title: Identification of Critical Paraoxonase 1 Residues Involved in High Density Lipoprotein Interaction.
abstract: Paraoxonase 1 (PON1) is a high density lipoprotein (HDL)-associated protein with atherosclerosis-protective and systemic anti-oxidant functions. We recently showed that PON1, myeloperoxidase, and HDL bind to one another in vivo forming a functional ternary complex (Huang, Y., Wu, Z., Riwanto, M., Gao, S., Levison, B. S., Gu, X., Fu, X., Wagner, M. A., Besler, C., Gerstenecker, G., Zhang, R., Li, X. M., Didonato, A. J., Gogonea, V., Tang, W. H., et al. (2013) J. Clin. Invest. 123, 3815-3828). However, specific residues on PON1 involved in the HDL-PON1 interaction remain unclear. Unambiguous identification of protein residues involved in docking interactions to lipid surfaces poses considerable methodological challenges. Here we describe a new strategy that uses a novel synthetic photoactivatable and click chemistry-taggable phospholipid probe, which, when incorporated into HDL, was used to identify amino acid residues on PON1 that directly interact with the lipoprotein phospholipid surface. Several specific PON1 residues (Leu-9, Tyr-185, and Tyr-293) were identified through covalent cross-links with the lipid probes using affinity isolation coupled to liquid chromatography with on-line tandem mass spectrometry. Based upon the crystal structure for PON1, the identified residues are all localized in relatively close proximity on the surface of PON1, defining a domain that binds to the HDL lipid surface. Site-specific mutagenesis of the identified PON1 residues (Leu-9, Tyr-185, and Tyr-293), coupled with functional studies, reveals their importance in PON1 binding to HDL and both PON1 catalytic activity and stability. Specifically, the residues identified on PON1 provide important structural insights into the PON1-HDL interaction. More generally, the new photoactivatable and affinity-tagged lipid probe developed herein should prove to be a valuable tool for identifying contact sites supporting protein interactions with lipid interfaces such as found on cell membranes or lipoproteins.
authors.affiliation: From the Department of Cellular and Molecular Medicine, Center for Cardiovascular Diagnostics and Prevention, and.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Gu
authors.affiliation: From the Department of Cellular and Molecular Medicine, Center for Cardiovascular Diagnostics and Prevention, and.
authors.forename: Ying
authors.initials: Y
authors.lastname: Huang
authors.affiliation: From the Department of Cellular and Molecular Medicine, Center for Cardiovascular Diagnostics and Prevention, and.
authors.forename: Bruce S
authors.initials: BS
authors.lastname: Levison
authors.affiliation: the Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115.
authors.forename: Gary
authors.initials: G
authors.lastname: Gerstenecker
authors.affiliation: From the Department of Cellular and Molecular Medicine, Center for Cardiovascular Diagnostics and Prevention, and.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: DiDonato
authors.affiliation: From the Department of Cellular and Molecular Medicine, Center for Cardiovascular Diagnostics and Prevention, and. Electronic address: hazens@ccf.org.
authors.forename: Leah B
authors.initials: LB
authors.lastname: Hazen
authors.affiliation: From the Department of Cellular and Molecular Medicine, Center for Cardiovascular Diagnostics and Prevention, and.
authors.forename: Joonsue
authors.initials: J
authors.lastname: Lee
authors.affiliation: From the Department of Cellular and Molecular Medicine, Center for Cardiovascular Diagnostics and Prevention, and; the Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115.
authors.forename: Valentin
authors.initials: V
authors.lastname: Gogonea
authors.affiliation: From the Department of Cellular and Molecular Medicine, Center for Cardiovascular Diagnostics and Prevention, and.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: DiDonato
authors.affiliation: From the Department of Cellular and Molecular Medicine, Center for Cardiovascular Diagnostics and Prevention, and; Department of Cardiovascular Medicine, Cleveland Clinic, Cleveland, Ohio 44195 and.
authors.forename: Stanley L
authors.initials: SL
authors.lastname: Hazen
mesh_terms: D020816:Amino Acid Motifs; D016632:Apolipoprotein A-I / Q000235:genetics / Q000378:metabolism; D043303:Aryldialkylphosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D018360:Crystallography, X-Ray; D006801:Humans; D008075:Lipoproteins, HDL / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding
chemical_list: D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human
keywords: apolipoprotein A-I; click chemistry; high density lipoprotein (HDL); paraoxonase 1; phospholipid; photoactivatable phospholipid; photoaffinity labeling; pon1; protein-lipid interaction; protein-lipid interactions