A H-REV107 Peptide Inhibits Tumor Growth and Interacts Directly with Oncogenic KRAS Mutants
Abstract
:1. Introduction
2. Results
2.1. The H-REV107 Peptide Bound to KRAS G12V with High Binding Affinity
2.2. The GTP Binding to the KRAS Mutants Was Greatly Decreased in the Presence of H-REV107 Peptide
2.3. Inhibitory Effect of H-REV107 Peptide Was Observed on Tumor Cells
2.4. H-REV107 Peptide Can Suppress Pancreatic Tumor Growth
2.5. The Crystal Structure of KRAS G12V-H-REV107 Peptide Complex Was Determined
2.6. H-REV107 Peptide Was Strongly Docking in the Switch I and II Binding Pockets and the P-Loop of KRAS G12V
2.7. H-REV107 Peptide Stabilized the KRAS G12V in An Irreversible Inactive GDP Binding as An Open Conformation
3. Discussion
4. Materials and Methods
4.1. Recombinant Protein Expression and Purification
4.2. Size-Exclusion Chromatography–Multi-Angle Light Scattering (SEC-MALS)
4.3. Peptide Synthesis
4.4. Biacore Biosensor Analysis
4.5. Auto-Isothermal Titration Calorimetry (Auto-ITC)
4.6. Circular Dichroism (CD) Spectrometer Analysis
4.7. Guanine Nucleotide Binding Assay
4.8. Cell Proliferation Assay and Western Blot Analysis
4.9. Tumor Xenograft Model
4.10. Crystallization, Data Collection, and Structure Determination
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Data Collection | KRAS G12V- H-REV107 Peptide (PDB ID: 7C41) | KRAS G12V (PDB ID: 7C40) | H-REV107 (PDB ID: 7C3Z) |
---|---|---|---|
Space group | P1 | P63 | P212121 |
Unit cell | |||
a, b, c (Å) | 41.907, 84.339, 84.335 | 82.547, 82.547, 40.804 | 42.935, 52.996, 62.796 |
α, β, γ (o) | 59.983, 89.961, 89.965 | 90.0, 90.0, 120.0 | 90.0, 90.0, 90.0 |
Wavelength (Å) | 0.97934 | 0.97934 | 0.97934 |
Resolution (Å) | 50.0–2.27 | 50.0–2.50 | 50.0–1.95 |
Completeness (%) | 97.7 (81.6) | 99.9 (100) | 99.9 (100) |
Observed reflections | 328,760 | 823,903 | 367,550 |
Unique reflections | 45,853 | 5543 | 10,814 |
I/σ (I) | 16.5 (5.7) | 27.2 (9.3) | 36.9 (8.0) |
Rmerge (%) a | 10.2 (20.0) | 12.8 (51.1) | 8.8 (21.8) |
Redundancy | 3.9 | 21.6 | 14.0 |
Rcryst/Rfree (%) b | 26.03/28.34 | 18.46/24.24 | 23.43/26.29 |
Protein atoms | 5348 | 1337 | 839 |
Solvent molecules | 83 | 14 | 56 |
Mg2+/GDP molecules | 4 | 1 | 0 |
Peptide | 1 | 0 | 0 |
Bond length (Å) | 0.009 | 0.009 | 0.008 |
Bond angle (o) | 1.160 | 1.077 | 0.890 |
Average <B> factor (Å2) Peptide | 31.3 | 0 | 0 |
Average <B> factor (Å2) Mg2+/GDP | 19.3 | 24.4 | 0 |
Most favored regions (%) | 97.2 | 94.0 | 97.0 |
Additional allowed regions (%) | 2.2 | 6.0 | 3.0 |
Disallowed regions (%) | 0.6 | 0 | 0 |
KRAS | H-REV107 Peptide | Interaction Distance (Å) | KRAS | H-REV107 Peptide | Interaction Distance (Å) |
---|---|---|---|---|---|
O (G12V) | N (K73) | 4.04 | O (N85) | NZ (K73) | 2.66 |
N (G12V) | O (Y74) | 3.91 | O2B (GDP) | O (G70) | 2.50 |
N (G13) | O (D72) | 3.89 | N (KRAS G13)–O1B (GDP) | 2.70 | |
OG (S17) | OH (Y66) | 2.81 | O (KRAS V29)–O2 (GDP) | 2.59 | |
O (D33) | OH (Y66) | 2.51 | O (KRAS D30)–O2 (GDP) | 2.91 | |
O (P34) | OD1 (D67) | 2.85 | O (KRAS D30)–O3 (GDP) | 2.76 | |
O (T35) | OD1 (D67) | 3.21 | OD1 (KRAS N116)–O6 (GDP) | 3.21 | |
O (T35) | OD2 (D67) | 3.16 | OD2 (KRAS D119)–N2 (GDP) | 3.14 | |
O (A59) | N (L65) | 3.10 | OG (KRAS S17)–Mg2+ | 2.71 | |
O (A59) | N (Y66) | 3.13 | O (KRAS T58)–Mg2+ | 3.03 | |
NE2 (Q61) | N (L65) | 2.74 | O3B (GDP)–Mg2+ | 2.99 |
KRAS Residues | RMSD (Å) | KRAS Residues | RMSD (Å) |
---|---|---|---|
M1 | 1.83 | E62 | 1.36 |
V12 | 0.48 | E63 | 2.15 |
G13 | 0.48 | Y64 | 2.31 |
D30 | 0.97 | S65 | 1.58 |
E31 | 0.84 | A66 | 1.26 |
Y32 | 0.78 | M67 | 1.08 |
D33 | 0.92 | M72 | 1.09 |
P34 | 1.37 | R73 | 1.25 |
T35 | 1.03 | T74 | 0.89 |
Q61 | 1.05 | D108 | 0.99 |
Cell Lines | Tissues | KRAS Mutations |
---|---|---|
SW480 | Colon | G12V |
AsPC-1 | Pancreas | G12D |
NCI-H23 | Lung | G12C |
NCI-H460 | Lung | Q61H |
HCT116 | Colon | G13D |
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Han, C.W.; Jeong, M.S.; Ha, S.C.; Jang, S.B. A H-REV107 Peptide Inhibits Tumor Growth and Interacts Directly with Oncogenic KRAS Mutants. Cancers 2020, 12, 1412. https://doi.org/10.3390/cancers12061412
Han CW, Jeong MS, Ha SC, Jang SB. A H-REV107 Peptide Inhibits Tumor Growth and Interacts Directly with Oncogenic KRAS Mutants. Cancers. 2020; 12(6):1412. https://doi.org/10.3390/cancers12061412
Chicago/Turabian StyleHan, Chang Woo, Mi Suk Jeong, Sung Chul Ha, and Se Bok Jang. 2020. "A H-REV107 Peptide Inhibits Tumor Growth and Interacts Directly with Oncogenic KRAS Mutants" Cancers 12, no. 6: 1412. https://doi.org/10.3390/cancers12061412