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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

Inhibitory effect of soluble head groups and divalent cations on binding of PAB-TP to vesicles containing PA.(A) Binding of PAB-TP to LUVs (31.25 μM total lipids) composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30) in the presence of indicated concentrations of ATP, glycerol 3-phosphate, phosphate, and LUVs composed of DOPA was examined by the SPVB assay. (mean ± SE, n = 3). (B) PAB-TP coated on the solid phase was incubated with MgCl2, CaCl2, CoCl2, NiCl2, ZnCl2, and CuCl2 (0.1, 1, 10 mM). After removal of unbound divalent cations, binding of PAB-TP to LUVs (31.25 μM total lipids) composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30) was examined by the SPVB assay. (mean ± SE, n = 3). The binding of LUVs to PAB-TP in the absence of competitor (A) or divalent cation (B) was represented as 100 (arbitrary units).
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pone.0131668.g004: Inhibitory effect of soluble head groups and divalent cations on binding of PAB-TP to vesicles containing PA.(A) Binding of PAB-TP to LUVs (31.25 μM total lipids) composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30) in the presence of indicated concentrations of ATP, glycerol 3-phosphate, phosphate, and LUVs composed of DOPA was examined by the SPVB assay. (mean ± SE, n = 3). (B) PAB-TP coated on the solid phase was incubated with MgCl2, CaCl2, CoCl2, NiCl2, ZnCl2, and CuCl2 (0.1, 1, 10 mM). After removal of unbound divalent cations, binding of PAB-TP to LUVs (31.25 μM total lipids) composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30) was examined by the SPVB assay. (mean ± SE, n = 3). The binding of LUVs to PAB-TP in the absence of competitor (A) or divalent cation (B) was represented as 100 (arbitrary units).

Mentions: The interaction between PAB-TP with the water-soluble head group of PA and other soluble compounds carrying the phosphomonoester group was examined by inhibition analysis of the SPVB assay. LUV composed of PA effectively inhibited the binding of PA-containing vesicles to PAB-TP, but no significant inhibition was observed with soluble compounds such as glycerol 3-phosphate and ATP (Fig 4A). This result suggests that a secondary interaction between PAB-TP and the hydrophobic fatty acyl chains of the PA molecules is required for the stable complex formation. The presence of divalent cations such as Cu2+, Zn2+, Ni2+, and Co2+ that associate with histidine also effectively inhibited the binding, while neither Ca2+ nor Mg2+ had a significant effect on the binding, suggesting that the histidine residues of PAB-TP may play a critical role in the binding to PA (Fig 4B).


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)

Inhibitory effect of soluble head groups and divalent cations on binding of PAB-TP to vesicles containing PA.(A) Binding of PAB-TP to LUVs (31.25 μM total lipids) composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30) in the presence of indicated concentrations of ATP, glycerol 3-phosphate, phosphate, and LUVs composed of DOPA was examined by the SPVB assay. (mean ± SE, n = 3). (B) PAB-TP coated on the solid phase was incubated with MgCl2, CaCl2, CoCl2, NiCl2, ZnCl2, and CuCl2 (0.1, 1, 10 mM). After removal of unbound divalent cations, binding of PAB-TP to LUVs (31.25 μM total lipids) composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30) was examined by the SPVB assay. (mean ± SE, n = 3). The binding of LUVs to PAB-TP in the absence of competitor (A) or divalent cation (B) was represented as 100 (arbitrary units).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131668.g004: Inhibitory effect of soluble head groups and divalent cations on binding of PAB-TP to vesicles containing PA.(A) Binding of PAB-TP to LUVs (31.25 μM total lipids) composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30) in the presence of indicated concentrations of ATP, glycerol 3-phosphate, phosphate, and LUVs composed of DOPA was examined by the SPVB assay. (mean ± SE, n = 3). (B) PAB-TP coated on the solid phase was incubated with MgCl2, CaCl2, CoCl2, NiCl2, ZnCl2, and CuCl2 (0.1, 1, 10 mM). After removal of unbound divalent cations, binding of PAB-TP to LUVs (31.25 μM total lipids) composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30) was examined by the SPVB assay. (mean ± SE, n = 3). The binding of LUVs to PAB-TP in the absence of competitor (A) or divalent cation (B) was represented as 100 (arbitrary units).
Mentions: The interaction between PAB-TP with the water-soluble head group of PA and other soluble compounds carrying the phosphomonoester group was examined by inhibition analysis of the SPVB assay. LUV composed of PA effectively inhibited the binding of PA-containing vesicles to PAB-TP, but no significant inhibition was observed with soluble compounds such as glycerol 3-phosphate and ATP (Fig 4A). This result suggests that a secondary interaction between PAB-TP and the hydrophobic fatty acyl chains of the PA molecules is required for the stable complex formation. The presence of divalent cations such as Cu2+, Zn2+, Ni2+, and Co2+ that associate with histidine also effectively inhibited the binding, while neither Ca2+ nor Mg2+ had a significant effect on the binding, suggesting that the histidine residues of PAB-TP may play a critical role in the binding to PA (Fig 4B).

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