Structure, stability and lipid binding of consensus sequences CSP-BABA and CSP-ABBA of exchangeable apolipoproteins

ApoA-I, the major apolipoprotein of high density lipoprotein, is a member of the exchangeable apolipoprotein family that plays critical roles in lipid and lipoprotein metabolism and the pathophysiology of dyslipoproteinemias. Arising by gene duplication, this class of apolipoproteins contains multiple tandem repeats of an 11/22 amino acid sequence that is thought to form an amphipathic helical lipid-binding motif. This work presents conformational and thermodynamic stability studies together with dynamics computer simulations of peptides representing this fundmental motif. CSP-BABA and CSP-ABBA are consensus sequence peptides for the exchangeable apolipoproteins with different combinations of the 11-amino acid sequences, "A" (PLAEELRARLR) and "B" (AQLEELRERLG). Far-UV CD spectroscopy demonstrated that the major secondary structure of the two peptides is alpha-helix that can be induced up to 55% and 45% respectively by increasing the peptide concentration, consistent with self-association. Addition of phospholipid (DMPC) or detergent (n-Octyl-beta-D-glucopyranoside) also results in alpha-helix formation for CSP-ABBA, but not for CSP-BABA. CSP-BABA displayed more cooperative unfolding than CSP-ABBA over the temperature range 5-95°C with a 20°C higher T1/2 for CSP-BABA vs. CSP-ABBA. Differential scanning calorimetry studies showed that CSP-BABA had narrower transitions with DeltaHv (∼70-90kcal/mol) double that of CSP-ABBA (∼20-50kcal/mol), and Tm ∼10°C higher. Multiple oligomeric states were confirmed for both peptides by gel electrophoresis after cross-linking by glutaraldehyde. Both peptides form homogeneous "nascent" HDL-like discoidal structures at low DMPC:peptide ratios (< 3:1) but form heterogeneous vesicle-like structures at high ratios (> 5:1). Molecular dynamic simulations of peptides with different combinations of sequences "A" and "B" confirmed that the sequence "AB" (PLAEELRARLRAQLEELRERLG), is a strong amphipathic alpha-helix-forming motif that is readily disrupted by proline in short peptides. Simulation of the folding of CSP-ABAB demonstrated that a stable helix-loop-helix structure is formed, stabilized by salt-bridge interactions consistent with crystallographic and other previous studies. Similar simulations of native sequence peptides showed that [1-44]apoA-I (no sequence homology to CSP-AB) is totally unfolded; [142-186]apoA-I (homology to CSP-ABAB) had ∼19% alpha helical content; while [198-243]apoA-I (homology to CSP-BAAB) maintained high alpha-helical content. The different structure and stability displayed by these peptides suggested different roles of each part of apoA-I in its function.