- Email: [email protected]
Structure–Function Analysis of the 7B2 CT Peptide
Biochemical and Biophysical Research Communications 267, 940 –942 (2000) doi:10.1006/bbrc.1999.2060, available online at http://www.idealibrary.com on
Structure–Function Analysis of the 7B2 CT Peptide Ekaterina V. Apletalina,* Maria A. Juliano,† Luiz Juliano,† and Iris Lindberg* ,1 *Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112; and †Department of Biophysics, Escola Paulista de Medicina, Rua Tres de Maio 100, 04044-020, Sao Paulo, Brazil Received December 22, 1999
Prohormone convertases play important roles in the proteolytic conversion of many protein precursors. The neuroendocrine protein 7B2 and its 31-residue carboxyl-terminal (CT) peptide potently and specifically inhibit prohormone convertase 2 (PC2). We have analyzed the residues contributing to inhibition using N-terminal truncation and alanine scanning. Removal of more than 3 residues from the amino-terminal end of CT1-18 resulted in a more than 190-fold drop in inhibitory activity, showing that most of the residues between 3 and 18 are required for inhibition. In agreement, an Ala scan indicated that only 4 residues could be replaced with Ala without losing mid-nanomolar inhibitory potency; in particular, Gln7, Gln9, and Asp12 could be Ala-substituted to yield peptides with a similar inhibitory potency to the starting peptide. The all-D-retro-inverso, all-L-inverso, and all-D analogues of CT peptide were completely inactive, indicating that amino acid side chains and the CT peptide main chain interact with PC2. CT peptide inhibition could not be competitively blocked by preincubation with truncated CT peptide forms, supporting an absolute requirement for the Lys–Lys pair in initial binding of the CT peptide to the active site. © 2000 Academic Press
The prohormone convertases are eukaryotic subtilisins believed to be involved in a variety of protein maturation processes within the secretory pathway (reviewed in 1). Within this family, the prohormone convertase 2 (PC2) is unique in its requirement for another neuroendocrine protein, 7B2, for maturation to an active enzyme species (reviewed in 2). In addition to its role in facilitating the maturation of proPC2, 7B2 Abbreviations used: CT peptide, 7B2 carboxy-terminal peptide; PC2, prohormone convertase 2; MCA, 4-methyl-coumaryl-7-amide; AMC, 7-amino-4-methylcoumarin; TFA, trifluoroacetic acid; ACN, acetonitrile; Fmoc, 9-fluorenylmethyloxycarbonyl. 1 To whom correspondence should be addressed at Department of Biochemistry and Molecular Biology, L.S.U. Health Sciences Center, 1901 Perdido Street, New Orleans, LA 70112. Fax: (504) 568-6598. E-mail: [email protected]. 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
also contains a 31-residue peptide which functions as a potent inhibitor of active PC2 (3–5). This peptide is internally hydrolyzed within cells to generate an 18residue fragment which is still highly inhibitory; however, removal of the C-terminal Lys–Lys pair results in complete loss of inhibition (6). In the present study, we have undertaken a structure-function analysis of the 18-residue human CT peptide fragment in an effort to understand the inhibitory mechanism of this unusual tight-binding inhibitory peptide. MATERIALS AND METHODS Enzyme assay. Recombinant PC2 used in the assay was purified to homogeneity from conditioned medium of CHO cells transfected with expression plasmids encoding cDNAs for rat 21-kDa 7B2 and mouse proPC2 as previously described (7). Duplicate reactions were performed in a polypropylene microtiter plate in a total volume of 50 l, containing 100 mM sodium acetate buffer, pH 5.0, 5 mM CaCl2, 0.4% n-octyl glucoside, 22 ng of preactivated mouse PC2, varying amounts of the human CT peptide derivatives, and 0.2 mM fluorogenic substrate pGluArg-Thr-Lys-Arg-MCA. Preactivated mouse PC2 was preincubated with the peptide to be tested for 30 min at room temperature prior to the addition of substrate. The reaction was conducted at 37°C for 30 – 45 min. Released AMC was measured with a Fluoroscan Ascent fluorometer (Labsystems) using an excitation and an emission wavelength of 355 and 460 nm, respectively. The IC50 values for the peptides were calculated using non-linear regression of the experimental data (fluorescence vs peptide concentration) using the program GraphPad (GraphPad Software, Inc., San Diego, CA). Peptide synthesis. An automated bench-top simultaneous multiple solid-phase peptide synthesizer (PSSM 8 system from Shimadzu) was used for the solid-phase synthesis of all the peptides by the Fmoc-procedure. The final deprotected peptides were purified by semipreparative HPLC using an Econosil C-18 column (10 m, 22.5 ⫻ 250 mm) and a two-solvent system: (A) trifluoroacetic acid (TFA)/H 2O (1:1000) and (B) TFA/acetonitrile (ACN)/H 2O (1:900:100). The column was eluted at a flow rate of 5 ml/min with a 10 (or 30)–50 (or 60)% gradient of solvent B over 30 or 45 min. Analytical HPLC was performed using a binary HPLC system from Shimadzu with a SPD-10AV Shimadzu uv–vis detector, coupled to an Ultrasphere C-18 column (5 m, 4.6 ⫻ 150 mm) which was eluted with solvent systems A1 (H 3PO 4/H 2O, 1:1000) and B1 (ACN/H 2O/H 3PO 4, 900: 100:1) at a flow rate of 1.7 ml/min and a 10 – 80% gradient of B1 over 15 min. The HPLC column eluates were monitored by their absorbance at 220 nm. The molecular weight and purity of synthesized peptides were checked by MALDI–TOF–mass spectrometry (TofSpec-E, Micromass, Manchester, UK) and by peptide sequencing (Sequencer PPSQ-23 Shimadzu Tokyo, Japan).
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE I
IC 50 Values of PC2 for Truncated CT Peptides Peptide
IC 50 (M)
SVNPYLQGQRLDNVVAKK-NH 2 VNPYLQGQRLDNVVAKK-NH 2 NPYLQGQRLDNVVAKK-NH 2 PYLQGQRLDNVVAKK-NH 2 YLQGQRLDNVVAKK-NH 2 LQGQRLDNVVAKK-NH 2
0.026 ⫾ 0.004 0.15 ⫾ 0.02 0.41 ⫾ 0.04 ⬎5 ⬎5 ⬎5
Note. Results indicate means ⫾ SD, determined from two to four independent experiments.
The all-D-retro-inverso, all-L-inverso analogues of CT peptide were synthesized as the other peptides, using Fmoc D- or L-amino acids, but the sequences in this case are inverted, i.e., the C-terminal amino acid was Ser, followed by all other amino acids of CT peptide till the N-terminal end Lys. Therefore, if these two peptides are aligned with the CT peptide they will have the same sequence, but with inverted peptide bonds. No effort was made to use a malonic derivative of Lys or a geminate amine derivative of Ser in order to correct the C- and N-terminal carboxyl and amino positions in these inverso analogues. However, peptides were amidated and acetylated at their ends. For the design concept of these peptides see Petit et al. (8), Carver et al. (9), and Carmona and Juliano (10).
RESULTS AND DISCUSSION Previous data have shown that the carboxy-terminal Lys–Lys pair is absolutely required for inhibition of PC2 (6). To obtain information on requirements at the amino terminus of this peptide, a series of N-terminally truncated peptides was synthesized and tested for inhibition against recombinant PC2. The results of this analysis are shown in Table I. Loss of one to two residues from the amino terminus resulted in peptides with 3-6 fold lower inhibitory potency than the starting peptide. However, truncation by three residues was found to eliminate inhibitory potency, indicating a strict requirement for the amino terminal Asn3. The lack of inhibitory potency of amino-terminally truncated peptides is in line with our failure to isolate a CT peptide-like sequence using a combinatorial hexapeptide library screen which consisted only of hexapeptides (11). An Ala scan was then performed in order to identify the residues, which contribute most to inhibition. Surprisingly, most of the amino acids within the 18residue peptide contributed heavily to inhibitory potency (Table II). Only four residues could be replaced with Ala without losing mid-nanomolar inhibitory potency; in particular, Gln7, Gln9, and Asp12 could be substituted with Ala to yield peptides with a similar inhibitory potency to the starting peptide. Substitutions of the Lys-Lys pair were extremely deleterious, in line with previous mutagenesis and carboxypeptidase results (5, 6). In addition to the Lys-Lys pair, Asn3 (mentioned above) and Tyr5 also appeared to contrib-
ute heavily to inhibitory potency, with IC 50 drops of more than 190-fold. Substitution of Leu6 and Gly8 resulted, respectively, in an 80- and 45-fold decrease in the IC 50 value, indicating that these residues are also very important for the inhibition potency of the CT peptide. Substitutions at other sites yielded peptides with intermediate potencies, i.e., 8- to 30-fold drops in potency. These results are supportive of recent evolutionary data which indicate that an amino terminal heptapeptide within the CT peptide, VNPYLQG, is one of the most conserved regions in the 186-residue 7B2 protein (12). To determine whether stabilization of the CT peptide structure would occur by introduction of a disulfide bridge, Cys residues were introduced at the least deleterious positions, Gln7 and Asp12. The resulting peptides exhibited good inhibitory potency but were not further stabilized by the disulfide bridge (IC 50 ⫽ 1.2 ⫾ 0.31 M for the disulfide-linked peptide compared to 0.10 ⫾ 0.1 M for peptide containing Cys[SMe] at the positions 7 and 12). The all-D-retro-inverso, all-L-inverso and all-D analogues of CT peptide were completely devoid of inhibitory activity, suggesting that both, the side chain of the amino acids as well as the CT peptide main chain are involved in its interaction with PC2. To evaluate the degree of protection of CT peptide binding by peptides containing the amino terminal heptapeptide, we preincubated PC2 with CT1-16 at concentrations of 1, 10, or 20 M and then assessed the remaining inhibitory potency of the 1–18 peptide. Surprisingly, CT1-16 was totally unable to protect the active site of PC2 from inhibition by the 1-18 peptide, even at a 100-fold molar excess (results not shown). These results indicate that the Lys–Lys pair is absoTABLE II
Alanine Scan (IC 50 Values for the CT Peptide Derivatives) Peptide
IC 50 (M)
SVNPYLQGQRLDNVVAKK-NH 2 SVNPYLQGQRLDNVVAKA-NH 2 SVNPYLQGQRLDNVVAAK-NH 2 SVNPYLQGQRLDNVAAKK-NH 2 SVNPYLQGQRLDNAVAKK-NH 2 SVNPYLQGQRLDAVVAKK-NH 2 SVNPYLQGQRLANVVAKK-NH 2 SVNPYLQGQRADNVVAKK-NH 2 SVNPYLQGQALDNVVAKK-NH 2 SVNPYLQGARLDNVVAKK-NH 2 SVNPYLQAQRLDNVVAKK-NH 2 SVNPYLAGQRLDNVVAKK-NH 2 SVNPYAQGQRLDNVVAKK-NH 2 SVNPALQGQRLDNVVAKK-NH 2 SVNAYLQGQRLDNVVAKK-NH 2 SVAAYLQGQRLDNVVAKK-NH 2
0.026 ⫾ 0.004 ⬎5 ⬎35 0.185 ⫾ 0.013 0.90 ⫾ 0.08 0.057 ⫾ 0.004 0.026 ⫾ 0.003 0.74 ⫾ 0.17 0.23 ⫾ 0.03 0.032 ⫾ 0.003 1.26 ⫾ 0.24 0.021 ⫾ 0.003 2.17 ⫾ 0.13 ⬎5 0.76 ⫾ 0.10 ⬎5
Note. Results indicate means ⫾ SD, determined from two to four independent experiments.
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lutely required for binding of the remainder of the peptide to PC2. We speculate that binding of this dibasic pair- which clearly occurs at the active site of PC2 since cleavage of the 31-residue peptide occurs at this position- generates conformational changes within the binding pocket of PC2 which then permit tight binding of the remainder of the molecule, in particular, of the highly conserved amino-terminal heptapeptide. ACKNOWLEDGMENTS This work was funded by DA05084 to I.L., who was supported by K02 Award DA00204. L.J. and M.A.J. were supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq). We thank Angus Cameron for critical commentary on the manuscript.
REFERENCES 1. Zhou, A., Webb, G., Zhu, X., and Steiner, D. F. (1999) J. Biol. Chem. 274, 20745–20748. 2. Muller, L., and Lindberg, I. (1999) Prog. Nucleic Acid Res. Mol. Biol. 63, 69 –108.
3. Martens, G. J. M., Braks, J. A. M., Eib, D. W., Zhou, Y., and Lindberg, I. (1994) Proc. Natl. Acad. Sci. USA 91, 5784 – 5785. 4. Lindberg, I., Van den Hurk, W. H., Bui, C., and Batie, C. J. (1995) Biochemistry 34, 5486 –5493. 5. Van Horssen, A. M., Van den Hurk, W. H., Bailyes, E. M., Hutton, J. C., Martens, G. J. M., and Lindberg, I. (1995) J. Biol. Chem. 270, 14292–14296. 6. Zhu, X., Rouille, Y., Lamango, N. S., Steiner, D. F., and Lindberg, I. (1996) Proc. Natl. Acad. Sci. USA 93, 4919 – 4924. 7. Lamango, N. S., Apletalina, E., Liu, J., and Lindberg, I. (1999) Arch. Biochem. Biophys. 362, 275–282. 8. Petit, M. C., Benkirane, N., Guichard, G., Du, A. P., Marraud, M., Cung, M. T., Briand, J. P., and Muller, S. (1999) J. Biol. Chem. 274, 3686 –3692. 9. Carver, J. A., Esposito, G., Viglino, P., Fogolari, F., Guichard, G., Briand, J. P., Van Regenmortel, M. H., Brown, F., and Mascageni, P. (1997) Biopolymers 41, 569 –590. 10. Carmona, A. K., and Juliano, L. (1996) Biochem. Pharmacol. 51, 1051–1060. 11. Apletalina, E., Appel, J., Lamango, N. S., Houghten, R. A., and Lindberg, I. (1998) J. Biol. Chem. 273, 26589 –26595. 12. Lindberg, I., Tu, B., Muller, L., and Dickerson, I. (1998) DNA Cell Biol. 17, 727–734.
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Structure–Function Analysis of the 7B2 CT Peptide Ekaterina V. Apletalina,* Maria A. Juliano,† Luiz Juliano,† and Iris Lindberg* ,1 *Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112; and †Department of Biophysics, Escola Paulista de Medicina, Rua Tres de Maio 100, 04044-020, Sao Paulo, Brazil Received December 22, 1999
Prohormone convertases play important roles in the proteolytic conversion of many protein precursors. The neuroendocrine protein 7B2 and its 31-residue carboxyl-terminal (CT) peptide potently and specifically inhibit prohormone convertase 2 (PC2). We have analyzed the residues contributing to inhibition using N-terminal truncation and alanine scanning. Removal of more than 3 residues from the amino-terminal end of CT1-18 resulted in a more than 190-fold drop in inhibitory activity, showing that most of the residues between 3 and 18 are required for inhibition. In agreement, an Ala scan indicated that only 4 residues could be replaced with Ala without losing mid-nanomolar inhibitory potency; in particular, Gln7, Gln9, and Asp12 could be Ala-substituted to yield peptides with a similar inhibitory potency to the starting peptide. The all-D-retro-inverso, all-L-inverso, and all-D analogues of CT peptide were completely inactive, indicating that amino acid side chains and the CT peptide main chain interact with PC2. CT peptide inhibition could not be competitively blocked by preincubation with truncated CT peptide forms, supporting an absolute requirement for the Lys–Lys pair in initial binding of the CT peptide to the active site. © 2000 Academic Press
The prohormone convertases are eukaryotic subtilisins believed to be involved in a variety of protein maturation processes within the secretory pathway (reviewed in 1). Within this family, the prohormone convertase 2 (PC2) is unique in its requirement for another neuroendocrine protein, 7B2, for maturation to an active enzyme species (reviewed in 2). In addition to its role in facilitating the maturation of proPC2, 7B2 Abbreviations used: CT peptide, 7B2 carboxy-terminal peptide; PC2, prohormone convertase 2; MCA, 4-methyl-coumaryl-7-amide; AMC, 7-amino-4-methylcoumarin; TFA, trifluoroacetic acid; ACN, acetonitrile; Fmoc, 9-fluorenylmethyloxycarbonyl. 1 To whom correspondence should be addressed at Department of Biochemistry and Molecular Biology, L.S.U. Health Sciences Center, 1901 Perdido Street, New Orleans, LA 70112. Fax: (504) 568-6598. E-mail: [email protected]. 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
also contains a 31-residue peptide which functions as a potent inhibitor of active PC2 (3–5). This peptide is internally hydrolyzed within cells to generate an 18residue fragment which is still highly inhibitory; however, removal of the C-terminal Lys–Lys pair results in complete loss of inhibition (6). In the present study, we have undertaken a structure-function analysis of the 18-residue human CT peptide fragment in an effort to understand the inhibitory mechanism of this unusual tight-binding inhibitory peptide. MATERIALS AND METHODS Enzyme assay. Recombinant PC2 used in the assay was purified to homogeneity from conditioned medium of CHO cells transfected with expression plasmids encoding cDNAs for rat 21-kDa 7B2 and mouse proPC2 as previously described (7). Duplicate reactions were performed in a polypropylene microtiter plate in a total volume of 50 l, containing 100 mM sodium acetate buffer, pH 5.0, 5 mM CaCl2, 0.4% n-octyl glucoside, 22 ng of preactivated mouse PC2, varying amounts of the human CT peptide derivatives, and 0.2 mM fluorogenic substrate pGluArg-Thr-Lys-Arg-MCA. Preactivated mouse PC2 was preincubated with the peptide to be tested for 30 min at room temperature prior to the addition of substrate. The reaction was conducted at 37°C for 30 – 45 min. Released AMC was measured with a Fluoroscan Ascent fluorometer (Labsystems) using an excitation and an emission wavelength of 355 and 460 nm, respectively. The IC50 values for the peptides were calculated using non-linear regression of the experimental data (fluorescence vs peptide concentration) using the program GraphPad (GraphPad Software, Inc., San Diego, CA). Peptide synthesis. An automated bench-top simultaneous multiple solid-phase peptide synthesizer (PSSM 8 system from Shimadzu) was used for the solid-phase synthesis of all the peptides by the Fmoc-procedure. The final deprotected peptides were purified by semipreparative HPLC using an Econosil C-18 column (10 m, 22.5 ⫻ 250 mm) and a two-solvent system: (A) trifluoroacetic acid (TFA)/H 2O (1:1000) and (B) TFA/acetonitrile (ACN)/H 2O (1:900:100). The column was eluted at a flow rate of 5 ml/min with a 10 (or 30)–50 (or 60)% gradient of solvent B over 30 or 45 min. Analytical HPLC was performed using a binary HPLC system from Shimadzu with a SPD-10AV Shimadzu uv–vis detector, coupled to an Ultrasphere C-18 column (5 m, 4.6 ⫻ 150 mm) which was eluted with solvent systems A1 (H 3PO 4/H 2O, 1:1000) and B1 (ACN/H 2O/H 3PO 4, 900: 100:1) at a flow rate of 1.7 ml/min and a 10 – 80% gradient of B1 over 15 min. The HPLC column eluates were monitored by their absorbance at 220 nm. The molecular weight and purity of synthesized peptides were checked by MALDI–TOF–mass spectrometry (TofSpec-E, Micromass, Manchester, UK) and by peptide sequencing (Sequencer PPSQ-23 Shimadzu Tokyo, Japan).
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Vol. 267, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE I
IC 50 Values of PC2 for Truncated CT Peptides Peptide
IC 50 (M)
SVNPYLQGQRLDNVVAKK-NH 2 VNPYLQGQRLDNVVAKK-NH 2 NPYLQGQRLDNVVAKK-NH 2 PYLQGQRLDNVVAKK-NH 2 YLQGQRLDNVVAKK-NH 2 LQGQRLDNVVAKK-NH 2
0.026 ⫾ 0.004 0.15 ⫾ 0.02 0.41 ⫾ 0.04 ⬎5 ⬎5 ⬎5
Note. Results indicate means ⫾ SD, determined from two to four independent experiments.
The all-D-retro-inverso, all-L-inverso analogues of CT peptide were synthesized as the other peptides, using Fmoc D- or L-amino acids, but the sequences in this case are inverted, i.e., the C-terminal amino acid was Ser, followed by all other amino acids of CT peptide till the N-terminal end Lys. Therefore, if these two peptides are aligned with the CT peptide they will have the same sequence, but with inverted peptide bonds. No effort was made to use a malonic derivative of Lys or a geminate amine derivative of Ser in order to correct the C- and N-terminal carboxyl and amino positions in these inverso analogues. However, peptides were amidated and acetylated at their ends. For the design concept of these peptides see Petit et al. (8), Carver et al. (9), and Carmona and Juliano (10).
RESULTS AND DISCUSSION Previous data have shown that the carboxy-terminal Lys–Lys pair is absolutely required for inhibition of PC2 (6). To obtain information on requirements at the amino terminus of this peptide, a series of N-terminally truncated peptides was synthesized and tested for inhibition against recombinant PC2. The results of this analysis are shown in Table I. Loss of one to two residues from the amino terminus resulted in peptides with 3-6 fold lower inhibitory potency than the starting peptide. However, truncation by three residues was found to eliminate inhibitory potency, indicating a strict requirement for the amino terminal Asn3. The lack of inhibitory potency of amino-terminally truncated peptides is in line with our failure to isolate a CT peptide-like sequence using a combinatorial hexapeptide library screen which consisted only of hexapeptides (11). An Ala scan was then performed in order to identify the residues, which contribute most to inhibition. Surprisingly, most of the amino acids within the 18residue peptide contributed heavily to inhibitory potency (Table II). Only four residues could be replaced with Ala without losing mid-nanomolar inhibitory potency; in particular, Gln7, Gln9, and Asp12 could be substituted with Ala to yield peptides with a similar inhibitory potency to the starting peptide. Substitutions of the Lys-Lys pair were extremely deleterious, in line with previous mutagenesis and carboxypeptidase results (5, 6). In addition to the Lys-Lys pair, Asn3 (mentioned above) and Tyr5 also appeared to contrib-
ute heavily to inhibitory potency, with IC 50 drops of more than 190-fold. Substitution of Leu6 and Gly8 resulted, respectively, in an 80- and 45-fold decrease in the IC 50 value, indicating that these residues are also very important for the inhibition potency of the CT peptide. Substitutions at other sites yielded peptides with intermediate potencies, i.e., 8- to 30-fold drops in potency. These results are supportive of recent evolutionary data which indicate that an amino terminal heptapeptide within the CT peptide, VNPYLQG, is one of the most conserved regions in the 186-residue 7B2 protein (12). To determine whether stabilization of the CT peptide structure would occur by introduction of a disulfide bridge, Cys residues were introduced at the least deleterious positions, Gln7 and Asp12. The resulting peptides exhibited good inhibitory potency but were not further stabilized by the disulfide bridge (IC 50 ⫽ 1.2 ⫾ 0.31 M for the disulfide-linked peptide compared to 0.10 ⫾ 0.1 M for peptide containing Cys[SMe] at the positions 7 and 12). The all-D-retro-inverso, all-L-inverso and all-D analogues of CT peptide were completely devoid of inhibitory activity, suggesting that both, the side chain of the amino acids as well as the CT peptide main chain are involved in its interaction with PC2. To evaluate the degree of protection of CT peptide binding by peptides containing the amino terminal heptapeptide, we preincubated PC2 with CT1-16 at concentrations of 1, 10, or 20 M and then assessed the remaining inhibitory potency of the 1–18 peptide. Surprisingly, CT1-16 was totally unable to protect the active site of PC2 from inhibition by the 1-18 peptide, even at a 100-fold molar excess (results not shown). These results indicate that the Lys–Lys pair is absoTABLE II
Alanine Scan (IC 50 Values for the CT Peptide Derivatives) Peptide
IC 50 (M)
SVNPYLQGQRLDNVVAKK-NH 2 SVNPYLQGQRLDNVVAKA-NH 2 SVNPYLQGQRLDNVVAAK-NH 2 SVNPYLQGQRLDNVAAKK-NH 2 SVNPYLQGQRLDNAVAKK-NH 2 SVNPYLQGQRLDAVVAKK-NH 2 SVNPYLQGQRLANVVAKK-NH 2 SVNPYLQGQRADNVVAKK-NH 2 SVNPYLQGQALDNVVAKK-NH 2 SVNPYLQGARLDNVVAKK-NH 2 SVNPYLQAQRLDNVVAKK-NH 2 SVNPYLAGQRLDNVVAKK-NH 2 SVNPYAQGQRLDNVVAKK-NH 2 SVNPALQGQRLDNVVAKK-NH 2 SVNAYLQGQRLDNVVAKK-NH 2 SVAAYLQGQRLDNVVAKK-NH 2
0.026 ⫾ 0.004 ⬎5 ⬎35 0.185 ⫾ 0.013 0.90 ⫾ 0.08 0.057 ⫾ 0.004 0.026 ⫾ 0.003 0.74 ⫾ 0.17 0.23 ⫾ 0.03 0.032 ⫾ 0.003 1.26 ⫾ 0.24 0.021 ⫾ 0.003 2.17 ⫾ 0.13 ⬎5 0.76 ⫾ 0.10 ⬎5
Note. Results indicate means ⫾ SD, determined from two to four independent experiments.
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lutely required for binding of the remainder of the peptide to PC2. We speculate that binding of this dibasic pair- which clearly occurs at the active site of PC2 since cleavage of the 31-residue peptide occurs at this position- generates conformational changes within the binding pocket of PC2 which then permit tight binding of the remainder of the molecule, in particular, of the highly conserved amino-terminal heptapeptide. ACKNOWLEDGMENTS This work was funded by DA05084 to I.L., who was supported by K02 Award DA00204. L.J. and M.A.J. were supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq). We thank Angus Cameron for critical commentary on the manuscript.
REFERENCES 1. Zhou, A., Webb, G., Zhu, X., and Steiner, D. F. (1999) J. Biol. Chem. 274, 20745–20748. 2. Muller, L., and Lindberg, I. (1999) Prog. Nucleic Acid Res. Mol. Biol. 63, 69 –108.
3. Martens, G. J. M., Braks, J. A. M., Eib, D. W., Zhou, Y., and Lindberg, I. (1994) Proc. Natl. Acad. Sci. USA 91, 5784 – 5785. 4. Lindberg, I., Van den Hurk, W. H., Bui, C., and Batie, C. J. (1995) Biochemistry 34, 5486 –5493. 5. Van Horssen, A. M., Van den Hurk, W. H., Bailyes, E. M., Hutton, J. C., Martens, G. J. M., and Lindberg, I. (1995) J. Biol. Chem. 270, 14292–14296. 6. Zhu, X., Rouille, Y., Lamango, N. S., Steiner, D. F., and Lindberg, I. (1996) Proc. Natl. Acad. Sci. USA 93, 4919 – 4924. 7. Lamango, N. S., Apletalina, E., Liu, J., and Lindberg, I. (1999) Arch. Biochem. Biophys. 362, 275–282. 8. Petit, M. C., Benkirane, N., Guichard, G., Du, A. P., Marraud, M., Cung, M. T., Briand, J. P., and Muller, S. (1999) J. Biol. Chem. 274, 3686 –3692. 9. Carver, J. A., Esposito, G., Viglino, P., Fogolari, F., Guichard, G., Briand, J. P., Van Regenmortel, M. H., Brown, F., and Mascageni, P. (1997) Biopolymers 41, 569 –590. 10. Carmona, A. K., and Juliano, L. (1996) Biochem. Pharmacol. 51, 1051–1060. 11. Apletalina, E., Appel, J., Lamango, N. S., Houghten, R. A., and Lindberg, I. (1998) J. Biol. Chem. 273, 26589 –26595. 12. Lindberg, I., Tu, B., Muller, L., and Dickerson, I. (1998) DNA Cell Biol. 17, 727–734.
942