Page 68 - TD-3-1

Tumor Discovery Missense mutations in CXCR1: Impact on stability and function

I-TASSER server was subsequently used with a multiple representation of the native structure, compatibility with
threading approach to construct the desired mutant a suitable force field, inclusion of the surrounding solvent
model. 47 and relevant environmental factors, and the ability to
undergo post-simulation analysis. Ensuring these aspects
53
2.6. Evaluation of the quality of the protein model enhances the reliability and biological relevance of insights
The CXCR1 protein is a complex molecule composed of gained from MDS.
amino acids, and its function is associated with its specific The mutant and normal model structures with the
3D structure. Understanding the structure of a protein can highest I-TASSER score, indicating their favorable
provide insights into its function, interactions with other conformational arrangement, were chosen for subsequent
molecules, and potential roles in biological processes, and investigation and analysis. To investigate the effect
47
the CXCR1 protein is a chemokine receptor involved in the of the identified mutations on the structure of CXCR1
48
immune system and inflammation. The CXCR1 mutant and at the mechanistic level, we conducted an MDS using
native protein models were designed through the I-TASSER Schrödinger’s Desmond software. 54,55 Specifically, we
47
server and subsequently subjected to quality validation analyzed the effects of the N57D, R135C, and P302S
using the RAMPAGE server (https://servicesn.mbi.ucla.edu/ mutations on the protein structure over a simulation
PROCHECK). The quality validation involved analyzing period of 100 ns. As a critical first step, protein docking
49
56
the stereochemical features of the models, including bond was performed before MDS, predicting the static binding
lengths, bond angles, and torsion angles, thereby ensuring position of the molecule at the active site of the protein
that the protein models have a geometrically and chemically and providing important information for the subsequent
plausible conformation. Significant deviations in the model’s analysis of the protein’s structure and function during
stereochemical features from expected values may indicate the simulation. MDS typically utilizes Newton’s classical
55
errors in the model. In addition, the protein models equation of motion to simulate the movements of atoms
49
were compared against experimental data to evaluate over time, providing predictions of ligand-binding status
their arrangement with known structural properties. This in various physiological environments. By modeling
51
comparison aimed to validate the accuracy of the predicted the behavior of individual particles based on physical
models by assessing how well they aligned with the actual laws, MDS can provide valuable insights into the
structure of the CXCR1 protein.
dynamic behavior and stability of proteins under diverse
The quality of the protein models refers to their conditions. Maestro’s Protein Preparation Wizard was
54
accuracy and reliability in representing the true 3D used to optimize, minimize, and replace missing residues in
structure of the CXCR1 protein. The validation process the CXCR1 receptor protein. In addition, the MDS system
ensures that the models have appropriate stereochemical was constructed using the System Builder tool, where
features and are consistent with the experimental data, the TIP3P (Intermolecular Interaction Potential 3 Points
enhancing confidence in their utility for further research Transferable) solvent model with 300 K temperature, 1
and analysis. Using the RAMPAGE server, we ensured atm pressure, and OPLS_2005 force field was applied.
57
50
the reliability of the mutant and normal protein models The models were neutralized by adding counterions and
by checking the stereochemical quality of the protein 0.15 M sodium chloride and subsequently used to simulate
structure by analyzing the residue-by-residue geometry physiological conditions. Before simulation, the models
and overall structural geometry. were equilibrated, and trajectories were saved for analysis
every 100 ps. The stability of the simulations was assessed
58
2.7. Molecular dynamics simulation by calculating the root mean square deviation (RMSD)
Molecular dynamics simulation (MDS) is a computational and root mean square fluctuation (RMSF) of the proteins
technique that simulates the movements and interactions during the simulation period. 58
of atoms and molecules over time. It provides a dynamic, RMSD determines the average change in the distance
detailed view of how biological molecules, such as of a set of atoms concerning a reference frame, and it is
proteins, behave at the atomic level for studying molecular computed for each frame of trajectory. The RMSD for
59
structures, dynamics, and interactions, offering insights frame x is:
that are often challenging to obtain through experimental
methods. In MDS, a protein model is a 3D representation n 2
51
of the protein’s structure that is used as the starting point RMSD X = 1/ N ( ∑ r`i(35) ) − (ri tref )) (I)
for simulations. The quality of the model is crucial i =1
51
for obtaining accurate simulation results. Key factors Where N is the number of atoms in atom selection; tref
52
for a high-quality protein model include an accurate is the reference time (typically, the first frame is used as

Volume 3 Issue 1 (2024) 4 https://doi.org/10.36922/td.2512