Protein structure is more conserved than sequence in nature. In this direction we developed a novel methodology that significantly improves conventional homology modelling when sequence identity is low, by taking into consideration 3D structural features of the template, such as size and shape. Herein, our new homology modelling approach was applied to the homology modelling of the RNA-dependent RNA polymerase (RdRp) of dengue (type II) virus. The RdRp of dengue was chosen due to the low sequence similarity shared between the dengue virus polymerase and the available templates, while purposely avoiding to use the actual X-ray structure that is available for the dengue RdRp. The novel approach takes advantage of 3D space corresponding to protein shape and size by creating a 3D scaffold of the template structure. The dengue polymerase model built by the novel approach exhibited all features of RNA-dependent RNA polymerases and was almost identical to the X-ray structure of the dengue RdRp, as opposed to the model built by conventional homology modelling. Therefore, we propose that the space-aided homology modelling approach can be of a more general use to homology modelling of enzymes sharing low sequence similarity with the template structures.
3D structural information provides invaluable insights into the organization, mode of action, folding, and utterly function of a given protein. The 3D structure of proteins is usually experimentally determined using X-ray crystallography, NMR, or microscopy [
Homology modelling is the current leading technique for
Conventional homology modelling methods are comprised of the following steps [
In the present work, a use case was sought that would be impossible to model using the previously described conventional homology techniques in an effort to put to the test and apply our novel proposed homology modelling technique. In this direction an example from the highly mutagenic field of RNA viruses was selected. In particular, we chose to model the three-dimensional structure of the RNA-dependent RNA polymerase (RdRp) of Dengue (type II) virus by using the crystal structures of other polymerases of the
The viral family
The NS5 proteins of hepatitis C virus (HCV), bovine viral diarrhoea virus (BVDV), and Dengue flavivirus type II have been attributed a RNA-dependent RNA polymerase (RdRp) function and thus constitute very good targets for the drug design approach [
Overall, it was found that the model derived from conventional homology modelling was poor in quality and structurally incapable for the
All computations and simulations were carried out on an Intel P4-based Microsoft Windows XP workstation mainly using MOE 2005.03 Package [
The amino acid sequence of dengue polymerase was obtained from the GenBank database (accession no. NC_001474, entry name: dengue virus type 2, complete genome) [
The 3D modelling of the Dengue RdRp was performed using the MOE package through the built in homology modelling module. The RCSB entries 1NB7 and 1S48 corresponding to the crystal structures of the HCV [
Due to low sequence identity, an enhanced approach to conventional homology modelling process was applied. The novel approach involves the exploitation of common 3D space on the template structures. For this purpose, the conformational space corresponding to the ssRNA, UTP, and rNTP tunnel regions was first calculated and subsequently filled by alpha spheres [
The initial models were further optimized by energy minimization using the conjugate gradient method as implemented within MOE and the CHARMM22 forcefield [
The quality of the final models was assessed using the PROCHECK suite of programs [
Electrostatic potential surfaces were calculated by solving nonlinear Poisson-Boltzmann equation using finite difference method [
Towards the modelling of the 3D structure of the RNA-dependent RNA polymerase (RdRp) of Dengue virus (type II) the known crystal structures of the RdRps of hepatitis C virus (HCV) [
(a) Stereoview of the superimposed crystal structures of the HCV [
The Dengue RdRp sequence was included to the above alignment guided by threading results through the program PHYRE [
The modelling of the Dengue RdRp structure was based on the sequence alignment shown in (Figure
The model produced by the conventional homology modelling procedure showed only few secondary structure elements and was largely unstructured (Figure
3D model of the Dengue RNA-dependent RNA polymerase produced by conventional homology modelling. This model exhibits only few secondary structure elements. Backbone clashes between the model and an ssRNA fragment (depicted in green sticks) copied from the HCV template structure indicate that the conventional homology modelling approach failed to model the ssRNA channel correctly. This figure demonstrates that conventional homology modelling of the Dengue polymerase was unsuccessful.
In order to overcome the deficiency of the conventional homology modelling, a novel approach based on additional information from the template structures has been developed. The approach takes advantage of the space occupied by ligands or substrates in the template structures to restrain the folding of the target protein. In the case of the Dengue RdRp, the model was enfolded up the 3D conformational space corresponding to the channel occupied by the ssRNA, the Mn++ ions, and the rNTP tunnel in the template structures. For this purpose, the abovementioned 3D space was first filled with alpha-spheres (see Methods) in both templates (Figure
The novel (3D space-aided) approach to homology modelling. Left: the template structures of the HCV (in green) and BVDV (in blue) are shown in ribbon representation and their 3D space corresponding to the substrate channel is filled with alpha-spheres (see methods). Middle: the sum of alpha-spheres from the templates used to restrict the modelling of the Dengue polymerase. Right: the dengue polymerase model produced by the space-aided approach. The new model is enfolded up the sphere-filled cavity space of the templates, which guarantees that the space corresponding to substrates will not be occupied by parts of the protein.
The quality of the produced model as assessed by PROCHECK [
Parameters reflecting the quality of the Dengue polymerase model calculated by PROCHECK [
Stereochemical parameters | Number of data points | Parameter value | Typical value | Band width | Number of band widths from mean | Quality compared to structures at 2.9 Å |
---|---|---|---|---|---|---|
A: main chain parameters | ||||||
Percentage residues in A, B, L | 490 | 71.0 | 68.7 | 10.0 | 0. | Inside |
Omega angle st dev | 545 | 9.0 | 6.0 | 3.0 | 1.0 | Inside |
Bad contacts/100 residues | 0 | 0.0 | 17.9 | 10.0 | −1.8 | BETTER |
Zeta angle st dev | 508 | 3.3 | 3.1 | 1.6 | 0.1 | Inside |
H-bond energy st dev | 290 | 0.7 | 1.0 | 0.2 | −1.7 | BETTER |
Overall G-factor | 547 | −1.0 | −0.8 | 0.3 | −0.9 | Inside |
| ||||||
B: side chain parameters | ||||||
Chi-1 gauche minus st dev | 86 | 11.5 | 26.3 | 6.5 | −2.3 | BETTER |
Chi-1 trans st dev | 168 | 11.3 | 25.7 | 5.3 | −2.7 | BETTER |
Chi-1 gauche plus st dev | 203 | 12.1 | 24.3 | 4.9 | −2.5 | BETTER |
Chi-1 pooled st dev | 457 | 12.9 | 25.1 | 4.8 | −2.5 | BETTER |
Chi-2 trans st dev | 109 | 13.5 | 25.3 | 5.0 | −2.3 | BETTER |
As expected from the sequence alignment (Figure
The Dengue polymerase model produced by the 3D space-aided approach (colour turquoise) superposed to the Dengue type II X-ray crystal structure (coloured red, PDB entry: 2BMF). All major
In order to evaluate the substrate binding site, the model was subjected to energy minimization in the presence of the RdRp substrates. The coordinates of the ssRNA or UTP/Mn++ were transferred to the model from the HCV RdRp template structure (entries 1NB7 and1NB6, resp.) for this purpose. The model could accommodate either substrate upon energy minimization in contrast to the model obtained by conventional homology modelling. Invariant residues of various motifs in the vicinity of either substrate in the HCV template structure were conserved structurally in the Dengue polymerase model (Figure
Closeup of the ssRNA (upper) and the UTP/Mn++ (lower) sites in (a) the dengue polymerase model produced by the 3D space-aided approach and (b) the Dengue X-ray polymerase structure, for comparison (PDB entry: 2BMF). Coordinates of the substrates were copied to the model from the template structure (see text). The substrates are depicted in sticks and dotted spheres, whereas spheres in magenta represent the catalytic Mn++ atoms. Invariant residues (labeled) of various motifs in the vicinity of either substrate are structurally conserved in this Dengue polymerase model demonstrating the efficiency of the novel approach as opposed to the conventional homology modeling.
To evaluate further the novel approach, the models obtained by both conventional homology modelling and the 3D space-aided homology modelling method were compared with the X-ray resolved structure of the Dengue RdRp by calculating the root mean square deviations (RMSd) between equivalent atoms. Large RMSd values are indicative of systems of poor quality. The
Summary of sequence and structural similarities between the homology models of dengue polymerase (novel and conventional approaches) and the RdRp known structures (RCSB codes in parenthesis) used as templates.
Template | Sequence comparison | RMSd (Å) | ||||
---|---|---|---|---|---|---|
Number of equivalent amino acids | Identity (%) | Similarity (%) | Number of atoms | Novel | Conventional | |
HCV RdRp (1NB7) | 351 | 18 | 34 | 139 ( |
1.19 | 3.57 |
BVDV RdRp (1S48) | 172 | 18 | 31 | 65 ( |
1.04 | 3.04 |
Equivalent invariant residues between the HCV template and the Dengue virus polymerase model in the vicinity of the ssRNA fragment or the UTP substrate.
Substrate | ssRNA | UTP | ||||
---|---|---|---|---|---|---|
Interacting invariant RdRp residues | HCV RdRp structure | Dengue virus Model | Motif | HCV RdRp structure | Dengue virus Model | Motif |
D220 | D312 | IV | D318 | D441 | VI | |
D318 | D441 | VI | D319 | D442 | VI | |
D319 | D442 | VI | D220 | D312 | IV | |
N291 | N388 | V | K155 | K247 | II | |
T287 | T384 | V | ||||
R158 | R250 | II | ||||
E143 | E327 | I | ||||
S282 | S379 | V | ||||
K141 | K235 | I | ||||
R168 | R260 | II | ||||
R394 | R515 | Conserved |
In order to analyze the molecular surface of the produced Dengue polymerase model, the electrostatic potential surface of the 3D space-aided model was calculated (see Section
Surface comparison of the crystal structures of the HCV, BVDV, and Dengue RdRp (PDB entry: 2BMF) versus the 3D model of the Dengue polymerase that was produced by the 3D space-aided homology modelling approach. The surfaces are colored according to their electrostatic potential.
The 3D crystal structure of Dengue has been determined by X-ray crystallography. Therefore, a direct comparison can be performed between the homology model and the crystal structure of the Dengue RdRp. The ultimate aim is to judge whether the space-aided homology modelling approach did make a significant difference and improvement to the conventionally built model using unrestrained and unbiased homology modelling. The RMSd between the two models is 4.6 Å. The RMSd between the conventionally built and the space-aided RdRp models is 6.3 Å and 1.4 Å respectively. The 3D structure of the space-aided model is very similar to that of the crystal structure (Figures
In the current study, a novel approach to conventional homology modelling has been developed. The approach takes advantage of the 3D conformational space corresponding to the template’s shape and size characteristics as well as the existence of ligands and substrates, which are used to restrain the folding of the target (query) protein. With the example of the successful modelling of the 3D structure of the RNA-dependent RNA polymerase of Dengue (type II) virus, which shared low sequence identity with the chosen templates, the new approach illustrated an efficient way to model 3D structures of enzymes sharing low sequence identity with the modelling templates.