Apolipoprotein E: structure-function relationships.
This chapter focuses on the role of apoE in lipoprotein metabolism and the most completely described function of the protein. Apolipoprotein E (apoE) is one of. Results: Apolipoprotein A-I and E mimetic peptides suppress Further exploring structure-function relationships, we studied the alternate apoA-I .. to date, being responsive to several ligands of varying structure and origin. Structure-function relationships of apolipoprotein A-I: a flexible protein with . of evidence to date favors the belt or hairpin belt models over the picket fence.
We identify suppression of chemokine-directed chemotaxis as a mechanism underlying the apolipoprotein effect. Pursuing the possibility that L-4F might suppress chemotaxis through heterologous desensitization i.
Further exploring structure-function relationships, we studied an alternate apoA-I mimetic peptide, LpA, a bihelical version of L-4F with two Leu-Phe substitutions Taken together, we report that apolipoprotein mimetic peptides are novel PMN and monocyte chemotactic agents that possess complex structure-function activity relationships to multiple receptors.
ApoA-I was purified as previously described Fura-2 was from Invitrogen. D-amino acid version of 37pA were synthesized by a solid-phase procedure as previously reported Mice Female mice 8—12 weeks old, weighing 15—22 g were used in all experiments. Bronchoalveolar Lavage Fluid Collection and Analysis Bronchoalveolar lavage fluid was collected immediately following sacrifice, and total leukocyte and differential counts were performed, as previously described Cytokines were quantified by a multiplex assay Bio-Plex, Bio-Rad per the manufacturer's instructions.
Neutrophil and Monocyte Isolation Mature murine bone marrow PMNs were isolated from mouse femurs and tibias by discontinuous Percoll gradient centrifugation, as previously described Human peripheral blood monocytes were isolated from buffy coats NIH Clinical Center, Transfusion Medicine Department, Bethesda, MD enriched for mononuclear cells by using an iso-osmotic Percoll gradient, and neutrophils by dextran sedimentation of the buffy coat, both as previously described Results are obtained from triplicate samples and are representative of at least 5 experiments.
The two molecules of apoA-I are shown in different colors with intermittent amino acids numbered in boxes and circles. The N-terminal 44 amino acids are proposed to double back to interact with the C-terminal region of the opposing apoA-I molecule on the disc edge.
With the question of helical orientation addressed, attention focused on determining the spatial relationships between molecules of apoA-I on a disc. Computer analysis predicted a registry between the monomers with similar intermolecular salt bridge connections implied by the Borhani crystal structure. An alternative belt model 11 proposed two possible hairpin orientations, where each molecule interacts with both leaflets after a turn.
This allows salt bridge interactions similar to the double belt, although they are intramolecular in the hairpin. The similarity in potential salt bridge patterns between the hairpin and belt models initially led to some difficulty in defining tertiary relationships.
Other studies using similar methodologies suggested mixtures of head-to-head and head-to-tail hairpins More recently, we applied a cross-linking approach to discoidal HDL particles containing two molecules of apoA-I The result was nine intermolecular distance constraints that strongly supported the double belt model in particles with two molecules of apoA-I.
The functional implications for such a conformational shift are intriguing and open the possibility that different rotamers may interact with distinct plasma factors to modulate HDL metabolism. Interestingly, the authors interpreted a third cross-link to indicate that the apoA-I N terminus forms a hairpin turn centered around residue 44 in order to interact with the C-terminal portion of the second apoA-I molecule that has also doubled back on itself Fig.
This organization differs in that it no longer forms a closed loop encapsulating the lipid bilayer. Instead, the two molecules form an open-ended structure reminiscent of a pair of earmuffs. This twist on the double-belt model may have implications for the addition of other apolipoproteins to HDL particles.
Apolipoprotein E: structure-function relationships.
Taking a different approach, Martin et al. The data clearly showed that apoA-I forms an extended antiparallel conformation with a registry consistent with the double belt model.
In the same study, electron paramagnetic resonance experiments further suggested that region — may be a flexible loop that may modulate changes in particle size, an observation consistent with previous proposals of a hinge domain near this sequence.
However, it is also clear that there is potential for significant conformational adaptability within this general framework, particularly at the termini and in the middle of the apoA-I molecules.
Although the picket fence model appears to have fallen out of favor, the hairpin models remain worthy of continued consideration.
The Structure of Apolipoprotein A-I in High Density Lipoproteins
If one assumes that there is room for only two helices lying parallel around the edge of a disc, then the addition of a third molecule of apoA-I to these discs requires that it adopt a distinct conformation from the first two.
A conceptually straightforward way to do this is to invoke the hairpin organization for at least one apoA-I molecule in discs that contain three apoA-I polypeptides More work will be required to tackle this intriguing issue. The apoA-I helices likely float among the phospholipid molecules with their hydrophobic faces penetrating past the phosphate group to interact with the acyl chains With no disc edge, the protein could be envisioned to spread out across the particle surface.
Thus, the protein-protein contacts in spheres may be profoundly different from those in the discs. Unfortunately, much less is known about the conformation of apoA-I in spherical particles versus discs, even though they make up the vast majority of HDL found in plasma.