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Development of a Novel Tetravalent Synthetic Peptide That Binds to Phosphatidic Acid.

Ogawa R, Nagao K, Taniuchi K, Tsuchiya M, Kato U, Hara Y, Inaba T, Kobayashi T, Sasaki Y, Akiyoshi K, Watanabe-Takahashi M, Nishikawa K, Umeda M - PLoS ONE (2015)

Bottom Line: Furthermore, a series of amino acid substitutions at positions 5 to 9 of PAB-TP revealed the involvement of consecutive histidine and arginine residues in recognition of the phosphomonoester head group of PA.Our results demonstrate that the recognition of PA by PAB-TP is achieved by a combination of hydrophobic, electrostatic and hydrogen-bond interactions, and that the tetravalent structure of PAB-TP contributes to the high affinity binding to PA in the membrane.The novel PA-binding tetravalent peptide PAB-TP will provide insight into the molecular mechanism underlying the recognition of PA by PA-binding proteins that are involved in various cellular events.

View Article: PubMed Central - PubMed

Affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto, Japan.

ABSTRACT
We employed a multivalent peptide-library screening technique to identify a peptide motif that binds to phosphatidic acid (PA), but not to other phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). A tetravalent peptide with the sequence motif of MARWHRHHH, designated as PAB-TP (phosphatidic acid-binding tetravalent peptide), was shown to bind as low as 1 mol% of PA in the bilayer membrane composed of PC and cholesterol. Kinetic analysis of the interaction between PAB-TP and the membranes containing 10 mol% of PA showed that PAB-TP associated with PA with a low dissociation constant of KD = 38 ± 5 nM. Coexistence of cholesterol or PE with PA in the membrane enhanced the PAB-TP binding to PA by increasing the ionization of the phosphomonoester head group as well as by changing the microenvironment of PA molecules in the membrane. Amino acid replacement analysis demonstrated that the tryptophan residue at position 4 of PAB-TP was involved in the interaction with PA. Furthermore, a series of amino acid substitutions at positions 5 to 9 of PAB-TP revealed the involvement of consecutive histidine and arginine residues in recognition of the phosphomonoester head group of PA. Our results demonstrate that the recognition of PA by PAB-TP is achieved by a combination of hydrophobic, electrostatic and hydrogen-bond interactions, and that the tetravalent structure of PAB-TP contributes to the high affinity binding to PA in the membrane. The novel PA-binding tetravalent peptide PAB-TP will provide insight into the molecular mechanism underlying the recognition of PA by PA-binding proteins that are involved in various cellular events.

No MeSH data available.


Related in: MedlinePlus

Analysis of binding of PAB-TP to phospholipids by the solid phase vesicle-binding assay.(A) Binding of PAB-TP to LUVs composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30), (5:63:2:30), (2:66:2:30), (1:67:2:30), and (0:68:2:30) was examined by the SPVB assay (mean ± SE, n = 3). (B) Binding of PAB-TP to LUVs composed of DOPC, biotin-DOPE, cholesterol, and indicated lipid (58:2:30:10) was examined by the SPVB assay (mean ±SE, n = 3). (A, B) The binding of LUVs composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30) at the concentration of 125 μM to PAB-TP was represented as 100 (arbitrary units).
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pone.0131668.g002: Analysis of binding of PAB-TP to phospholipids by the solid phase vesicle-binding assay.(A) Binding of PAB-TP to LUVs composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30), (5:63:2:30), (2:66:2:30), (1:67:2:30), and (0:68:2:30) was examined by the SPVB assay (mean ± SE, n = 3). (B) Binding of PAB-TP to LUVs composed of DOPC, biotin-DOPE, cholesterol, and indicated lipid (58:2:30:10) was examined by the SPVB assay (mean ±SE, n = 3). (A, B) The binding of LUVs composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30) at the concentration of 125 μM to PAB-TP was represented as 100 (arbitrary units).

Mentions: In this study, we employed a peptide library composed of tetravalent peptides containing a polylysine core bifurcating at both ends with six randomized residues and fixed methionine, alanine, and arginine at positions, 1, 2, and 6, respectively [29] (Fig 1A). The peptide library was screened for its ability to bind to vesicles containing PA but not to vesicles without PA. As previously reported for screening of the Stx2-binding peptide [29], we determined the relative amino acid preference at each position, and identified four candidate sequence motifs: MAKKMRRRM, MAKHRRTWY, MARWHRHHH, and MAMWHRTWR (Fig 1A). The tetravalent forms of these peptides with the same core structure were synthesized and were evaluated for their ability to bind to vesicles containing PA by the solid phase vesicle-binding (SPVB) assay. In this assay, the peptides coated on the solid phase were incubated with LUVs composed of DOPA, DOPC, biotin-DOPE, and cholesterol at the molar ratio of 10:58:2:30, followed by quantification of the binding of vesicle to the peptide using HRP-streptavidin. As shown in Fig 1B, the tetravalent peptide 3 with the sequence motif of MARWHRHHH bound effectively to the PA-containing vesicle, while only weak or no significant bindings were observed with other peptides. We designated the tetravalent peptide 3 with the sequence of MARWHRHHH as PAB-TP (phosphatidic acid-binding tetravalent peptide) and employed it for further analyses. In the SPVB assay, PAB-TP did not bind to the vesicles without PA, but bound effectively to the vesicles containing as low as 1 mol% of PA (Fig 2A). PAB-TP did not bind to the vesicles containing PE, PG, or ganglioside GM3, but exhibited a weak cross-reactivity with vesicles containing PS or PI (Fig 2B).


Development of a Novel Tetravalent Synthetic Peptide That Binds to Phosphatidic Acid.

Ogawa R, Nagao K, Taniuchi K, Tsuchiya M, Kato U, Hara Y, Inaba T, Kobayashi T, Sasaki Y, Akiyoshi K, Watanabe-Takahashi M, Nishikawa K, Umeda M - PLoS ONE (2015)

Analysis of binding of PAB-TP to phospholipids by the solid phase vesicle-binding assay.(A) Binding of PAB-TP to LUVs composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30), (5:63:2:30), (2:66:2:30), (1:67:2:30), and (0:68:2:30) was examined by the SPVB assay (mean ± SE, n = 3). (B) Binding of PAB-TP to LUVs composed of DOPC, biotin-DOPE, cholesterol, and indicated lipid (58:2:30:10) was examined by the SPVB assay (mean ±SE, n = 3). (A, B) The binding of LUVs composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30) at the concentration of 125 μM to PAB-TP was represented as 100 (arbitrary units).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4493020&req=5

pone.0131668.g002: Analysis of binding of PAB-TP to phospholipids by the solid phase vesicle-binding assay.(A) Binding of PAB-TP to LUVs composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30), (5:63:2:30), (2:66:2:30), (1:67:2:30), and (0:68:2:30) was examined by the SPVB assay (mean ± SE, n = 3). (B) Binding of PAB-TP to LUVs composed of DOPC, biotin-DOPE, cholesterol, and indicated lipid (58:2:30:10) was examined by the SPVB assay (mean ±SE, n = 3). (A, B) The binding of LUVs composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30) at the concentration of 125 μM to PAB-TP was represented as 100 (arbitrary units).
Mentions: In this study, we employed a peptide library composed of tetravalent peptides containing a polylysine core bifurcating at both ends with six randomized residues and fixed methionine, alanine, and arginine at positions, 1, 2, and 6, respectively [29] (Fig 1A). The peptide library was screened for its ability to bind to vesicles containing PA but not to vesicles without PA. As previously reported for screening of the Stx2-binding peptide [29], we determined the relative amino acid preference at each position, and identified four candidate sequence motifs: MAKKMRRRM, MAKHRRTWY, MARWHRHHH, and MAMWHRTWR (Fig 1A). The tetravalent forms of these peptides with the same core structure were synthesized and were evaluated for their ability to bind to vesicles containing PA by the solid phase vesicle-binding (SPVB) assay. In this assay, the peptides coated on the solid phase were incubated with LUVs composed of DOPA, DOPC, biotin-DOPE, and cholesterol at the molar ratio of 10:58:2:30, followed by quantification of the binding of vesicle to the peptide using HRP-streptavidin. As shown in Fig 1B, the tetravalent peptide 3 with the sequence motif of MARWHRHHH bound effectively to the PA-containing vesicle, while only weak or no significant bindings were observed with other peptides. We designated the tetravalent peptide 3 with the sequence of MARWHRHHH as PAB-TP (phosphatidic acid-binding tetravalent peptide) and employed it for further analyses. In the SPVB assay, PAB-TP did not bind to the vesicles without PA, but bound effectively to the vesicles containing as low as 1 mol% of PA (Fig 2A). PAB-TP did not bind to the vesicles containing PE, PG, or ganglioside GM3, but exhibited a weak cross-reactivity with vesicles containing PS or PI (Fig 2B).

Bottom Line: Furthermore, a series of amino acid substitutions at positions 5 to 9 of PAB-TP revealed the involvement of consecutive histidine and arginine residues in recognition of the phosphomonoester head group of PA.Our results demonstrate that the recognition of PA by PAB-TP is achieved by a combination of hydrophobic, electrostatic and hydrogen-bond interactions, and that the tetravalent structure of PAB-TP contributes to the high affinity binding to PA in the membrane.The novel PA-binding tetravalent peptide PAB-TP will provide insight into the molecular mechanism underlying the recognition of PA by PA-binding proteins that are involved in various cellular events.

View Article: PubMed Central - PubMed

Affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto, Japan.

ABSTRACT
We employed a multivalent peptide-library screening technique to identify a peptide motif that binds to phosphatidic acid (PA), but not to other phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). A tetravalent peptide with the sequence motif of MARWHRHHH, designated as PAB-TP (phosphatidic acid-binding tetravalent peptide), was shown to bind as low as 1 mol% of PA in the bilayer membrane composed of PC and cholesterol. Kinetic analysis of the interaction between PAB-TP and the membranes containing 10 mol% of PA showed that PAB-TP associated with PA with a low dissociation constant of KD = 38 ± 5 nM. Coexistence of cholesterol or PE with PA in the membrane enhanced the PAB-TP binding to PA by increasing the ionization of the phosphomonoester head group as well as by changing the microenvironment of PA molecules in the membrane. Amino acid replacement analysis demonstrated that the tryptophan residue at position 4 of PAB-TP was involved in the interaction with PA. Furthermore, a series of amino acid substitutions at positions 5 to 9 of PAB-TP revealed the involvement of consecutive histidine and arginine residues in recognition of the phosphomonoester head group of PA. Our results demonstrate that the recognition of PA by PAB-TP is achieved by a combination of hydrophobic, electrostatic and hydrogen-bond interactions, and that the tetravalent structure of PAB-TP contributes to the high affinity binding to PA in the membrane. The novel PA-binding tetravalent peptide PAB-TP will provide insight into the molecular mechanism underlying the recognition of PA by PA-binding proteins that are involved in various cellular events.

No MeSH data available.


Related in: MedlinePlus