ApoB-100 Has a Pentapartite Structure Composed of Three Amphipathic ar-Helical Domains Alternating With Two Amphipathic /3-Strand Domains

Due to the great length of apolipoprotein (apo) B-100, the localization of lipid-associating domains in this protein has been difficult. To address this question, we developed a computer program called LOCATE that searches amino acid sequences to identify potential amphipathic a-helixes and /3-strands by using sets of rules for helix and strand termination. A series of model chimeric protein test datasets were created by tandem linking of amino acid sequences of multiple proteins containing four different secondary structural motifs: motif A (exchangeable plasma apolipoproteins); motif G (globular a-helical proteins); motif C (coiled-coil a-helical proteins); and motif B (0 pleated-sheet proteins). These four test datasets, as well as randomly scrambled sequences of each dataset, were analyzed by LOCATE using increasingly stringent parameters. Using intermediately stringent parameters under which significant numbers of amphipathic helixes were found only in the unscrambled motif A, two dense clusters of putative lipid-associating amphipathic helixes were located precisely in the middle and at the C-terminal end of apoB-100 (a sparse cluster of class G* helixes is located at the N-terminus). The dense clusters are located between residues 2103 through 2560 and 4061 through 4338 and have densities of 2.4 and 2.2 amphipathic helixes per 100 residues, respectively; under P lasma apolipoproteins can be grouped into two general classes, the nonexchangeable apolipoproteins (apolipoprotein [apo] B-100 and apoB48), and the exchangeable apolipoproteins (all other apolipoproteins). The B apolipoproteins, present in chylomicrons, very-low-density lipoprotein, intermediate-density lipoprotein, low-density lipoprotein (LDL), and lipoprotein(a), are highly insoluble in aqueous solutions and thus remain with the lipoprotein particle throughout its metabolism. Because of their size and insoluble nature, it has been difficult to deduce the structural motif(s) responsible for the lipid-associating properties of the B apolipoproteins. On the other Received January 4, 1994; revision accepted Jury 21, 1994. From the Departments of Medicine and Biochemistry and the Atherosclerosis Research Unit, UAB Medical Center, Birmingham, Ala. Correspondence to Jere P. Segrest, MD, PhD, Atherosclerosis Research Unit, Department of Medicine, 630 DREB, No. 3, UAB Medical Center, Birmingham, AL 35294-0012. •LOCATE is available on request. It presently runs only on VAX computers. O 1994 American Heart Association, Inc. these conditions, motif A has a density of 1.4 amphipathic helixes per 100 residues. These two domains correspond closely to the two major apoB-100 lipid-associated domains at residues 2100 through 2700 and 4100 through 4500 using the principle of releasability of tryptic peptides from trypsintreated intact low-density lipoprotein. The classes of amphipathic helixes identified within these two putative lipid-associating domains are considerably more diverse than those found in the exchangeable plasma apolipoproteins. Interestingly, apoB-48 terminates at the TV-terminal edge of the middle cluster. By using a similar strategy for analysis of amphipathic ^-strands, we discovered that the two gap regions between the three amphipathic helix clusters are highly enriched in putative amphipathic /3-strands, while the three amphipathic helical domains are essentially devoid of this putative lipidassociating motif. We propose, therefore, that apoB-100 has a pentapartite structure, NH2-a1-/3,-a2-ft-a3-COOH, with a, representing a globular domain. (ArterioscUr Thromb. 1994; 14:1674-1685.)