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Cite this: Chem. Commun., 2013, 49, 11086 Received 28th August 2013, Accepted 8th October 2013
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Exchangeability of amino acid residues with similar physicochemical properties in coiled-coil interactions† Guiying Zhang,za Kun Wang,zab Baohua Zheng,zac Maosheng Cheng,c Yanni Li,*b Keliang Liu*a and Lifeng Cai*a
DOI: 10.1039/c3cc46560h www.rsc.org/chemcomm
Systematic exchange of amino acid residues of similar physicochemical properties maintains specific coiled-coil interaction between two heptad repeats of HIV-1 gp41, as well as the biological activity of related peptide fusion inhibitors. This exchangeability can greatly degenerate sequence space of peptides thus making ab initio design of coiled-coil interaction feasible.
Coiled-coil is an important protein–protein interaction model.1 In human immunodeficiency virus type 1 (HIV-1) infection, the formation of a coiled-coil six helical bundle (6-HB) between the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) of gp41, a transmembrane subunit of the HIV-1 envelop glycoprotein (Env), provides the energy necessary for virus-cell membrane fusion and entry into host cells (Fig. 1).2,3 Peptides derived from NHR and CHR sequences can bind to their counterparts in gp41 to form a hetero 6-HB and prevent fusogenic 6-HB formation, thereby inhibiting membrane fusion and viral infection and being used as fusion inhibitors.4–6 Peptide engineering has been used to design highly mutated peptides derived from HIV-1 gp41 wild type sequences, which showed high potency against drugresistant HIV-1 isolates with improved pharmacokinetic and pharmacodynamic profiles.7,8 Ab initio design of a highly potent fusion inhibitor, which can provide fundamental insight into coiled-coil interaction, is still unfeasible due to the huge sequence space of peptides comprised of 20 proteinogenic amino acids, however. Among the 20 proteinogenic amino acids, some have similar physiochemical properties based on their hydrophobicity, polarity, and electric charge. In protein engineering and evolution, it is well
Fig. 1 HIV-1 gp41 NHR–CHR interaction and experimental design. (A) HIV-1 gp41 functional domains and the peptide sequences in our study. FP, fusion peptide; NHR, N-terminal heptad repeat; CHR, C-terminal heptad repeat; MPER, membrane proximal external region; TM, transmembrane region; CT, cytoplasmic tail. (B) Coiled-coil interactions between N36 and C34. Residues at ‘a’ and ‘d’ positions of the heptad register in the NHR (in red) associated with each other to form a trimeric inner core, and ‘a’ and ‘d’ residues in the CHR interact with ‘e’ and ‘g’ residues in the NHR (in underscore bold) to stabilize the 6-HB. These residues are considered as ‘key residues’ in coiled-coil interaction and in peptide fusion inhibitor design. (C) Peptide sequences used in this study. C34 and N36 are native sequences based on HIV-1HXB2 Env sequences. ‘Key residues’ are highlighted as in (B), mutated residues are highlighted in red.
a
Department of Medicinal Chemistry, Beijing Institute of Pharmacology & Toxicology, 27 Taiping Rd, Haidian District, Beijing 100850, China. E-mail: [email protected], [email protected]; Fax: +86-10-68211656 b Key Laboratory of Systems Bioengineering, Ministry of Education, Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China. E-mail: [email protected] c Key Lab. Struc. Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China † Electronic supplementary information (ESI) available: Experiments, CD spectra and thermal denaturation data. See DOI: 10.1039/c3cc46560h ‡ G. Y. Zhang, K. Wang and B. H. Zheng contributed equally to this work.
11086
Chem. Commun., 2013, 49, 11086--11088
evidenced that amino acid residues with similar properties are exchangeable for maintaining basic structure and function of a protein.9,10 However, due to the complexity of protein folding and function, systematically applying this exchangeability to protein and peptide design is problematic. Generally, each of the 20 proteinogenic amino acids is unique in protein function. If, in certain protein folding and interaction systems, such This journal is
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The Royal Society of Chemistry 2013
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Communication exchangeability between amino acid residues with similar properties is applicable, the sequence space of proteins and peptides will be greatly degenerated thus making ab initio protein and peptide design feasible. Therefore, we investigated the exchangeability of amino acid residues with similar physicochemical properties in coiled-coil interactions. We chose a HIV-1 gp41 N36/C34 6-HB as model,11 making systematic exchange of amino acid residue pairs of similar properties, and studied the effects of such exchanges on the coiled-coil interaction and biological activities of corresponding peptides. N36/C34 6-HB has an available crystal structure and is widely used to represent the HIV-1 gp41 fusion core.2,11,12 We introduced mutations throughout the N36 and C34 sequences by making systematic exchanges between amino acid residue pairs with similar physicochemical properties, including E–D, I–L, Q–N, or R–K pairs. These residues occupy more than two thirds of the sequences of the two peptides. The peptide sequences are shown in Fig. 1C. The exchangeabilities of these amino acid residue pairs were evaluated based on the effects of the mutations on the stability of the coiledcoil structure formed between mutated N36- and C34-peptides, as well as the inhibitory activities of the C34 peptides against HIV-1 Env mediated cell–cell fusion. We first tested the effects of these mutations on the coiled-coil interactions by circular dichroism (CD) spectroscopic analysis. C34 and N36 form random or partial a-helices in solution when separated, and form a typical 6-HB structure with high a-helical content when they are mixed in a 1 : 1 molar ratio, which is characterized by a large signal increase at 222 nm in the CD spectra (Fig. S1A, ESI†).11,12 As shown in Fig. 2A, the systematic exchange between any pair of E–D, I–L, Q–N or R–K residues throughout C34 largely retained the coiled-coil interaction, and the mutated C34-peptides interacted with N36 to form complexes with high a-helical contents.
Fig. 2 CD spectra of complexes formed between mutated C34-peptides and N36 (A), and mutated N36-peptides and C34 (B).
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ChemComm Similar mutations in N36 had bigger effects on the coiled-coil interaction than those in C34, and only the N36EDKR peptide, which had a total of four mutations, interacted with C34 to form the 6-HB structure (Fig. 2B). N36 plays dual roles in the coiled-coil interactions, i.e. stabilizes the inner trimeric core and interacts with C34 to stabilize the 6-HB structure. In contrast, C34 only interacts with the N36 trimer to stabilize the coiled coil interaction, which may account for the higher sensitivities of exchangeable mutations in the N36 trimer to the coiled-coil interaction than those of C34. The accumulation of such mutations weakened the coiledcoil interaction. The C34mut peptide (6), which had a total of 25 mutations of a total of 34 residues, could not form a 6-HB with N36 (Fig. 2A), and the N36Mut peptide (12) with 26 mutations of a total of 36 residues was insoluble and difficult to purify. We analyzed the relationship between the number of total mutations and the a-helical content of the mutated 6-HBs. Based upon the contributions of C34 and N36 to the 6-HB interaction, we doubled the number of N36 mutations in the analysis. A good linear relationship between the total number of mutations in C34 or N36 and the a-helical content of related 6-HBs was observed (Fig. 3A). We then tested the effects of these mutations on the biological activities of the C34-peptides, using a HIV-1 Env-mediated cell–cell fusion assay.13 The activities of the C34-peptides were evaluated based on the concentrations that achieved 50% inhibition in the assay (IC50). Peptides derived from the HIV-1 gp41 CHR are potent fusion inhibitors.5,6 C34 showed an IC50 of 0.6 nM in the cell–cell fusion assay. Exchanges between any amino acid residue pair of similar physicochemical properties retained the high biological activity of C34, and most of these mutated C34-peptides showed an IC50 in the low nanomolar range in the cell–cell fusion assay (Table 1). The exchange between Q and N had a bigger effect on the biological activity of C34 than other mutations, and the C34QN (5) peptide showed a significantly 85-fold potent decrease compared
Fig. 3 Correlation between the total number of mutations and the a-helical content of C34/N36 6-HB (A), the inhibitory activities of the mutated C34-peptides against HIV-1 Env mediated cell–cell fusion (B). Error bars are standard deviations from at least duplicate measurements.
Chem. Commun., 2013, 49, 11086--11088
11087
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Table 1
Communication
Biological and biochemical properties of the peptides a
b
Peptides
Total mutations
IC50 (nM)
C34 (1) C34IL (2) C34ED (3) C34KR (4) C34QN (5) C34Mut (6) N36 (7) N36IL (8) N36EDKR (9) N36QN (10) N36ILV (11)
0 7 8 2 8 25 0 11 4 10 13
0.60 1.68 9.8 0.46 51 410 2090 2100 3100 1420 1170
(3) (1) (2) (6) (9) (0) (4) (11)
0.43 0.19 2.4 0.39 11 150 390 590 1460 410 85
c
6-HB helix (%) 91.2 74.7 70.4 87.1 63.4 23.9 91.2 24.5 72.6 4.2 6.8
0.8 7.6 7.8 4.1 4.6 1.3 0.8 2.8 10.6
1.0
a
The numbers in parentheses represent the total number of mutations of ‘key residues’ as shown in Fig. 1B. b The concentrations of peptides that achieved 50% inhibition in cell–cell fusion assay. c The 6-HBs were formed between mutated C34-peptides and native N36, or between mutated N36-peptides and C34. Numbers after ‘’ are standard deviations from at least duplicate measurements.
to C34. The CD thermal denaturation of the C34QN–N36 complex showed a gradual loss of secondary structure instead of a cooperatively two-state conformational transition, indicating a partly folded, molten globule form instead of an ordered coiled-coil structure (Fig. S2, ESI†). This may be attributed to the low biological activity of the C34QN peptide. Other complexes with high a-helical content showed typical S-shape thermal denaturation curves, indicating ordered coiled-coil structures (Fig. S2, ESI†). Consistent with the CD results, C34Mut (6) showed an IC50 of 410 nM, an approximately 700-fold potent decrease compared to the native peptide (Table 1). It is noteworthy that C34Mut (6) still showed clear inhibitory activity in the cell–cell fusion assay compared to unrelated peptides, which were usually inactive at concentrations even higher than 200 mM. Consistent with the biochemical results, the biological activities of the mutated C34-peptides showed a good linear correlation with their total mutation numbers (Fig. 3B). In a 6-HB, three NHRs form a trimeric inner core, and three CHRs are packed in an antiparallel manner in the hydrophobic grooves of the NHR trimer.11 Residues at the ‘a’ and ‘d’ positions of the heptad register in the N36 interact with each other to form the inner trimeric core, and its ‘e’ and ‘g’ residues interact with the ‘a’ and ‘d’ residues of C34 (Fig. 1B).11 These residues are considered ‘key residues’ in the coiled-coil interaction.1,7 The abovementioned exchangeable mutations included ‘key residues’ as well as other residues, and the total number of mutations of ‘key residues’ is presented in Table 1. However, compared to Fig. 3, no better correlation between the number of mutations of ‘key residues’ and the biochemical and biological activities of the mutated C34- and N36-peptides was observed. The clear linear relationship between the total number of mutations and the biochemical and biological activities of the mutated peptides indicated that no single mutation between the amino acid residues of similar physiochemical properties had a significant effect on the coiled-coil interaction. Based on Fig. 3B, each exchangeable mutation caused an average activity decrease of C34 with a negligible factor of 0.23 in cell–cell fusion assay. This suggested that amino acid residues of similar physiochemical
11088
Chem. Commun., 2013, 49, 11086--11088
properties are largely exchangeable in coiled-coil interactions. However, the accumulation of such mutations weakened the interaction between these peptides and resulted in the loss of the secondary structure of the coiled-coil, thereby reducing the biological activity of the peptide. The fact that the exchangeable mutations at ‘key residues’ had no bigger effect on the biochemical and biological activity of C34 and N36 than those at other positions suggested that the exchangeable mutations did not weaken the coiled-coil interaction through disruption of specific interactions between C34 and N36. The gradually reduced interaction between C34 and N36 by accumulated mutations may be due to differences in side chain length and orientation between these amino acid residue pairs, which may therefore affect coiled-coil packing and disrupt subtle hydrogen bond and salt bridge networks, and consequently weaken the coiled-coil interaction. The large exchangeability between these amino acid residue pairs with different side chain lengths also implies that interactive peptide partners can adjust their conformations to each other and adapt proper folding to stabilize the coiled-coil interaction. In conclusion, using an HIV-1 gp41 6-HB model, we experimentally demonstrated that amino acid residues of similar physicochemical properties were largely exchangeable in coiled-coil interaction. This exchangeability can be used in ab initio protein and peptide design, although fine tuning is required to precisely match the subtle interacting network between two protein interfaces. It is worth investigating similar exchangeability in certain protein– protein interactions in order to degenerate the sequence space of proteins and peptides thus improving protein and peptide design targeting these interactions. This research is supported, in part, by the National Natural Science Foundation of China Grant (81072581, 81273434), and by the National Science and Technology Major Project of China grant (2012ZX09301003).
Notes and references 1 B. Apostolovic, M. Danial and H.-A. Klok, Chem. Soc. Rev., 2010, 39, 3541–3575. 2 L. F. Cai and S. B. Jiang, ChemMedChem, 2010, 5, 1813–1824. 3 D. M. Eckert and P. S. Kim, Annu. Rev. Biochem., 2001, 70, 777–810. 4 C. Wild, T. Oas, C. McDanal, D. Bolognesi and T. Matthews, Proc. Natl. Acad. Sci. U. S. A., 1992, 89, 10537–10541. 5 C. T. Wild, D. C. Shugars, T. K. Greenwell, C. B. McDanal and T. J. Matthews, Proc. Natl. Acad. Sci. U. S. A., 1994, 91, 9770–9774. 6 S. B. Jiang, K. Lin, N. Strick and A. R. Neurath, Nature, 1993, 365, 113. 7 A. Otaka, M. Nakamura, D. Nameki, E. Kodama, S. Uchiyama, S. Nakamura, H. Nakano, H. Tamamura, Y. Kobayashi, M. Matsuoka and N. Fujii, Angew. Chem., Int. Ed., 2002, 41, 2938–2940. 8 J. J. Dwyer, K. L. Wilson, D. K. Davison, S. A. Freel, J. E. Seedorff, S. A. Wring, N. A. Tvermoes, T. J. Matthews, M. L. Greenberg and M. K. Delmedico, Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 12772–12777. 9 S. B. Needleman and C. D. Wunsch, J. Mol. Biol., 1970, 48, 443–453. 10 L. Y. Yampolsky and A. Stoltzfus, Genetics, 2005, 170, 1459–1472. 11 D. C. Chan, D. Fass, J. M. Berger and P. S. Kim, Cell, 1997, 89, 263–273. 12 L. Cai, M. Gochin and K. Liu, Curr. Top. Med. Chem., 2011, 11, 2959–2984. 13 Y. Wexler-Cohen and Y. Shai, FASEB J., 2007, 21, 3677–3684.
This journal is
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Published on 08 October 2013. Downloaded by University of California - Irvine on 27/10/2014 11:10:36.
COMMUNICATION
Cite this: Chem. Commun., 2013, 49, 11086 Received 28th August 2013, Accepted 8th October 2013
View Journal | View Issue
Exchangeability of amino acid residues with similar physicochemical properties in coiled-coil interactions† Guiying Zhang,za Kun Wang,zab Baohua Zheng,zac Maosheng Cheng,c Yanni Li,*b Keliang Liu*a and Lifeng Cai*a
DOI: 10.1039/c3cc46560h www.rsc.org/chemcomm
Systematic exchange of amino acid residues of similar physicochemical properties maintains specific coiled-coil interaction between two heptad repeats of HIV-1 gp41, as well as the biological activity of related peptide fusion inhibitors. This exchangeability can greatly degenerate sequence space of peptides thus making ab initio design of coiled-coil interaction feasible.
Coiled-coil is an important protein–protein interaction model.1 In human immunodeficiency virus type 1 (HIV-1) infection, the formation of a coiled-coil six helical bundle (6-HB) between the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) of gp41, a transmembrane subunit of the HIV-1 envelop glycoprotein (Env), provides the energy necessary for virus-cell membrane fusion and entry into host cells (Fig. 1).2,3 Peptides derived from NHR and CHR sequences can bind to their counterparts in gp41 to form a hetero 6-HB and prevent fusogenic 6-HB formation, thereby inhibiting membrane fusion and viral infection and being used as fusion inhibitors.4–6 Peptide engineering has been used to design highly mutated peptides derived from HIV-1 gp41 wild type sequences, which showed high potency against drugresistant HIV-1 isolates with improved pharmacokinetic and pharmacodynamic profiles.7,8 Ab initio design of a highly potent fusion inhibitor, which can provide fundamental insight into coiled-coil interaction, is still unfeasible due to the huge sequence space of peptides comprised of 20 proteinogenic amino acids, however. Among the 20 proteinogenic amino acids, some have similar physiochemical properties based on their hydrophobicity, polarity, and electric charge. In protein engineering and evolution, it is well
Fig. 1 HIV-1 gp41 NHR–CHR interaction and experimental design. (A) HIV-1 gp41 functional domains and the peptide sequences in our study. FP, fusion peptide; NHR, N-terminal heptad repeat; CHR, C-terminal heptad repeat; MPER, membrane proximal external region; TM, transmembrane region; CT, cytoplasmic tail. (B) Coiled-coil interactions between N36 and C34. Residues at ‘a’ and ‘d’ positions of the heptad register in the NHR (in red) associated with each other to form a trimeric inner core, and ‘a’ and ‘d’ residues in the CHR interact with ‘e’ and ‘g’ residues in the NHR (in underscore bold) to stabilize the 6-HB. These residues are considered as ‘key residues’ in coiled-coil interaction and in peptide fusion inhibitor design. (C) Peptide sequences used in this study. C34 and N36 are native sequences based on HIV-1HXB2 Env sequences. ‘Key residues’ are highlighted as in (B), mutated residues are highlighted in red.
a
Department of Medicinal Chemistry, Beijing Institute of Pharmacology & Toxicology, 27 Taiping Rd, Haidian District, Beijing 100850, China. E-mail: [email protected], [email protected]; Fax: +86-10-68211656 b Key Laboratory of Systems Bioengineering, Ministry of Education, Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China. E-mail: [email protected] c Key Lab. Struc. Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China † Electronic supplementary information (ESI) available: Experiments, CD spectra and thermal denaturation data. See DOI: 10.1039/c3cc46560h ‡ G. Y. Zhang, K. Wang and B. H. Zheng contributed equally to this work.
11086
Chem. Commun., 2013, 49, 11086--11088
evidenced that amino acid residues with similar properties are exchangeable for maintaining basic structure and function of a protein.9,10 However, due to the complexity of protein folding and function, systematically applying this exchangeability to protein and peptide design is problematic. Generally, each of the 20 proteinogenic amino acids is unique in protein function. If, in certain protein folding and interaction systems, such This journal is
c
The Royal Society of Chemistry 2013
View Article Online
Published on 08 October 2013. Downloaded by University of California - Irvine on 27/10/2014 11:10:36.
Communication exchangeability between amino acid residues with similar properties is applicable, the sequence space of proteins and peptides will be greatly degenerated thus making ab initio protein and peptide design feasible. Therefore, we investigated the exchangeability of amino acid residues with similar physicochemical properties in coiled-coil interactions. We chose a HIV-1 gp41 N36/C34 6-HB as model,11 making systematic exchange of amino acid residue pairs of similar properties, and studied the effects of such exchanges on the coiled-coil interaction and biological activities of corresponding peptides. N36/C34 6-HB has an available crystal structure and is widely used to represent the HIV-1 gp41 fusion core.2,11,12 We introduced mutations throughout the N36 and C34 sequences by making systematic exchanges between amino acid residue pairs with similar physicochemical properties, including E–D, I–L, Q–N, or R–K pairs. These residues occupy more than two thirds of the sequences of the two peptides. The peptide sequences are shown in Fig. 1C. The exchangeabilities of these amino acid residue pairs were evaluated based on the effects of the mutations on the stability of the coiledcoil structure formed between mutated N36- and C34-peptides, as well as the inhibitory activities of the C34 peptides against HIV-1 Env mediated cell–cell fusion. We first tested the effects of these mutations on the coiled-coil interactions by circular dichroism (CD) spectroscopic analysis. C34 and N36 form random or partial a-helices in solution when separated, and form a typical 6-HB structure with high a-helical content when they are mixed in a 1 : 1 molar ratio, which is characterized by a large signal increase at 222 nm in the CD spectra (Fig. S1A, ESI†).11,12 As shown in Fig. 2A, the systematic exchange between any pair of E–D, I–L, Q–N or R–K residues throughout C34 largely retained the coiled-coil interaction, and the mutated C34-peptides interacted with N36 to form complexes with high a-helical contents.
Fig. 2 CD spectra of complexes formed between mutated C34-peptides and N36 (A), and mutated N36-peptides and C34 (B).
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ChemComm Similar mutations in N36 had bigger effects on the coiled-coil interaction than those in C34, and only the N36EDKR peptide, which had a total of four mutations, interacted with C34 to form the 6-HB structure (Fig. 2B). N36 plays dual roles in the coiled-coil interactions, i.e. stabilizes the inner trimeric core and interacts with C34 to stabilize the 6-HB structure. In contrast, C34 only interacts with the N36 trimer to stabilize the coiled coil interaction, which may account for the higher sensitivities of exchangeable mutations in the N36 trimer to the coiled-coil interaction than those of C34. The accumulation of such mutations weakened the coiledcoil interaction. The C34mut peptide (6), which had a total of 25 mutations of a total of 34 residues, could not form a 6-HB with N36 (Fig. 2A), and the N36Mut peptide (12) with 26 mutations of a total of 36 residues was insoluble and difficult to purify. We analyzed the relationship between the number of total mutations and the a-helical content of the mutated 6-HBs. Based upon the contributions of C34 and N36 to the 6-HB interaction, we doubled the number of N36 mutations in the analysis. A good linear relationship between the total number of mutations in C34 or N36 and the a-helical content of related 6-HBs was observed (Fig. 3A). We then tested the effects of these mutations on the biological activities of the C34-peptides, using a HIV-1 Env-mediated cell–cell fusion assay.13 The activities of the C34-peptides were evaluated based on the concentrations that achieved 50% inhibition in the assay (IC50). Peptides derived from the HIV-1 gp41 CHR are potent fusion inhibitors.5,6 C34 showed an IC50 of 0.6 nM in the cell–cell fusion assay. Exchanges between any amino acid residue pair of similar physicochemical properties retained the high biological activity of C34, and most of these mutated C34-peptides showed an IC50 in the low nanomolar range in the cell–cell fusion assay (Table 1). The exchange between Q and N had a bigger effect on the biological activity of C34 than other mutations, and the C34QN (5) peptide showed a significantly 85-fold potent decrease compared
Fig. 3 Correlation between the total number of mutations and the a-helical content of C34/N36 6-HB (A), the inhibitory activities of the mutated C34-peptides against HIV-1 Env mediated cell–cell fusion (B). Error bars are standard deviations from at least duplicate measurements.
Chem. Commun., 2013, 49, 11086--11088
11087
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Published on 08 October 2013. Downloaded by University of California - Irvine on 27/10/2014 11:10:36.
Table 1
Communication
Biological and biochemical properties of the peptides a
b
Peptides
Total mutations
IC50 (nM)
C34 (1) C34IL (2) C34ED (3) C34KR (4) C34QN (5) C34Mut (6) N36 (7) N36IL (8) N36EDKR (9) N36QN (10) N36ILV (11)
0 7 8 2 8 25 0 11 4 10 13
0.60 1.68 9.8 0.46 51 410 2090 2100 3100 1420 1170
(3) (1) (2) (6) (9) (0) (4) (11)
0.43 0.19 2.4 0.39 11 150 390 590 1460 410 85
c
6-HB helix (%) 91.2 74.7 70.4 87.1 63.4 23.9 91.2 24.5 72.6 4.2 6.8
0.8 7.6 7.8 4.1 4.6 1.3 0.8 2.8 10.6
1.0
a
The numbers in parentheses represent the total number of mutations of ‘key residues’ as shown in Fig. 1B. b The concentrations of peptides that achieved 50% inhibition in cell–cell fusion assay. c The 6-HBs were formed between mutated C34-peptides and native N36, or between mutated N36-peptides and C34. Numbers after ‘’ are standard deviations from at least duplicate measurements.
to C34. The CD thermal denaturation of the C34QN–N36 complex showed a gradual loss of secondary structure instead of a cooperatively two-state conformational transition, indicating a partly folded, molten globule form instead of an ordered coiled-coil structure (Fig. S2, ESI†). This may be attributed to the low biological activity of the C34QN peptide. Other complexes with high a-helical content showed typical S-shape thermal denaturation curves, indicating ordered coiled-coil structures (Fig. S2, ESI†). Consistent with the CD results, C34Mut (6) showed an IC50 of 410 nM, an approximately 700-fold potent decrease compared to the native peptide (Table 1). It is noteworthy that C34Mut (6) still showed clear inhibitory activity in the cell–cell fusion assay compared to unrelated peptides, which were usually inactive at concentrations even higher than 200 mM. Consistent with the biochemical results, the biological activities of the mutated C34-peptides showed a good linear correlation with their total mutation numbers (Fig. 3B). In a 6-HB, three NHRs form a trimeric inner core, and three CHRs are packed in an antiparallel manner in the hydrophobic grooves of the NHR trimer.11 Residues at the ‘a’ and ‘d’ positions of the heptad register in the N36 interact with each other to form the inner trimeric core, and its ‘e’ and ‘g’ residues interact with the ‘a’ and ‘d’ residues of C34 (Fig. 1B).11 These residues are considered ‘key residues’ in the coiled-coil interaction.1,7 The abovementioned exchangeable mutations included ‘key residues’ as well as other residues, and the total number of mutations of ‘key residues’ is presented in Table 1. However, compared to Fig. 3, no better correlation between the number of mutations of ‘key residues’ and the biochemical and biological activities of the mutated C34- and N36-peptides was observed. The clear linear relationship between the total number of mutations and the biochemical and biological activities of the mutated peptides indicated that no single mutation between the amino acid residues of similar physiochemical properties had a significant effect on the coiled-coil interaction. Based on Fig. 3B, each exchangeable mutation caused an average activity decrease of C34 with a negligible factor of 0.23 in cell–cell fusion assay. This suggested that amino acid residues of similar physiochemical
11088
Chem. Commun., 2013, 49, 11086--11088
properties are largely exchangeable in coiled-coil interactions. However, the accumulation of such mutations weakened the interaction between these peptides and resulted in the loss of the secondary structure of the coiled-coil, thereby reducing the biological activity of the peptide. The fact that the exchangeable mutations at ‘key residues’ had no bigger effect on the biochemical and biological activity of C34 and N36 than those at other positions suggested that the exchangeable mutations did not weaken the coiled-coil interaction through disruption of specific interactions between C34 and N36. The gradually reduced interaction between C34 and N36 by accumulated mutations may be due to differences in side chain length and orientation between these amino acid residue pairs, which may therefore affect coiled-coil packing and disrupt subtle hydrogen bond and salt bridge networks, and consequently weaken the coiled-coil interaction. The large exchangeability between these amino acid residue pairs with different side chain lengths also implies that interactive peptide partners can adjust their conformations to each other and adapt proper folding to stabilize the coiled-coil interaction. In conclusion, using an HIV-1 gp41 6-HB model, we experimentally demonstrated that amino acid residues of similar physicochemical properties were largely exchangeable in coiled-coil interaction. This exchangeability can be used in ab initio protein and peptide design, although fine tuning is required to precisely match the subtle interacting network between two protein interfaces. It is worth investigating similar exchangeability in certain protein– protein interactions in order to degenerate the sequence space of proteins and peptides thus improving protein and peptide design targeting these interactions. This research is supported, in part, by the National Natural Science Foundation of China Grant (81072581, 81273434), and by the National Science and Technology Major Project of China grant (2012ZX09301003).
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