Analysis of Structural Changes in the Protein near the Phosphorylation Site
Abstract
:1. Introduction
2. Methods
2.1. Study Design
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- The amino acid sequence of the protein must include at least 15 residues (excluding small peptides);
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- The protein pair must have a common unique Uniprot ID (excluding comparisons with homologous proteins from different organisms in the intact/modified structure pair);
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- The intact form of the pair should not contain any non-canonical amino acid residues (unless the proteins containing non-canonical amino acid variants are matched to the intact protein structure);
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- The length of the proteins in a pair should not differ by more than five amino acid residues (excluding comparisons between proteins with significantly different lengths in the intact/modified structure pair);
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- The amino acid sequence of the proteins (FASTA) in a pair should match at least 90% (selecting identical sequences in the intact/modified structure pair);
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- If several structures are found in the PDB for the modified form, the three-dimensional structure with the lowest RMSD, regarding the intact protein, is selected (reducing the number of comparison variants);
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- The number of phosphate groups in the modified form should be ≤3;
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- Each protein chain in one crystal with a modifying group is considered a separate structure;
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- Structure is identified by a unique combination of UniProt ID and PTM locus (to avoid redundancy in the dataset).
2.2. Calculating Geometric Indicators
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- Before calculating the geometric parameters, all the atoms not included in the structure of polymer chains were removed from the structures;
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- Hydrogen atoms were removed and then added using PyMol (h_add command) to avoid the error caused by the possible presence of hydrogen atoms in one of the compared structures while being absent in the other one.
- root-mean square distance between Cα-atoms in the intact and modified protein forms (RMSD, Å);
- solvent-accessible surface area (SASA, Å2);
- radius of gyration (Rg, nm);
- displacement between Cα-atoms in individual amino acid residues (Cα displacement, Å).
2.3. Analysis of Geometric Indicators
3. Results
3.1. Phosphorylated Proteins in PDB
3.2. Comparative Analysis of the Geometry of Intact and Modified Protein Forms
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- Group N1 includes protein pairs that are characterized by RMSD > 2Å. Due to significant differences, it is impossible to compare these forms of proteins (see Supplementary Materials Table S2—proteins with RMSD > 2 Å). Fifteen such protein pairs were found. This group of protein pairs was excluded from further consideration;
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- Group N2 includes protein pairs of high similarity for which the RMSD value is <2 Å. The protein pairs of this group are characterized by significant spatial differences in the neighborhood of the modification site: the RMSD value of at least one of the studied neighborhoods of the modification site exceeds 2 Å. Nineteen such protein pairs were selected;
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- Group N3 also includes protein pairs of high similarity with RMSD <2 Å. The neighborhoods of the modification site are also characterized by high similarity: the calculated RMSD values do not exceed 2 Å for the intact and modified forms. This group included 29 protein pairs.
4. Discussion
5. Limitations
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- Filtering without considering differences in structure length with alignment based on the smaller structure;
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- Selecting modified and intact protein forms based on sequence homology without considering UniProt ID;
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- Disregarding the presence of amino acid substitutions and non-canonical amino acids outside the binding site;
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- Considering ligands present in the structures;
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- Identifying gaps in the amino acid sequence;
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- Selecting protein pairs where both structures were obtained using the same experimental method (X-Ray, NMR, or EM);
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- Averaging atomic positions across all three-dimensional structures of a given protein;
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- Analyzing local and global structural changes in a protein while considering b-factor values instead of RMSD.
6. Conclusions
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- unweaving or stretching of the α-helix turn, changes in β-strand length, and, therefore, changes in length and conformation of the unstructured region;
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- increased Rg values in the studied neighborhood;
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- significant surge in Cα displacement values for amino acid residues located near the modification site;
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- the calculated RMSD values for the neighborhood of the modification site exceed those for the whole protein.
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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PTM | Number of Protein Structures (PDB) | Mean Resolution, Å | Method | Origin |
---|---|---|---|---|
SEP | 1394 | 2.22 ± 0.8 | X-ray: 92% NMR: 3% EM: 5% | Homo sapiens: 53.5% Other eukaryote: 33.2% Bacteria: 6.8% Virus: 0.3% Archaea: 0.3% Other: 1.3% No data: 4.6% |
TPO | 999 | 2.21 ± 0.6 | X-ray: 93% NMR: 3% EM: 4% | Homo sapiens: 49.1% Other eukaryote: 38.9% Bacteria: 3.9% Virus: 0.5% Archaea: 0.5% Other: 1.7% No data: 5.4% |
PTR | 659 | 2.25 ± 0.5 | X-ray: 92% NMR: <8% EM: <0.5% | Homo sapiens: 72.9% Other eukaryote: 6.5% Bacteria: 2.4% Virus: 1.7% Other: 0.9% No data: 15.6% |
Neighborhood | Min, (Å) | Max, (Å) | Mean, (Å) | Median, (Å) | Std, (Å) |
---|---|---|---|---|---|
3 | 5.14 | 7.61 | 6.55 | 6.74 | 0.57 |
6 | 8.77 | 17.87 | 13.64 | 13.86 | 2.21 |
9 | 10.47 | 26.17 | 17.8 | 17.8 | 3.43 |
12 | 11.23 | 32.86 | 21 | 20.14 | 4.4 |
15 | 13.11 | 37.79 | 23 | 20.77 | 5.78 |
Position | Group N2 | Group N3 |
---|---|---|
α-helix | 1 | 9 |
Coil (middle) | 13 | 7 |
β-strand | 3 | 2 |
Coil (edge) | 2 | 11 |
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Nikolsky, K.S.; Kulikova, L.I.; Petrovskiy, D.V.; Rudnev, V.R.; Malsagova, K.A.; Kaysheva, A.L. Analysis of Structural Changes in the Protein near the Phosphorylation Site. Biomolecules 2023, 13, 1564. https://doi.org/10.3390/biom13111564
Nikolsky KS, Kulikova LI, Petrovskiy DV, Rudnev VR, Malsagova KA, Kaysheva AL. Analysis of Structural Changes in the Protein near the Phosphorylation Site. Biomolecules. 2023; 13(11):1564. https://doi.org/10.3390/biom13111564
Chicago/Turabian StyleNikolsky, Kirill S., Liudmila I. Kulikova, Denis V. Petrovskiy, Vladimir R. Rudnev, Kristina A. Malsagova, and Anna L. Kaysheva. 2023. "Analysis of Structural Changes in the Protein near the Phosphorylation Site" Biomolecules 13, no. 11: 1564. https://doi.org/10.3390/biom13111564