Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein
Abstract
:1. Introduction
2. Materials and Methods
2.1. Oligopeptide Synthesis and Characterization
2.2. Enzyme Assay with Synthetic Peptide Substrates
2.3. Molecular Modeling
2.4. Design of Recombinant Fluorescent Protein Substrates
2.5. Expression, Purification and Cleavage of Protein Substrates
3. Results
3.1. Probing the S1′ Binding Site of HIV-1 Protease Using Substituted Peptides Representing the Proximal Cleavage Site of HIV-1 NC
3.2. Design of Nucleocapsid Cleavage Site “Revertants”
3.3. Cleavage Reactions Using Recombinant Protein Substrates
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primer Name | Cleavage Site Sequence/Oligonucleotide Primer Sequence |
---|---|
Wild-type | KIVKCFNCGKEGHTARNCRAPR |
FWD | 5′-TAAAAAAATTGTGAAATGCTTTAACTGCGGCAAAGAAGGCCATACCGCGCGTAACTGCCGTGCGCCGCGTG-3′ |
REV | 5′-GATCCACGCGGCGCACGGCAGTTACGCGCGGTATGGCCTTCTTTGCCGCAGTTAAAGCATTTCACAATTTTTTTAAT-3′ |
N17F | KIVKCFFCGKEGHTARNCRAPR |
FWD | 5′-TAAAAAAATTGTGAAATGCTTTTTTTGCGGCAAAGAAGGCCATACCGCGCGTAACTGCCGTGCGCCGCGTG-3′ |
REV | 5′-GATCCACGCGGCGCACGGCAGTTACGCGCGGTATGGCCTTCTTTGCCGCAAAAAAAGCATTTCACAATTTTTTTAAT-3′ |
N17L | KIVKCFLCGKEGHTARNCRAPR |
FWD | 5′-TAAAAAAATTGTGAAATGCTTTCTGTGCGGCAAAGAAGGCCATACCGCGCGTAACTGCCGTGCGCCGCGTG-3′ |
REV | 5′-GATCCACGCGGCGCACGGCAGTTACGCGCGGTATGGCCTTCTTTGCCGCACAGAAAGCATTTCACAATTTTTTTAAT-3′ |
N17T | KIVKCFTCGKEGHTARNCRAPR |
FWD | 5′-TAAAAAAATTGTGAAATGCTTTACCTGCGGCAAAGAAGGCCATACCGCGCGTAACTGCCGTGCGCCGCGTG-3′ |
REV | 5′-GATCCACGCGGCGCACGGCAGTTACGCGCGGTATGGCCTTCTTTGCCGCAGGTAAAGCATTTCACAATTTTTTTAAT-3′ |
K14I-N17G | KIVICFGCGKEGHTARNCRAPR |
FWD | 5′-TAAAAAAATTGTGATTTGCTTTGGCTGCGGCAAAGAAGGCCATACCGCGCGTAACTGCCGTGCGCCGCGTG-3′ |
REV | 5′-GATCCACGCGGCGCACGGCAGTTACGCGCGGTATGGCCTTCTTTGCCGCAGCCAAAGCAAATCACAATTTTTTTAAT-3′ |
I14_ins-N17G | KIVKICFGCGKEGHTARNCRAPR |
FWD | 5′-TAAAAAAATTGTGAAAATTTGCTTTGGCTGCGGCAAAGAAGGCCATACCGCGCGTAACTGCCGTGCGCCGCGTG-3′ |
REV | 5′-GATCCACGCGGCGCACGGCAGTTACGCGCGGTATGGCCTTCTTTGCCGCAGCCAAAGCAAATTTTCACAATTTTTTTAAT-3′ |
sequencing primer | 5′-GATGAAGCCCTGAAAGACGCGCAG-3′ |
Name | Sequence | Cleavage Site | Km (mM) | kcat (s−1) | kcat/Km (mM−1 s−1) | kcat/Km Fold-Change | |
---|---|---|---|---|---|---|---|
1. | wild-type | KIVKCFNCGK | CF↓NC | 0.43 1 | 0.017 1 | 0.040 1 | - |
2. | N17F | KIVKCFFCGK | CF↓FC | 0.02 1 | 0.900 1 | 59.6 1 | 1490.00 |
3. | N17L | KIVKCFLCGK | CF↓LC | 0.17 1 | 0.196 1 | 1.153 1 | 28.83 |
4. | N17V | KIVKCFVCGK | CF↓VC | 0.45 | 0.284 | 0.632 | 15.80 |
5. | N17A | KIVKCFACGK 2 | CF↓AC | 0.32 1 | 0.064 1 | 0.200 1 | 5.00 |
6. | N17C | KIVKCFCCGK 2 | CF↓CC | 1.52 | 0.247 | 0.162 1 | 4.05 |
7. | N17T | KIVKCFTCGK | CF↓TC | 0.31 | 0.017 | 0.054 | 1.35 |
8. | N17Q | KIVKCFQCGK | CF↓QC | 0.67 | 0.035 | 0.052 | 1.30 |
9. | N17S | KIVKCFSCGK 2 | CF↓SC | 0.63 | 0.004 | 0.006 1 | 0.15 |
10. | N17R | KIVKCFRCGK 2 | CF↓RC | 1.05 | 0.020 | 0.019 1 | 0.48 |
11. | N17K | KIVKCFKCGK | CF↓KC | 0.86 1 | 0.005 1 | 0.006 1 | 0.15 |
12. | N17E | KIVKCFECGK | CF↓EC | 1.21 | 0.013 | 0.011 | 0.28 |
13. | N17D | KIVKCFDCGK | not hydrolysed | - | |||
14. | N17G | KIVKCFGCGK | CF↓GC | 1.34 1 | 0.002 1 | 0.001 1 | 0.03 |
Name | Sequence | Cleavage Site | Km (mM) | kcat (s−1) | kcat/Km 2 (mM−1 s−1) | kcat/Km 2 Fold-Change | kcat/Km 3 (mM−1 s−1) | |
---|---|---|---|---|---|---|---|---|
1. | wild-type | KIVKCFNCGK 1 | CF↓NC | 0.43 | 0.017 | 0.040 | - | - |
14. | N17G | KIVKCFGCGK 1 | CF↓GC | 1.34 | 0.002 | 0.001 | 0.03 | - |
15. | K14T-N17G | KIVTCFGCGK | n.d. 4 | - | <0.010 | - | - | - |
16. | K14I-N17G | RKIVICFGCGKR | IC↓FG | 0.035 | 0.034 | 0.97 5 | 24.25 | 0.51 |
17. | I14_ins-N17G | KIVKICFGCGKR | IC↓FG | 0.070 | 0.025 | 0.35 | 8.75 | 0.48 |
18. | N17G-G19I | RKIVKCFGCIKR | n.d. 4 | - | <0.010 | - | - | - |
19. | K14I-N17G-G19I | RKIVICFGCIKR | IC↓FG | <0.010 | 0.015 | n.d. 4 | - | 0.48 |
20. | K14L-N17G | RKIVLCFGCGKR | VL↓CF/LC↓FG | - | n.d. 4 | - | - | - |
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Mótyán, J.A.; Miczi, M.; Oroszlan, S.; Tőzsér, J. Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein. Viruses 2021, 13, 1092. https://doi.org/10.3390/v13061092
Mótyán JA, Miczi M, Oroszlan S, Tőzsér J. Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein. Viruses. 2021; 13(6):1092. https://doi.org/10.3390/v13061092
Chicago/Turabian StyleMótyán, János András, Márió Miczi, Stephen Oroszlan, and József Tőzsér. 2021. "Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein" Viruses 13, no. 6: 1092. https://doi.org/10.3390/v13061092
APA StyleMótyán, J. A., Miczi, M., Oroszlan, S., & Tőzsér, J. (2021). Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein. Viruses, 13(6), 1092. https://doi.org/10.3390/v13061092