In silico characterisation, homology modelling and structure-based functional annotation of blunt snout bream (Megalobrama amblycephala) Hsp70 and Hsc70 proteins
© Tran et al. 2015
Received: 15 May 2015
Accepted: 28 November 2015
Published: 15 December 2015
Heat shock proteins play an important role in protection from stress stimuli and metabolic insults in almost all organisms.
In this study, computational tools were used to deeply analyse the physicochemical characteristics and, using homology modelling, reliably predict the tertiary structure of the blunt snout bream (Ma-) Hsp70 and Hsc70 proteins. Derived three-dimensional models were then used to predict the function of the proteins.
Previously published predictions regarding the protein length, molecular weight, theoretical isoelectric point and total number of positive and negative residues were corroborated. Among the new findings are: the extinction coefficient (33725/33350 and 35090/34840 - Ma-Hsp70/ Ma-Hsc70, respectively), instability index (33.68/35.56 – both stable), aliphatic index (83.44/80.23 – both very stable), half-life estimates (both relatively stable), grand average of hydropathicity (−0.431/-0.473 – both hydrophilic) and amino acid composition (alanine-lysine-glycine/glycine-lysine-aspartic acid were the most abundant, no disulphide bonds, the N-terminal of both proteins was methionine). Homology modelling was performed by SWISS-MODEL program and the proposed model was evaluated as highly reliable based on PROCHECK’s Ramachandran plot, ERRAT, PROVE, Verify 3D, ProQ and ProSA analyses.
The research revealed a high structural similarity to Hsp70 and Hsc70 proteins from several taxonomically distant animal species, corroborating a remarkably high level of evolutionary conservation among the members of this protein family. Functional annotation based on structural similarity provides a reliable additional indirect evidence for a high level of functional conservation of these two genes/proteins in blunt snout bream, but it is not sensitive enough to functionally distinguish the two isoforms.
KeywordsHsp70 Hsc70 Physicochemical characteristics Homology modelling Structural similarity Functional annotation
Heat shock (or stress) proteins (HSPs) are a family of highly conserved cellular proteins that play an important role in protection from stress stimuli and metabolic insults in almost all organisms [1–4]. They include three major families: Hsp90 (85–90 kDa), Hsp70 (68–73 kDa) and low molecular-weight Hsps (16–47 kDa) . The Hsp70 family is encoded by two different genes: a constitutive, “housekeeping” heat shock cognate (hsc) 70 gene, which is predominantly associated with physiological processes, and stress-inducible hsp70. Hsc70 protein plays a key role as molecular chaperone in a wide range of cellular processes, such as protein assembly, folding, transport through membrane channels, translocation and denaturation [4–7]. Hsp70 protein is mainly responsible for the maintenance of cellular homeostasis during the stress response, thus protecting cells from the damage caused by environmental stress agents, such as heat shock, chemical exposure and UV or γ-irradiation [4, 8, 9]. Hsp70 is also a potential activator of the innate immune system mechanisms [10–12]. Hsp70 is considered to be by far the most evolutionary conserved protein, found in all organisms from archaebacteria and plants to humans . Both proteins have a modular structure consisting of a highly conserved N-terminal ATPase domain, an adjacent well-conserved substrate-binding domain (SBD) that contains a hydrophobic pocket with a lid-like structure over it, and a conserved but more variable C-terminal domain, which plays an important role in Hsp70 functions required for cell growth. In the ATP-bound state, the substrate-binding pocket is open and rapidly exchanges substrate. ATP hydrolysis induces closing of the lid over the pocket, which stabilises substrate binding. Return to the ATP-bound state restores the open conformation, facilitating substrate release [14, 15].
In aquaculture, fish are often exposed to stressful situations, such as sudden temperature changes, high stocking density, trauma, hypoxia, as well as viral and bacterial infections, which often results in high fish mortality. Both genes have been identified and their expression characterised in many fish species, e.g., rainbow trout (Oncorhynchus mykiss), zebrafish (Danio rerio), Korean rockfish (Sebastes schlegeli), Nile tilapia (Oreochromis niloticus), mandarin fish (Siniperca chuatsi) [16–20] and both are known to have a crucial role in response to heat shock, hypoxia, crowding stress and bacterial pathogens in fish [3, 4, 7, 19]. However, according to our best knowledge, a three-dimensional (3-D) model of any fish Hsp70 family protein has not been published so far.
Blunt snout bream (Megalobrama amblycephala Yih, 1955), native to the middle portions of the Yangtze River basin, is becoming an increasingly important freshwater aquaculture species in China. Due to its successful artificial propagation and high economic value, the total output of the blunt snout bream aquaculture industry reached 652 215 tons in 2010 [21–23]. Previously, Ming, Xie  used bioinformatics tools to analyse some physicochemical characteristics of the two Ma-Hsp70 family proteins, such as molecular weight, isoelectric point, solubility (as hydrophilic property) and richness in B cells antigenic sites. Their results indicated that Ma-Hsp70 shares more than 85 % identity with its homologs in other vertebrates, has no signal peptide or transmembrane region, contains many protein kinase C phosphorylation sites, N-myristoylation sites, casein kinase II phosphorylation sites and N-glycosylation sites, while the predominant elements of the secondary structure are α-helix and random coil. However, as the previous study left many questions regarding the physicochemical and structural properties (particularly regarding the tertiary structure) open, this study, as a successive work, aims to fill this gap. Several different computational tools and available web servers were used to deeply analyse the physicochemical characteristics and, using homology modelling, reliably predict the tertiary structure of the blunt snout bream Hsp70 and Hsc70 proteins. Additionally, rapidly increasing number of known gene sequences in many organisms has prompted the need for new procedures and techniques for the high-throughput functional annotation of genes. While most of those traditionally used remain rather costly and work-intensive, with rapidly growing number of protein structures deposited in the Protein Data Bank (PDB), computational structural genomics is becoming an increasingly promising tool for fast and cheap insight into protein structures, functions and interactions [24–27]. As Ming, Xie  analysed the expression of Ma-hsp70 and Ma-hsc70 genes in order to gain insight into their functions, this study further aims to provide a supplementary evidence for conserved function of these two genes by in-deep structure analysis and functional annotation of their polypeptide products on the basis of the similarity of their tertiary structures to the available templates from other organisms. As structure-based functional annotation has seldom been used in study of fish proteins, the aim of this study is also to test the applicability of this approach for functional annotation of fish gene sequences.
Amino acid sequences of the blunt snout bream Hsp70 (Accession number: ACG63706.2) and Hsc70 (Accession number: GQ214528.1)  were obtained from the NCBI protein database (http://www.ncbi.nlm.nih.gov/) in FASTA format as the target template and used for further analyses. Physicochemical properties of the proteins, including molecular weight, amino acid composition, theoretical isoelectric point (pI), the total number of positive and negative residues, extinction coefficient (EC), instability index (II), aliphatic index (AI) and grand average of hydropathicity (GRAVY) were analysed using Expasy’s ProtParam prediction server . SOSUI server  was used to determine whether it is a soluble or a transmembrane protein, while CYS_REC (http://linux1.softberry.com) was used to predict the presence of cysteine residues and their bonding patterns.
Comparative homology modelling
Homology modelling of the proteins was performed by the SWISS-MODEL server [30, 31], which aligns an input target with pre-existing templates to generate a series of predicted models. The most suitable template to build the 3-D model was selected on the basis of sequence identity . Multiple amino acid sequence alignment was performed with ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2). Stereochemical quality and accuracy of the predicted models were analysed using PROCHECK’s Ramachandran plot analysis, ERRAT, PROVE, Verify3D (all four available from the SAVES server at http://nihserver.mbi.ucla.edu), ProQ  and ProSA [34, 35]. Structural analysis was performed and model figures generated by Swiss PDB Viewer .
Structural similarity and functional annotation
COFACTOR web server was used to perform the global structure match using TM-align algorithm and render the TM-score was calculated to assess the global structural similarity: values range from 0 to 1, where TM-score = 1 indicates the perfect match between two structures. Scores below 0.17 correspond to randomly chosen unrelated proteins, whereas a score higher than 0.5 implies generally the same fold . Annotations on ligand-binding sites, gene ontology and enzyme commission were performed by the I-TASSER suite, which structurally matches the 3-D model of Ma-Hsp70 and Ma-Hsc70 to the known templates in protein function databases [38–40].
Results and discussion
Amino acid composition of Ma-Hsp70 and Ma-Hsc70
Cysteine occurrence pattern and probability of cysteine residue pairing in Ma-Hsp70 and Ma-Hsc70 proteins
probably no SS-bond
Comparative homology modelling
Assessment of the predicted three-dimensional structures of Ma-Hsp70 and Ma-Hsc70 proteins
Residues in most favoured regions
Residues in additional allowed regions
Residues in generously allowed regions
Residues in disallowed regions
Structure similarity analysis
Top five identified structural analogs in the Protein Data Bank (PDB) library
Function prediction on the basis of structural similarity
Residue-specific ligand binding probability
Ligand-binding site residues
14, 15, 16, 17, 203, 204, 205, 206, 232, 270, 273, 274, 277, 340, 341, 342, 344, 345, 368
14, 15, 73, 149, 177, 231
12, 13, 14, 15, 17, 201, 202, 203, 204, 230, 268, 271, 272, 338, 339, 340, 342, 343, 366
12, 13, 71, 147, 175, 229
Enzyme Commission (EC) predictions for Ma-Hsp70 and Ma-Hsc70 proteins
Similarly, consensus prediction of GO terms also suggested ATP binding, interacting selectively and non-covalently with adenosine 5'-triphosphate (GO = 0005524; ontology = molecular function) as the main function for both proteins, with very high GO-scores (0.98 for Hsc70 and 0.99 for Hsp70).
All these results are in accordance with the previously described functioning mechanisms: Hsp70s in the ATP-bound state catch and release their substrates rapidly, while Hsp70s in the ADP-bound state seize them firmly. By cycling between the ATP- and ADP-bound states, Hsp70s exert their chaperone activity [14, 49]. However, the analysis was not sensitive enough to distinguish between the functions of the constitutive (Hsc) and inducible (Hsp) isoforms. This is not a major setback, as it has been suggested that the functional difference appears to lie more in regulation of the SBD-substrate interactions than in the physical properties of the two ATPase domains .
To help better understand the functional biology of Hsp70 and Hsc70 in blunt snout bream, several computational tools were used to analyse the physicochemical properties, generate valid homology models of both proteins and predict their functions on the basis of structural similarity to other protein templates. Apart from presenting the first published homology models of Hsp70 and Hsc70 proteins in fish, this research also revealed a high structural similarity to Hsp70/Hsc70 proteins from several taxonomically distant animal species, corroborating a remarkably high level of evolutionary conservation among the members of this protein family. Functional annotation based on structure similarity provides a reliable additional indirect evidence for a high level of functional conservation of these two genes/proteins in blunt snout bream, but it is not sensitive enough to distinguish between the two isoforms. In conclusion, even though gene function assignment based on protein structure similarity is at present somewhat limited by the number of available protein structures deposited in the PDB, it has a strong potential to become a very fast, cheap and relatively reliable technique for high-throughput gene function assignment in fish.
grand average of hydropathicity
heat shock cognate
heat shock protein
protein data bank
The first author Tran Ngoc Tuan would like to thank the China Scholarship Council for providing scholarship of doctoral program in Huazhong Agricultural University, Wuhan, Hubei, P.R. China.
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