Molecular dynamics study of tropical calcific pancreatitis (TCP) associated calcium-sensing receptor single nucleotide variation

Abstract

Tropical Calcific Pancreatitis (TCP) is a chronic non-alcoholic pancreatitis characterised by extensive calcification. The disease usually appears at a younger age and is more common in tropical regions. This disease's progression can lead to pancreatic diabetes, which can subsequently lead to pancreatic cancer. The CASR gene encodes a calcium-sensing receptor (CaSR), which is a GPCR protein of class C. It is expressed in the islets of Langerhans, the parathyroid gland, and other tissues. It primarily detects small gradients in circulating calcium concentrations and couples this information to intracellular signalling, which helps to regulate PTH (parathyroid hormone) secretion and mineral ion homeostasis. From co-leading insulin release, CaSR modulates ductal HCO_3- secretion, Ca²⁺ concentration, cell-cell communication, β-cell proliferation, and intracellular Ca²⁺ release. In pancreatic cancer, the CaSR limits cell proliferation. TCP-related four novel missense mutations P163R, I427S, D433H and V477A, found in CaSR extracellular domain (ECD) protein, which were reported in the mutTCPdb Database (https://lms.snu.edu.in/mutTCPDB/index.php). P163R mutation occurs in ligand-binding domain 1 (LBD-1) of the CaSR ECD. To investigate the influence of these variations on protein function and structural activity multiple in-silico prediction techniques such as SIFT, PolyPhen, CADD scores, and other methods have been utilized. A 500 ns molecular dynamic simulation was performed on the CaSR ECD crystal structure and the corresponding mutated models. Furthermore, Principal Component Analysis (PCA) and Essential Dynamics analysis were used to forecast collective motions, thermodynamic stabilities, and the critical subspace crucial to CaSR functions. The results of molecular dynamic simulations showed that the mutations P163R, I427S, D433H, and V477A caused conformational changes and decreased the stability of protein structures. This study also demonstrates the significance of TCP associated mutations. As a result of our findings, we hypothesised that the investigated mutations may have an effect on the protein's structure and ability to interact with other molecules, which may be related to the protein's functional impairment.

Keywords: CaSR; calcium sensing receptor; molecular dynamics simulation; mutTCPdb; pancreatitis; single nucleotide variants; tropical calcific pancreatitis.

FIGURE 1

(A) Domain organization of CaSR protein showing extracellular, transmembrane, and intracellular domain boundaries, (B) 3D structure of CaSR Extra Cellular Domain (ECD) homodimer (PDB ID: 5FBK), (C) Monomer 3D structure of CaSR ECD, (D) Monomer 3D structure of CaSR ECD lateral view (90°), and (E) Monomer 3D structure of CaSR ECD posterior view (180°). Figure (B) shows CaSR protein structure bound with metal ions Mg2+ (magenta) in LBD1 and at the interface of homodimernear LBD2. Figure (B) also shows Co-agonist L-tryptophan (TCR) (green) bound at the interface of LBD1 and LBD2. Domains in figure (B), (C), (D), and (E) are represented as per color scheme used in figure 1 (A).

FIGURE 2

Evolutionary conservation analysis using Consurf webserver to predict the importance and impact of amino acid mutation at the respective site. A conservation scale depicted in figure ranging from score 1 (variable) to 9 (Conserved) with color variation. Mutation site highlighted with Yellow (P163R), Red (I427S), Blue (D433H), and Cyan (V477A) color.

FIGURE 3

(A) RMSD plot of CaSR ECD protein C-alpha (Prot-CA) atoms throughout the simulation for WT and mutant proteins. (B–F) shows the RMSD plot for Protein-Ligand complex for WT, P163R, I427S, D433H, and V477A, respectively, throughout the simulation.

FIGURE 4

(A) RMSF plot of CaSR ECD protein C-alpha (Prot-CA) atoms throughout the simulation for WT and mutant proteins. (B–E) shows the RMSF plot for mutant P163R, I427S, D433H, and V477A, respectively.

FIGURE 5

Plot (A) describing the radius of gyration (Rg), and (B) solvent accessible surface area (SASA), measurement of WT and mutant proteins throughout the simulation.

FIGURE 6

(A) H-bond interactions, and (B) Salt bridge interactions of WT and mutant proteins, measured throughout the simulation.

FIGURE 7

Statistical measure analyzed using MD simulation results. Plots showing triplicate average and standard deviations of RMSD, RMSF, Rg, SASA, H-bonds, and Salt bridges calculations for WT and mutants. (A) Average RMSD, (B) Standard deviation in RMSD, (C) Average RMSF, (D) Standard deviation in RMSF, (E) Average Rg, (F) Standard deviation in Rg, (G) Average SASA, (H) Standard deviation in SASA, (I) Average h-bond interactions, (J) Standard deviation h-bond interactions, (K) Average salt bridge interactions, (L) Standard deviation in salt bridge interactions.

FIGURE 8

Projection of C-alpha atoms in essential subspace along with the first two principal components (PC1 and PC2) obtained from WT, P163R, I427S, D433H, and V477A. Figure (A) shows PC1 and PC2 of WT only. In contrast, figures (B–E) shows first two principal components of mutants, projected over WT principal components. (F) shows an overlay of WT and all four mutants PC1 and PC2 projection.

FIGURE 9

Cross correlations matrix obtained from essential dynamics of MD simulation trajectories for individual amino acid of (A) WT, (B) P163R, (C) I427S, (D) D433H, and (E) V477A, respectively. The color gradient represents correlated motion, where blue indicates highly correlated motion, and red indicates negatively correlated motion.

FIGURE 10

Porcupine plots for the first two principal components from essential dynamics of WT, and mutant P163R, I427S, D433H, and V477A MD simulation trajectories. Spikes for PC1 (Black) and PC2 (Red) originate from C-alpha atoms and represent the directionality of motions. The magnitude of spikes indicates the strength of motion.