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.
Highlights
Experimental ProceduresGeneral Methods—All solvents used for synthesis were distilled under a nitrogen atmosphere prior to use, and all materials were obtained from Sigma unless specified otherwise
Ever since the Framingham heart study revealed the inverse relationship between high density lipoprotein (HDL) cholesterol levels and the risk of coronary artery disease, the structure and biological functions of HDL have been under intense scrutiny [1,2,3,4,5,6]
We showed that HDL forms a functional ternary complex with paraoxonase 1 (PON1) and myeloperoxidase [31], an oxidant-generating enzyme linked to development of atherosclerosis [42,43,44,45,46,47,48]
Summary
General Methods—All solvents used for synthesis were distilled under a nitrogen atmosphere prior to use, and all materials were obtained from Sigma unless specified otherwise. (ii) The cross-linking between pac-PC and PON1/rHDL with pac-PC (human apoA-I concentration of 70 M) in 0.5 ml of 50 mM PBS buffer at pH 7.0 was incubated with recombinant Histagged PON1 (rPON1, 30 M, in 0.5 ml of 50 mM Tris buffer with 1 mM CaCl2, pH 7.0) that was expressed and purified from Escherichia coli, as described previously [60]. Photoactivatable lipid probe (pac-PC) cross-linked and biotin-tagged proteins (apoA-I or rPON1) were incubated with neutravidin beads (200 l, 1:1 slurry, pre-washed and equilibrated in PBS buffer) for 3 h at room temperature. To determine the Kd value between distinct rPON1 and rHDL, rPON1 ranging from 500 to 2000 nM were prepared in PON1 activity buffer (50 mM Tris, 50 mM NaCl, 1 mM CaCl2, pH 8.0) and flowed over the surface of the sensor chip at a rate of 20 l/min. Aliquots were taken at various time points, and the arylesterase activity was examined as described previously [39]
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