Role of Secondary Structure in Protein−Phospholipid Surface Interactions:  Reconstitution and Denaturation of Apolipoprotein C-I:DMPC Complexes

Abstract

Protein binding to phospholipid surface is commonly mediated by amphipathic α-helices. To understand the role of α-helical structure in protein-lipid interactions, we used discoidal lipoproteins reconstituted from dimyristoyl phosphatidylcholine (DMPC) and human apolipoprotein C-I (apoC-I, 6 kD) or its mutants containing single Pro substitutions along the sequence and differing in their α-helical content in solution (0-48%) and on DMPC (40-75%). Thermal denaturation revealed that lipoprotein stability correlates weakly with the protein helix content: proteins with higher α-helical content on DMPC may form more stable complexes. Lipoprotein reconstitution upon cooling from the heat-denatured state and DMPC clearance studies revealed that protein secondary structure in solution and on DMPC correlates strongly with the maximal temperature of lipoprotein reconstitution: more helical proteins can reconstitute lipoproteins at higher temperatures. Interestingly, at T c =24 °C of the DMPC gel-to-liquid crystal transition, the clearance rate is independent of the protein helical content. Consequently, if the packing defects at the phospholipid surface are readily available (e.g. at the lipid phase boundary), protein insertion into these defects is independent of the secondary structure in solution. However, if hydrophobic defects are limited, protein binding and insertion is aided by other surface-bound proteins and depends on their helical propensity: the larger the propensity the faster the binding and the broader its temperature range. This positive cooperativity in α-helical binding to phospholipid surface, which may result from direct and/or lipid-mediated protein-protein interactions, may be important for lipoprotein metabolism and for protein-membrane binding. KeywordsHigh-density lipoprotein; kinetic stability; lipid fusion; vesicle clearance; atherosclerosis Protein binding to phospholipid surface is an important step in many biological reactions including apolipoprotein exchange among plasma lipoproteins, activation of lipid-regulated enzymes such as phosphoglycerate kinase (PGK) or CTP:phosphocholine cytidylyltransferase (CCT), synuclein aggregation in Parkinson's disease, lipid storage in adypocytes, and binding of antimicrobial peptides to cell surfaces. The binding depends on the structural and physicochemical properties of both proteins and lipids and is facilitated by the hydrophobic defects on the lipid surface that form primary protein binding sites (1-4). The common lipid surface-binding motif found in many proteins, including apolipoproteins, perilipin, synucleins, CTT and PGK, is amphipathic α-helix comprised of 11-mer sequence repeats ((5,6) and references therein). The extended apolar face of such a helix is optimized for interactions with Corresponding author: Dr. Olga Gursky, Department of Physiology and Biophysics, W329, Boston University School of Medicine, 715 Albany Street, Boston MA 02118, E-mail: gursky@bu.edu, Phone: (617) FAX: (617) NIH-PA Author ManuscriptNIH-PA Author Manuscri...

Changes in helical content or net charge of apolipoprotein C-I alter its affinity for lipid/water interfaces

“…Due to the analogous position of Glu19 in the N-terminal helix of apoC-I to glutamate residues in 22-residue amphipathic ␣ -helices of apoA-I that activate lecithin:cholesterol acyltransferase ( 29 ), the apolar face of the N-terminal helix was extended to include Glu19 and Leu8. The phospholipid-bound structure of apoC-I in proline mutagenesis studies is more consistent with the C-terminal helix, spanning residues 34-47 ( 38,39 ) instead of residues 38-52 predicted via NMR ( 18 ).…”

“…Stock solutions were prepared by dissolving lyophilized peptides in 2 mM sodium phosphate buffer, pH 7.4 (standard buffer), to a peptide concentration of 1.0 mg/ml, as measured by Lowry assays. The helical content of aqueous peptides was verifi ed by far-UV circular dichroism (CD) ( 38 ).…”

“…On binding to phospholipid, the average helical content of apoC-I increases to 65-75%. The helical content of apoC-I in solution and on phospholipid can be altered by single Ala or Pro substitutions in either ␣ -helix ( Table 1 ) ( 37,38 ). We speculate that this is unique to small apolipoproteins, as point mutations in single helices of larger proteins would likely have little effect on protein helical content.…”

“…Studies using apoC-I point mutants and discoidal lipoproteins reconstituted from 1, 2-dimyristoylphosphatidylcholine (DMPC) showed that, as the aqueous ␣ -helical content of apoC-I increased, the rate and temperature range of DMPC clearance increased (37)(38)(39)(40)(41). However, these studies were limited by a nonphysiologic interface [DMPC is found only in trace amounts on lipoproteins ( 42,43 )] and the inability to monitor real-time surface modifi cations induced by protein binding.…”

“…ApoC-I is predicted to contain two class A, amphipathic ␣ -helices spanning the residues 7-29 and 34-47 ( 18,37,38 ). Fig.…”