motifs 387039 165967 2320373 853009
Intrachain 84591 36473 — —
Interchain 4013 1900 — —
NH: Nonhomologous unique sequences which have been affected by 100%
similarity removal.
3. Data Update
Using RCSB and UniProt API’s, the software will retrieve
all the Protein IDs available in the mentioned resources.
A query will list all the existing Protein IDs in our local
dataset. All new Protein IDs will be identified using both
above references. Using RCSB and UniProt ftp services, all
the newly identified protein files will be downloaded using
the Protein ID’s to our local server. As in our method of
preprocessing and data set preparation, all SEQRESS and
SSBONDtagswillbeextractedfromthedownloadedfiles.
All cysteine motifs based on the 1st, 2nd, 3rd, 4th, and
5th number of flanking residue on each side (neighboring
residues) will be captured and extracted to the records of
data with cysteine at the meddle. Each record contains the
motif sequence, Chain ID, cysteine residue position in the
sequence, bonding status of cysteine residue and the Protein
ID as the reference. Each record will be inserted into our
database. A log will be generated for the successful procedure
or any run time error.
4. Utility and Discussion
4.1. User Interface. The CMD website features an interactive
and comprehensive cysteine Motif query engine by support-
ing different search keywords, such as Protein IDs and motif
sequences in the FASTA format. Users can filter according
to proteins which are mutated and engineered proteins. All
results can be downloaded as text and CSV for further
analysis (Figures 1,2,and3).
4.2. Utility: Example Applications. CMD facilitate studies
focused on cysteine disulphide bonding status prediction
and analysis by processing the data. Here we present two
applications of our system that illustrate the potential of
CMD in greater details.
4.2.1. Application 1: Statistical Analysis of Bonding State.
To analyze the predictive power of CFMD, we investigated
the cysteine bonding pattern of human protein disulphide
isomerase (PDII, P07237 [UniParc]). PDI catalyses the
formation (oxidation) and rearrangement (isomerisation)
of disulphide bonds during the folding of secretory and

Advances in Bioinformatics 3
Figure 1: Annotated diagram describing the search options for
“Search By ID” section. (A) Users can choose either PDB or Swis-
sProt. (B) Users can enter single or multiple ProteinIDs separated
by comma (,) as keyword. (C) Users can choose which of the results
to appear in the output.
membrane-bound proteins (for review see [12]), thus sta-
bilising the native structure of these proteins. PDI contains
two domains with high sequence homology to thioredoxin.
One of these thioredoxin motives is found at position 52–
55, while the second motif is located at position 396–399.
The active site cysteine residues in the thioredoxin motives
are essential for the oxidase/isomerase activity of PDI. In
each motif the two cysteine residues within the sequence—
WCGHC—can potentially form a disulphide bond.
To investigate whether both thioredoxin motives have
similar disulphide bond propensities, that is, whether both
thioredoxin motives are in the same bonded form, we
analysed the disulphide bonding pattern with the CFMD
(Figure 4 and Tab l e 2). Our analysis predicted that the first
thioredoxin motif around residues 52–55 indeed forms an
intradomain disulphide bond; the second cysteine residue
in the sequence CGHCKAL has a very high propensity of
forming a disulphide bond with the first cysteine residue.
However, the second thioredoxin motif is not predicted to
be disulphide bonded, since the second cysteine residue in
the sequence CGHCKQL has zero propensity of forming a
disulphide bond with the first cysteine residue in this motif.
We therefore predict that the two thioredoxin motives in PDI
are in different bonding states; while the first—WCGHC—
motif is in the oxidized and thus disulphide bonded form,
the second thioredoxin motif is in the reduced form. From
this analysis we conclude that the two thioredoxin motives
inPDIhavedifferent reduction potentials. This result is in
excellent agreement with the findings of Chambers and co-
workers [13], who showed that the two thioredoxin motives
react differently to Ero1a, the in vivo oxidant of PDI.
4.2.2. Application 2: Protein Identification and Motif Explo-
ration. Catalytic functionalities of some enzymatic proteins
are dependant on the oxidation and reduction of state of
their cysteine residues. The oxidation of cysteine residues
and formation of disulphide bonds take place in a reducing
environment. In prokaryotes, disulphide bonds are mainly
formed in the periplasmic space outside the membrane.
In contrast, the formation of disulphide bonds takes place
in endoplasmic reticulum (ER) in eukaryotes. As a result,
proteins with stable disulfide bonds rarely reside in the
Figure 2: Annotated diagram of “Search By FASTA Sequence”
section showing all search options and filtering criteria. (A) Users
can choose either PDB or SwissProt. (B) Users can enter single or
multiple FASTA sequences to be investigated for each motif inside.
(C) Users can also upload a FASTA format file to be investigated.
(D) Users can choose the number of amino acid residues on each
side of cysteine for motif extraction process within the FASTA
sequence. (E) Users can filter the proteins in which the motif will be
investigated. User can specify whether the protein was engineered or
mutated and choose whether the protein contains any DNA or RNA
link. They can also filter out the similar proteins and keep only one
identical copy of them for advanced investigations.
Figure 3: Annotated diagram describing the result’s annotation
for the “Search By Molecule Name” section. (A) Showing the
motifs, secondary structure, cysteine position in the sequence, and
the chain name. (B) Showing the propensity values of the motif
sequence. (C) The navigation pane facilitating accessing ProteinIDs
having common and similar features. (D) Listing the pair patterns
existing in the protein in details. (E) The summary of bonding for
the selected protein.
cytoplasm. This knowledge would apply on a larger scale,
making the local and global profile of each protein environ-
ment, its folding localization, and classification becoming a
potential contribution on the disulphide bonding prediction
mechanism.
CMD offers the user a unique ability to identify and
mine all known proteins using specific motif sequence, and
explore their classification, motif sequences, structure, and
bonding status. During the creation of the datasets, we
discovered 15875 unique motifs that are always bonded

4Advances in Bioinformatics
Figure 4: Quer y forfull length human protein disulphide isomerase
(PDII, P07237 [UniParc]). (A) Screenshot of parameters for CFMD.
Tab l e 2: Edited output from (A). The bold rows indicate the
second active site cysteine residues in the respective thioredoxin
motif. Column 1 (Thioredoxin motif) was added for additional
clarification. The cysteine residue in italics indicates the queried
cysteine residue, the respective position of which is given in the
second column.
Thioredoxin
motif Position Motif Total Bond Coefficient
00 0
152 APWCGHC 12 5 0.417
155 CGHCKAL 1 1 1
00 0
00 0
2396 APWCGHC 12 5 0.417
2399 CGHCKQL 2 0 0
(EATLRCWALGF with the highest occurrence) and 41577
unique patterns that are always nonbonded (ALSVPCSDSKA
with the highest occurrence) for the five flanking residues
that can be utilized for cysteine state prediction. The number
of these unique motifs is considerably higher than prior
number of motifs used in cysteine bond prediction [3,14]
and not limited to specific genomes [15].
4.3. Data Availability. The CMD databases are accessible
through a web portal at http://birg4.fbb.utm.my/cmd.The
entire database with annotations is available for download
in the SQL format, describing the relations between classes
and fragments. As an additional service for programmers and
third party developers, all queries available in CMD are freely
accessible using available web services and web application
programming interfaces (API). Also for automated high-
throughput querying, all information contained in the CMD
database can be downloaded using ftp services.
5. Discussion
The CMD combined data of bonded and free cysteine motifs
aims to fill a gap in the knowledge query that will allow in-
depth characterization of the composition propensity, and its
role in determining the bonding state. Despite the bonding
information regarding cysteine residues in proteins available
in many databases and several applications focused on
disulphide bridge formation prediction, there is no complete
reference with a proper form of representation and analysis
available at the moment. This database is automatically
updated from the PDB and UniProt that currently contain
878000 cysteine motifs with more than 77,000 unique
cysteine motifs and cysteine pairing motifs. Compilation
of these cysteine motifs together with their secondary
structures and propensity value assignments, and the ability
to query using Protein IDs and motif sequences is a novel
and significant feature over prior prediction works which
use considerably smaller datasets [3]. In addition to the
novelty of the motif query tool, CMD has several novelties
such as inclusion of UniProt data, the distinction between
inter or intrachain disulphide bonds, inter or intradomain
bonds, and an application programming interfaces (APIs)
for interfacing with other bioinformatics tools.
6. Conclusion
The creation of CMD is useful when analyzing cysteine/
disulfide bond formation and its motif sequence compo-
sition analysis by providing (1) a query tool for cysteine
motifs based upon a comprehensive cysteine motif database
curated from PDB and UniProt, (2) secondary structure
and propensity values assignments of each motif sequence,
and (3) datasets of detailed information of the motifs such
as occurrence frequency and their amino acids propensity
value. We believe that CMD’s usefulness will be the query
tool that will complement other protein 3D structural
databases and similarly motif-based prediction tools.
Availability and Requirements
The CMD database is available to the public for free at
http://birg4.fbb.utm.my/cmd/. Contact: shahir@utm.my.
Funding
Ministry of Science, Technology and Innovation (MOSTI)
Grant no. 07-05-MGI-GMB007.
Conflict of Interests
The authors declare that they have no conflict of interests.
Acknowledgment
The authors would like to acknowledge Chew Teong Han for
the support throughout the development of CMD.
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