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Tumor Discovery Bioinformatics insights into CCL2 mutations

variants, including their potential association with cancer, The reliability of the mutant and native CCL2 protein
where scores below the default threshold of −0.75 indicate models was ensured by validating their stereochemical
a potential cancer link. FATHMM helps distinguish quality using the Rampage server (https://servicesn.
between cancer-related mutations and non-pathogenic mbi.ucla.edu/PROCHECK). This involved analyzing
germline polymorphisms. 25,49 Using FATHMM, we aimed the residue-by-residue geometry and overall structure
to deepen our understanding of the genetic basis of cancer geometry to assess the accuracy of models. 54
by evaluating the impact of harmful mutations on disease
progression, identifying clinically significant mutations, 2.8. MD simulation
and advancing our knowledge of cancer development. 25 MD simulation is a computational technique used to
simulate the dynamic movement of atoms and molecules
2.5. Sequence conservation analysis of CCL2 throughout a period. By simulating movements and
The ConSurf web server (http://consurf.tau.ac.il) analyzes interactions of biological molecules, such as proteins, MD
the evolutionary patterns of amino acids in proteins to provides detailed insights into their molecular structures,
identify regions critical for structure and function. Using dynamics, and interactions, which are often difficult to
a query sequence or structure, the server automatically obtain through experimental methods alone. 55,56 The
gathers homologous sequences, performs multiple accuracy of MD simulation results relies heavily on the
sequence alignment, and constructs a phylogenetic tree that quality of the protein model. Key factors for a high-quality
57
illustrates their evolutionary relationships. This information protein model include an accurate representation of the
is then utilized within a probabilistic framework to estimate native structure, selection of a suitable force field, inclusion
the evolutionary rates for each position in the sequence. 50 of surrounding solvent and relevant environmental factors,
and facilitation of post-simulation analysis. 58,59 Addressing
2.6. Structural modeling of CCL2 point mutation sites these aspects improves the credibility and biological
To create a mutant model of CCL2 featuring the targeted significance of the findings derived from MD simulations.
mutation C59C (native) and C59G (mutant), the crystal The native and mutant models of CCL2 proteins with
structure of the native peptides was initially downloaded the highest I-TASSER score, indicating their optimal
from the PDB. Subsequently, the I-TASSER server (https:// conformation, were selected for further analysis. To
29
zhanglab.ccmb.med.umich.edu/I-TASSER/), which uses a examine the effects of the identified mutations on the CCL2
multiple threading approach, was employed to construct protein structure at the molecular level, we performed an
the intended mutant model. 29 MD simulation using WebGro software. This revealed
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2.7. Quality evaluation of the protein model how native C59C and mutant C59G influenced the
structure of CCL2 during a 100-ns simulation, and the MD
The CCL2 protein is a complex molecule composed of simulation system was neutralized by introducing sodium
amino acids, with its function intricately linked to its specific or chlorine ions to balance the total charges. Before MD
3D structure. Understanding CCL2’s structure can provide simulation, the steepest descent algorithm was used for
valuable insights into its function, interactions with other system minimization through 5000 iterations (The
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molecules, and potential roles in biological processes, such simulations were performed at a constant temperature
as the immune system and inflammation. The I-TASSER of 300 K and a pressure of 1.0 bar, with a 0.15 M NaCl
51
server was used to generate protein models of mutant and concentration. Approximately 1000 frames were generated
native CCL2. The quality assessment process included per 100-ns simulation. 62
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an analysis of the stereochemical characteristics of the
models, such as the bond lengths, bond angles, and torsion As a crucial initial step, we performed protein docking
angles. This evaluation aimed to confirm that the protein to predict the binding location of the molecule within
models exhibit a conformation that is both geometrically the protein’s active site. This process provided essential
and chemically reasonable. Deviations from expected insights into the subsequent MD simulations, allowing
values in the stereochemical features of the model could for the detailed examination of the protein’s structure
63,64
suggest potential errors in the model. The protein models and function. MD simulations produced multiple
52
were validated by comparing them with experimental trajectories, including root mean square deviation
data to ensure that they aligned with the known structural (RMSD), root mean square fluctuation (RMSF), radius of
properties of CCL2 protein, which verified the accuracy gyration (Rg), solvent-accessible surface area (SASA), and
65,66
of the predicted models by examining how accurately hydrogen bonds.
they matched with the actual protein structure, thereby The RMSD profiles of protein–ligand complexes were
ensuring the reliability of the models. 53 analyzed using the WebGRO server. The best-docked

Volume 3 Issue 4 (2024) 5 doi: 10.36922/td.3891