Table 4 Key findings from studies investigating Tat-TAR binding affinities
From: HIV-1 Tat amino acid residues that influence Tat-TAR binding affinity: a scoping review
Reference | Binding technique | Amino acids (Being investigated) | Mutation introduced | In silico, In vitro/In vivo | Key findings |
|---|---|---|---|---|---|
[86] | Surface Plasmon Resonance (SPR) | Tat peptides Wild type: RVRTRKGRRIRIPP Tat peptide 1 RVRTKKGRRIRIPP | Tat peptide 1: R5K | In vitro | 1. Dissociation constant (Kd) values of Tat-TAR binding: Tat wild type = 0.2 × 10−7 M Tat peptide 1 = 6.7 × 10−7 M 2. R5K mutation in Tat peptide 1 can destabilize Tat-TAR interaction, reducing the rate of Tat release from TAR, decreasing overall affinity of Tat for TAR 3. When TAR binds to Tat peptide 1 the Kd value increased significantly (+ 15-fold) compared to the Tat wild type |
[69] | Electrophoretic mobility shift assay (EMSA) | Tat peptides (47–58) Tat wild type: YGRKKRRQRRRP Tat peptide 1: YGRKKARQRRRP Tat peptide 2: YGRKKRAQRRRP Tat peptide 3: YGRKKRRARRRP Tat peptide 4: YGRKKRRQARRP Tat peptide 5: YGRKKRRQRARP Tat peptide 6: YGRKKAAQRRRP Tat peptide 7: YGRKKRRQAARP Tat peptide 8: YGRKKRRQKKRP Tat peptide 9: YGRKKKKQRRRP Tat peptide 10: YGRKKARQARRP Tat peptide 11: YGRKKARQRARP Tat peptide 12: YGRKKRAQARRP Tat peptide 13: YGRRRRAQRARP Tat peptide 14: FITKALGISYGRKKRRQRRRP Tat peptide 15: SGQPPRRRQRRKKRG Tat peptide 16: YRKRRQRRGKRP Tat peptide 17: YRKRGRQRRKRP | Tat peptide 1: R52A Tat peptide 2: R53A Tat peptide 3: Q54A Tat peptide 4: R55A Tat peptide 5: R56A Tat peptide 6: R52AR53A Tat peptide 7: R55AR56A Tat peptide 8: R55KR56K Tat peptide 9: R52KR53K Tat peptide 10: R52AR55A Tat peptide 11: R52AR56A Tat peptide 12: R53AR55A Tat peptide 13: R53AR56A Tat peptide 14: Addition of N-terminal “FITKALGIS” Tat peptide 15: Addition of C-terminal “SGQ” Tat peptide 16: G48R, K50R, R52Q, Q54R, R55K, R56K, P58G Tat peptide 17: G48R, K50R, K51G, R53Q, Q54R, R56K | In vitro | 1. Kd values of Tat-TAR binding: Tat wild type = 6 X 10−9 M 2. When single mutations were introduced into Tat, the following Kd values were observed: Tat peptide 1 = 10 × 10−9 M Tat peptide 2 = 12 X 10−9 M Tat peptide 3 = 5 X 10−9 M Tat peptide 4 = 12 × 10−9 M Tat peptide 5 = 12 × 10−9 M 3. When Double mutations were introduced in Tat, the following Kd values were observed: Tat peptide 8 = 4 × 10−9 M Tat peptide 9 = 13 X 10−9 M Tat peptide 6, Tat peptide 7, Tat peptide 10, Tat peptide 11, Tat peptide 12, Tat peptide 13 = Kd = > 100 X 10−9 M 4. The Tat mutation Q54A in Tat peptide 3 had no significant effect on Tat-TAR binding 5. The Tat peptide 14 had a binding affinity of 6 × 10−9 M to TAR whereas the Tat peptide 15 had a binding affinity of 3 × 10−9 M Tat peptide 16 = 4 × 10−9 M Tat peptide 17 = 3 × 10−9 M 6. The Arginine (R)-rich region 47–58 of the Tat protein binds to 3’ nucleotide bulge of TAR-RNA 7. Substituting pairs of positions R52AR53A (Tat peptide 6), R55AR56A (Tat peptide 7), R52AR55A (Tat peptide 10), R52AR56A (Tat peptide 11), R53AR55A (Tat peptide 12), R53AR56A (Tat peptide 13) reduced binding affinity to TAR > 20 fold 8. Substituting a single R on Tat positions R52A (Tat peptide 1), R53A (Tat peptide 2), R55A (Tat peptide 4), R56A (Tat peptide 5) reduced binding to TAR by twofold 9. Therefore, double substitutions of R with A in the 47–58 region resulted in the highest Kd values, showing the lowest Tat-TAR binding affinity 10. Multiple double substitutions were made on Tat peptide 16 – G48R, K50R, R52Q, Q54R, R55K, R56K, P58G Tat peptide 17- G48R, K50R, K51G, R53Q, Q54R, R56K Tat peptides 15, 16, 17 had lower Kd values compared to Tat wild type |
[76] | Fluorescence resonance energy transfer (FRET) | Tat peptides (47–58 and 38–72) Tat 38–72: FTKKALGISYGRKKRRQRRRAPEDSQTHQVSLPKQ Tat peptide 1: N-terminus rh-Tat YGRKKRRQRRRP Tat peptide 2: N-terminus rh-Tat FTKKALGISYGRKKRRQRRRAPEDSQTHQVSLPK Tat peptide 3: FTKKALGISYGRKKRRQRRRAPEDSQTHQVSLPKQC C terminus rh-Tat (38–72) | Comparing Tat fragments/regions | In vitro | 1. Kd values of binding to TAR RNA 2. Tat peptide 1 = 2.1 × 10–9 (± 0.2 × 10–10) M; 3. Tat peptide 2 = Rh-Tat (38–72) = 1.0 × 10–9(± 0.1 × 10–10) M 4. Tat peptide 3 = Tat (38–72) C-Rh = 3.1 × 10–10 (± 0.2 × 10–10) M, and 5. The reason for Tat peptides consisting of 38–72 having higher binding affinity is because the amino acids from the core region makes the most contacts with TAR RNA and stabilize the structure of the TAR-Tat complex to enhance the affinity and specificity of Tat for TAR 6. The reason for the Kd value of Tat peptide 2 being threefold greater than Tat peptide 3 was that in Tat peptide 3, rhodamine moiety is relatively far from the basic binding region of Tat and will not influence binding affinity significantly |
[81] | EMSA and SPR | Tat peptide 1 Tat full length (1–86) EPVDPRLEPWKHPGSQPKTACTTCYCKKCCFHCQVCFTTKALGISYGRKKRRQRRRPPQGSQTHQVSLSKQPTSQPRGDPTGPKE Tat peptide 2 Biot-Tat (1–86): EPVDPRLEPWKHPGSQPKTACTTCYCKKCCFHCQVCFTTKALGISYGRKKRRQRRRPPQGSQTHQVSLSKQPTSQPRGDPTGPKE Tat peptide 3 Biot-Tat (30–86): CFHCQVCFTTKALGISYGRKKRRQRRRPPQGSQTHQVSLSKQPTSQPRGDPTGPKE | Comparing Tat fragments/regions | In vitro/in silico | 1. Tat peptide 3 (30–86) did not bind to TAR 2. This suggested that if the N terminus of Tat is deleted, no binding to TAR will take place 3. Tat peptide 2 (1–86) bound to TAR at a Kd of 1.85 × 10−9 M 4. Tat peptide 1 (1–86) bound to TAR at a Kd of 6.71 × 10−9 M |
[36] | EMSA and dual-label filter binding assay | Full length Tat, HIV-1BRU (1–86) Tat peptides (32–86): Tat peptide 1 (37–72): CFTTKALGISYGRKKRRQRRRPPQGSQTHQVSLSKQ Tat peptide 2 (43–72): LGISYGRKKRRQRRRPPQGSQTHQVSLSKQ Tat peptide 3 (48–72): GRKKRRQRRRPPQGSQTHQVSLSKQ Tat peptide 4 (32- 72): FHCQVCFTTKALGISYGRKKRRQRRRPPQGSQTHQVSLSKQ Tat peptide 5 (49–86): RKKRRQRRRPPQGSQTHQVSLSKQPTSQSRGDPTGPKE Tat peptide 6 (32–62): FHCQVCFTTKALGISYGRKKRRQRRRPPQGS | Comparing Tat fragments/regions | In vitro | 1. Kd values for full length Tat peptides to TAR were as follow Full length Tat: 6.7 × 10−9 M, Tat peptide 1: 8.4 × 10–8 (± 1.3 × 10–8) M, Tat peptide 2: 7.6 × 10–8 (± 8 × 10–9) M, Tat peptide 3: 3.0 × 10–7 (± 2.0 × 10–8) M, Tat peptide 4: 2.1 × 10–7 (± 2.1 × 10–8) M, Tat peptide 5: 5.3 × 10–8 (± 2 × 10–9) M 2. No findings were reported for Tat peptide 6 3. Tat affinity and specificity for TAR RNA are increased when core region (40–48) was present in the peptide, with K41 playing a key role in TAR recognition 4. Peptides which contain only the basic region & C terminal bind to TAR RNA weakly and non-specifically |
[72] | Electron paramagnetic resonance spectroscopy (EPR) | Tat peptides (47–57) Tat wild type: YGRKKRRQRRR Tat peptide 1 YKKKKRKKKKA | Tat peptide 1: G48K, R49K, R52K, R53K, Q54K, R55K, R56K, R57A | In vitro/In silico | 1. Kd values for Tat peptides binding TAR were as follow: Tat wild type -TAR = 4.0 × 10–7 M and Tat peptide 1-TAR = 1.5 × 10–6 M 2. The Presence of R52 in both peptides has similar binding affinity to TAR RNA 3. This suggest that other amino acids in wild type peptides account for difference in binding affinity and the development of a more rigid complexes between the wild type and peptide 1 |
[84] | EMSA Anisotropy assay | Tat peptides (47–57) Tat wild type peptide = YGRKKRRQRRR Tat peptide 1 = YGKKKKKQKKK | Tat peptide 1: R49K, R52K, R53K, R55K, R56K, R57K | In vitro | 1. Kd values for Tat-TAR binding was as follow: Tat wild type peptide = 2.1 × 10–9 (± 8 × 10–10) M, Tat peptide 1 = 3.2 × 10–8 (± 4 × 10–9) M 2. Tat affinity for RNA depends on side chain of R in basic region – and cationic side chains providing a polyelectrolyte-like affinity 3. A cluster of cationic residues may also provide a polyelectrolyte affinity of Tat for TAR |
[83] | Gel electrophoresis Circular dichroism (CD) | Tat peptide (49–57) Tat wild type—RKKRRQRRR Tat peptide 1-KKKRKQKKK Tat peptide 2- KKKRKKKKK Tat peptide 3- KKKKRQKKK Tat peptide 4- KKKRRQKKK Tat peptide 5—KKKRRKKKK Tat peptide 6- KKKKKQRKK Tat peptide 7- KKKKKQKRK | Tat peptide 1 = R49K, R53K, R55K, R56K, R57K Tat peptide 2 = R49K, R53K, Q54K, R55K, R56K, R57K Tat peptide 3 = R49K, R52K, R54K, R55K, R56K, R57K Tat peptide 4 = R49K, R55K, R56K, R57K Tat peptide 5 = R49K, Q54K, R55K, R56K, R57K Tat peptide 6 = R49K, R52K, R53K, R56K, R57K Tat peptide 7 = R49K, R52K, R53K, R55K, R57K | In vitro | 1. Kd values for Tat-TAR binding were as follow: Tat wild type = 4 × 10−10 M, Tat peptide 1 = 9 × 10−10 M, Tat peptide 2 = 7 × 10−10 M, Tat peptide 3 = 3.5 × 10−9 M, Tat peptide 4 = 5 × 10−10 M, Tat peptide 5 = 1.8 × 10−9 M, Tat peptide 6 = > 5 × 10−8 M and Tat peptide 7 = > 1 × 10−7 M 2. Therefore, Tat wild type had the highest binding affinity 3. Tat peptide 7 had the lowest binding affinity and this may be as a result of the absence of arginine in this amino acid sequence |
[82] | Fluorescence resonance energy transfer (FRET) MALDI-TOFMS Fluorescence binding assay | Tat peptides (46–61) Tat wild type: 46AAARKKRRQRRRAAA60 Tat peptide 1: 46AAARKKRRARRRAAA61 Tat peptide 2: 46AAAAKKRRARRRAAA61 Tat peptide 3: 46AAARAARRARRRAAA61 Tat peptide 4: 46AAAAARRRRRAAAAAA61 Tat peptide 5: 46AAAAARRARRRAAAAA61 Tat peptide 6: 46AAAAARRARRAAAAAA61 Tat peptide 7: 46AAAAARRARAAAAAAA61 Tat peptide 1: 46AAAAARRARAAAAAAA61 Tat peptide 2: 46AAAAARRARRAAAAAA61 Tat peptide 3: 46AAAAARRARRRAAAAA61 Tat peptide 4: 46AAAAARRRRRAAAAAA61 Tat peptide 5: 46AAARAARRARRRAAA61 Tat peptide 6: 46AAAAKKRRARRRAAA61 Tat peptide 7: 46AAARKKRRARRRAAA61 | Tat peptides Tat peptide 1—Q54A Tat peptide 2- R49A, Q54A Tat peptide 3—K50A, K51A, Q54A Tat peptide 4—R49A, K50A, K51A, Q54A, Tat peptide 5 – R49A, K50A, Q54A, K51R, R53A, Q54R, R57A Tat peptide 6—R49A K50A, R51R, R53A, Q54R. R56A, R 57A Tat peptide 7 – R49A, K50A, K51R, R53A, Q54R R55A, R56A, R57A | In vitro | 1. Kd Values between Tat peptides and TAR were as follow: Tat wild type: 7 × 10−8 M; Tat peptide 1 (Kd = 8 × 10−8 M); Tat peptide 2 (Kd = 1.7 × 10−7 M); Tat peptide 3 (Kd = 2.9 × 10−6 M); Tat peptide 4 (Kd = 1.8 × 10−5 M); Tat peptide 5 (Kd = 6.6 × 10−5 M); Tat peptide 6 = No binding, Tat peptide 7 = No binding, 2. The Tat wild type peptide with Kd 7 × 10−8 M had the highest affinity for TAR |
[70] | Nuclear Magnetic Resonance Spectroscopy (NMR) Fluorescence spectroscopy | Tat peptide (32–72) Tat wild type YHCQVCFITKGLGISYGRKKRRQRRRPSQGGQTHQDPIPKQ Tat peptide 1: YHSQVWFITKGLGISYGRKKRRQRRRPSQGGQTHQDPIPKQ | Tat peptide 1: C34S, C37W | In vitro | 1. Kd values of Tat wild type -TAR and Tat peptide 1 – TAR was as follows 2. Tat peptide 1 bound to TAR RNA was 7.16 × 10−8 M and Tat wild type – TAR binding = 5.75 × 10−8 M 3. By comparing Kd values of Tat wild type and Tat peptide 1 the introduced mutations, C34S and C37W in Tat peptide 1 indicate that C34 and C37 in core region are not involved in high affinity to HIV-1 TAR RNA |
[71] | Absorption spectroscopy Gel shift assays CD Spectroscopy | Tat peptides (49–55): Tat wild type: KKKRKKKK Tat peptide 1: AKKRKKKK Tat peptide 2: KAKKRKKK Tat peptide 3: KKARKKK Tat peptide 4: KKKRAKK Tat peptide 5: KKKRKAK Tat peptide 6: KKKRKKA | Tat peptide 1: K49A Tat peptide 2: K50A Tat peptide 3: K51A Tat peptide 4: K53A Tat peptide 5: K54A Tat peptide 6: K56A | In vitro | 1. Kd values for Tat binding TAR were as follow: Tat wild type = 2 × 10−7 M, Tat peptide 1 = 4 × 10−6 M, Tat peptide 2 = 2 × 10–5 M, Tat peptide 3 = 4 × 10−6 M, Tat peptide 4 = 4 × 10−6 M, Tat peptide 5 = 2 × 10–5 and Tat peptide 6 = 4 × 10−6 M 2. The specificity of a Tat amino acid sequence in binding to TAR is mediated by the R52 located in the middle of basic region and surrounding positive charges to R52 overall binding affinity to TAR |
[77] | Gel shift assay CD Spectroscopy, SPR | Tat peptides Tat peptide wild type YGRKKRRQRRRP Tat peptide 1 YGRKKRRQRRRPPQGSQT Tat peptide 2 YGRKKRRQRRRPPQGSQTHR | Comparing Tat fragments/regions | In vitro | 1. The binding of Tat peptides to TAR were as follow: Tat peptide wild type -TAR = 2.6 × 10–6 M, Tat peptide 1-TAR = 8.7 × 10–7 M, Tat peptide 2-TAR = 9.3 × 10–7 M, 2. The kinetic stabilities of Tat peptide 1- TAR and Tat peptide 2 -TAR complexes cannot be differentiated: it may be due to minimal difference between the two peptides being only two (Q and H) amino acids on the C terminal region of the Tat peptide 2 Increased kinetic stability in Tat peptides 1 and 2 – TAR complexes indicates that other amino acid residues outside the basic region contribute to overall binding affinity and kinetic stability of the Tat-TAR complex |
[78] | Gel retardation assays High performance liquid chromatography (HPLC) | Tat peptides: Tat peptide 1: RKKRRQRRRPPQGS Tat peptide 2: RKKRRQRRRPPQGSQTHQVSLSKQ Tat peptide 3: RKKRRQRRRPPQGSQTHQVSLSKQPTSQPRGDPTGPKE | Comparing Tat fragments/regions | In vitro | 1. Tat peptide 3 – TAR binding was approximately 5 × 10−9 M and Tat peptide 2-TAR binding approximately 5 × 10−9 M 2. Tat 1- TAR—dissociation occurs very fast and could not be measured 3. TAR sequence and structure can contribute to specific recognition by -COOH terminal domain of Tat 4. Residue -COOH terminal to the basic region are not necessary for binding to TAR but their presence contributes to the kinetic stability of Tat-TAR complex 5. Possible hydrogen bond acceptors on Q54 sidechain and peptide backbones on Tat peptide promote a more specific interaction with TAR RNA |