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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

Identification of amino acid residues involved in the PA-binding.(A, B) Binding of alanine mutants of PAB-TP to LUVs (125 μM total lipids) composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30) was examined by the SPVB assay. Substituted amino acids were indicated by outline characters. (mean ± SE, n = 3). The binding of LUVs to PAB-TP (MARWHRHHH) was represented as 100 (arbitrary units).
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pone.0131668.g005: Identification of amino acid residues involved in the PA-binding.(A, B) Binding of alanine mutants of PAB-TP to LUVs (125 μM total lipids) composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30) was examined by the SPVB assay. Substituted amino acids were indicated by outline characters. (mean ± SE, n = 3). The binding of LUVs to PAB-TP (MARWHRHHH) was represented as 100 (arbitrary units).

Mentions: To evaluate the possible involvement of each amino acid residue in the binding, the effect of alanine replacement of PAB-TP on its binding to PA-containing vesicles was examined. Alanine replacement of the Trp residue at position 4 significantly reduced the binding, while no significant change in the binding was observed with the replacement of other residues (Fig 5A). Since the binding was not significantly affected by the alanine replacements of a single His or Arg residue, we next examined the binding of double replacement mutants to PA-containing vesicles. As shown in Fig 5B, double replacement of the consecutive His and Arg residues (H5/R6 and R6/H7) significantly diminished the binding, while those of His residues (H5/H7) had no significant effect. Triple replacement of either H5/R6/H7 or R6/H7/H8 also strongly affected the binding. The wavelength of maximum fluorescence of triple replacement mutants (excitation 280 nm) in the absence of lipids is comparable to that of PAB-TP, indicating that abolishment of binding properties in triple replacement mutants was not due to the self-segregation of the peptide chains (data not shown) (see below). These results suggest that the Trp residue and the couplings of consecutive Arg and His residues play a critical role in the association of TAB-TP to the PA molecules in the membrane.


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)

Identification of amino acid residues involved in the PA-binding.(A, B) Binding of alanine mutants of PAB-TP to LUVs (125 μM total lipids) composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30) was examined by the SPVB assay. Substituted amino acids were indicated by outline characters. (mean ± SE, n = 3). The binding of LUVs to PAB-TP (MARWHRHHH) was represented as 100 (arbitrary units).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131668.g005: Identification of amino acid residues involved in the PA-binding.(A, B) Binding of alanine mutants of PAB-TP to LUVs (125 μM total lipids) composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30) was examined by the SPVB assay. Substituted amino acids were indicated by outline characters. (mean ± SE, n = 3). The binding of LUVs to PAB-TP (MARWHRHHH) was represented as 100 (arbitrary units).
Mentions: To evaluate the possible involvement of each amino acid residue in the binding, the effect of alanine replacement of PAB-TP on its binding to PA-containing vesicles was examined. Alanine replacement of the Trp residue at position 4 significantly reduced the binding, while no significant change in the binding was observed with the replacement of other residues (Fig 5A). Since the binding was not significantly affected by the alanine replacements of a single His or Arg residue, we next examined the binding of double replacement mutants to PA-containing vesicles. As shown in Fig 5B, double replacement of the consecutive His and Arg residues (H5/R6 and R6/H7) significantly diminished the binding, while those of His residues (H5/H7) had no significant effect. Triple replacement of either H5/R6/H7 or R6/H7/H8 also strongly affected the binding. The wavelength of maximum fluorescence of triple replacement mutants (excitation 280 nm) in the absence of lipids is comparable to that of PAB-TP, indicating that abolishment of binding properties in triple replacement mutants was not due to the self-segregation of the peptide chains (data not shown) (see below). These results suggest that the Trp residue and the couplings of consecutive Arg and His residues play a critical role in the association of TAB-TP to the PA molecules in the membrane.

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