Discovery of Klotho peptide antagonists against Wnt3 and Wnt3a target proteins using combination of protein engineering, protein-protein docking, peptide docking and molecular dynamics simulations - PubMed (original) (raw)
Discovery of Klotho peptide antagonists against Wnt3 and Wnt3a target proteins using combination of protein engineering, protein-protein docking, peptide docking and molecular dynamics simulations
Shaher Bano Mirza et al. J Enzyme Inhib Med Chem. 2017 Dec.
Abstract
The Klotho is known as lifespan enhancing protein involved in antagonizing the effect of Wnt proteins. Wnt proteins are stem cell regulators, and uninterrupted exposure of Wnt proteins to the cell can cause stem and progenitor cell senescence, which may lead to aging. Keeping in mind the importance of Klotho in Wnt signaling, in silico approaches have been applied to study the important interactions between Klotho and Wnt3 and Wnt3a (wingless-type mouse mammary tumor virus (MMTV) integration site family members 3 and 3a). The main aim of the study is to identify important residues of the Klotho that help in designing peptides which can act as Wnt antagonists. For this aim, a protein engineering study is performed for Klotho, Wnt3 and Wnt3a. During the theoretical analysis of homology models, unexpected role of number of disulfide bonds and secondary structure elements has been witnessed in case of Wnt3 and Wnt3a proteins. Different in silico experiments were carried out to observe the effect of correct number of disulfide bonds on 3D protein models. For this aim, total of 10 molecular dynamics (MD) simulations were carried out for each system. Based on the protein-protein docking simulations of selected protein models of Klotho with Wnt3 and Wnt3a, different peptides derived from Klotho have been designed. Wnt3 and Wnt3a proteins have three important domains: Index finger, N-terminal domain and a patch of ∼10 residues on the solvent exposed surface of palm domain. Protein-peptide docking of designed peptides of Klotho against three important domains of palmitoylated Wnt3 and Wnt3a yields encouraging results and leads better understanding of the Wnt protein inhibition by proposed Klotho peptides. Further in vitro studies can be carried out to verify effects of novel designed peptides as Wnt antagonists.
Keywords: Homology modeling; Klotho; Wnt3; Wnt3a; molecular dynamics (MD) simulations; peptide design; protein engineering; protein–protein docking.
Figures
Figure 1.
Distribution of 24 Cysteine residues over different domains of Wnt3 protein structure. The finger like domain, including Cysteine knot and thumb domain are Cysteine hotspots. The position of Cysteine residues in the protein structure gives the clue about disulfide bonds importance in the stability of critical Wnt protein domains (NTD: N-terminal domain; CTD: C-terminal domain).
Figure 2.
RMSD and RMSF analysis of xWnt8 protein MD simulations. Average RMSD for xWnt8 is 4.2 Å.
Figure 3.
Wnt3 comparison of change in domains in all three experiments from initial structure. Alignment of these structures have been done by Needleman–Wunsch Alignment algorithm and BLOSUM-62 matrix in Chimera molecular visualization tool by keeping initial stage structure as reference. Right side 90° angle view of finger domain has been presented. Cysteine knot shows huge difference among all three models like other domains. The thumb domain appears comparatively stable. (Yellow and red colored cartoons show before (yellow) and after (red) MD simulations of Wnt3 with 12 disulfide bonded model (Wnt3-M3). Blue and green-colored cartoons show after MD simulations of model with nine disulfide bonds (Wnt3-M2) and model with three disulfide bonds (Wnt3-M1), respectively).
Figure 4.
RMSD and RMSF analysis of three Wnt3 protein homology models (Wnt3-M1: Wnt3 homology model with two disulfide bonds; Wnt3-M2: Wnt3 homology model with nine disulfide bonds; Wnt3-M3: Wnt3 homology model with 12 disulfide bonds). Average RMSDs for Wnt3-M1, Wnt3-M2 and Wnt3-M3 are calculated as 4.7 Å, 7.6 Å and 4.3 Å, respectively.
Figure 5.
Wnt3a comparison of change in domains in all three experiments from initial structure. Alignment of these structures have been done by Needleman–Wunsch Alignment algorithm and BLOSUM-62 matrix in Chimera molecular visualization tool by keeping initial stage structure as reference. Right side 90° angle view of finger domain has been presented. Cysteine knot shows huge difference among all three models like other domains. The thumb domain appears comparatively stable. (Yellow and red colored cartoons show before (yellow) and after (red) MD simulations of Wnt3a with 12 disulfide bonded model (Wnt3a-M3). Blue and green-colored cartoons show after MD simulations of model with nine disulfide bonds (Wnt3a-M2) and model with three disulfide bonds (Wnt3a-M1), respectively).
Figure 6.
RMSD and RMSF analysis of three Wnt3a protein homology models (Wnt3a-M1: Wnt3a homology model with five disulfide bonds; Wnt3a-M2: Wnt3a homology model with nine disulfide bonds; Wnt3a-M3: Wnt3a homology model with 12 disulfide bonds). Average RMSDs for Wnt3a-M1, Wnt3a-M2 and Wnt3a-M3 are 6.8, 5.4 and 5.5 Å, respectively.
Figure 7.
Interaction of peptide with all three considered domains of Wnt3 and Wnt3a using induced fit docking (IFD) docking approach. (a) Docked complex of Wnt3 (green) with peptide 4 (magenta) against three binding pockets (cyan). (b) Docked complex of Wnt3a (cyan) with peptide 11 (magenta) against three binding pockets (yellow). Blue-colored lines show palmitoleic acid attached to Wnt proteins.
Figure 8.
2D-ligand interactions diagram of Wnt3 with peptide-4 derived by IFD docking. (a) Top-docking pose against thumb domain residues, (b) top-docking pose against finger domain and (c) top-docking pose against a patch of palm domain.
Figure 9.
2D-ligand interactions diagram of Wnt3a with peptide-11 derived by IFD docking (a) top-docking pose against thumb domain residues, (b) top-docking pose against finger domain and (c) top-docking pose against a patch of palm domain.
References
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- Rando TA. Stem cells, ageing and the quest for immortality. Nature 2006;441:1080–6. - PubMed
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