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The Structure of Bovine Brain Myelin Proteolipid and Its Organization In Proc. Nati. Acad. Sci. USA Vol. 81, pp. 2912-2916, May 1984 Neurobiology The structure of bovine brain myelin proteolipid and its organization in myelin (myelin basic protein/membrane proteins/protein folding/amino acid sequence/secondary structure prediction) RICHARD A. LAURSEN*, MOHAMMED SAMIULLAH*, AND MARJORIE B. LEESt *Department of Chemistry, Boston University, Boston, MA 02215; and tEunice Kennedy Shriver Center, Waltham, MA 02254 Communicated by Francis 0. Schmitt, December 27, 1983 ABSTRACT A model, based on amino acid sequence data, in the proteolipid are defined in terms of polarity, proposed is proposed for the organization of the myelin proteolipid in orientation in the membrane, and sequence homology. Thus, myelin membrane. The model has three distinctive features: Ti (residues 59-90), T2 (residues 151-177) and T3 (residues three trans-membrane segments that traverse the lipid bilayer, 238-267) are hydrophobic trans-membrane segments that two cis-membrane domains that enter and exit the same side of span the bilayer; Cl (residues 1-35) and C3 (residues 206- the membrane, and a highly charged segment resembling my- 216) contain hydrophobic cis-membrane segments that enter elin basic protein on the cytoplasmic side of the membrane. It and exit the same side of the bilayer; El, E2, and E3 are is proposed that the cis-membrane domain(s) can promote the intervening extra-membrane sequences that contain nearly formation and stabilization of the multilamellar myelin struc- all of the charged amino acids (Figs. 1 and 2). The segments ture by hydrophobic interaction with the apposite bilayer C1', C2, and C3' are designated as cis-membrane segments across the extracellular space. based on homology (10) but probably are located outside of the lipid bilayer. As we have already reported (10), the pro- In the central nervous system, myelin is formed as an ex- teolipid shows a striking degree of internal sequence homol- tended, modified oligodendroglial plasma membrane that ogy, with various domains (cis, trans, or extra) mutually ho- spirals around the axon to form a multilamellar structure (1). mologous within a domain type-e.g., Ti, T2, and T3 are During maturation, the membranes become compacted with homologous. a close apposition of both the internal and external faces of Orientation of Polar Domains in the Membrane. Assuming the membrane. The relative thicknesses of the bilayer, cyto- that the proteolipid polypeptide chain is threaded through plasmic, and extracellular spaces are 47 A, 30 A, and 30 A, the membrane in a regular fashion (Fig. 1), we propose that respectively (2, 3). the basic segments, El + C2 and E3, are located on the cyto- Although the myelin sheath is characterized by a relatively plasmic face of the membrane and that the other polar seg- high proportion of lipid (70-80%), it is the myelin proteins ments, Cl', E2, and C3', are on the extracellular side. Near- that provide specificity and perform an important, albeit ill- ly all of the trans-membrane proteins studied so far have defined role in the maintenance of myelin structure. The pre- clusters of predominantly positively charged amino acids on dominant proteins are the water-soluble extrinsic myelin ba- the cytoplasmic side of the membrane (14). It has been pro- sic protein (Mr 18,000), characterized by a high proportion of posed that these charged groups interact electrostatically basic and other polar amino acids (4, 5), and the hydropho- with the negatively charged inner face of the membrane dur- bic, chloroform/methanol-soluble proteolipid (Mr 30,000) (4, ing insertion of the protein into the membrane, thereby an- 6). The basic protein is well-characterized (4, 5); however, choring the cluster and preventing it from passing through the proteolipid has resisted structure elucidation, and only (14). A second argument is that El and E3 are remarkably recently has its amino acid sequence been determined (7-9). similar, in terms of amino acid sequence (36% homology) In this article we propose a model (Fig. 1) for bovine brain and predicted secondary structure to the myelin basic pro- myelin proteolipid, based on amino sequence data and analo- tein, which appears to be located entirely on the cytoplasmic gy with other proteins, and suggest how the proteolipid may side of the membrane (5, 15, 16). Finally, there is evidence help to stabilize the myelin sheath. (see below) that Cl and the region E2 + C3 + C3' contain disulfide bonds and are therefore likely to be located in and on the outer leaflet of the cell membrane bilayer (17). RESULTS AND DISCUSSION Oxidation State of Cysteine Residues. Myelin proteolipid General Structural Features. The most notable characteris- contains 14 half-cystine residues, the oxidation states of tic of the proteolipid sequence (Fig. 2) is the clustering of which have not been completely established. Half of the cys- hydrophobic amino acids into distinct domains. A plot of teine residues are found in the polar and half in the hydro- amino acid hydropathy (11) (Fig. 3) shows four hydrophobic phobic domains: 6 in Cl, 1 in T2, 3 in El + C2, and 4 in E2 + domains of about 30 amino acids each, which alternate with C3 + C3'. We have proposed that El + C2 is located on the polar segments containing most of the charged and neutral cytoplasmic side of the membrane Cl', E2, and C3' are on hydrophilic residues. The polar domains also contain all of the exterior. Since intracellular proteins containing cysteine the predicted ,B-turns (12), suggesting that these regions are generally exist in the thiol form because of the reducing envi- extensively folded. These features suggest a model, such as ronment of the cell (17), we predict that the three cysteine has been proposed for bacteriorhodopsin (13) and other in- residues in El + C2, as well as the single cysteine in T2 trinsic membrane proteins (14), wherein the polypeptide (which presumably is isolated in the bilayer), are in the re- chain passes repeatedly through the lipid bilayer, the hydro- duced state. Conversely, the 4 cysteine residues in E2 + C3 phobic regions being embedded in the lipid and the polar do- + C3' on the external face probably exist as disulfides, in mains exposed on the external and internal faces ofthe mem- analogy with the majority of extracellular proteins (17). We brane (Fig. 1). For purposes of discussion, various domains further propose (see below) that the 6 cysteine residues in Cl occur as disulfides. Only 25-30% of the cysteine residues in even under The publication costs of this article were defrayed in part by page charge proteolipid can be carboxymethylated, forcing payment. This article must therefore be hereby marked "advertisement" conditions (18). This suggests that 4 of the 14 cysteine resi- in accordance with 18 U.S.C. §1734 solely to indicate this fact. dues are in the thiol form. The sequence studies of Jolles et 2912 Downloaded by guest on September 23, 2021 Neurobiology: Laursen et aL Proc. NatL. AcadJ Sci USA 81 (1984) 2913 MEMBRANE EXTRACELLULAR SPACE 30A 47A 30A RESIDUE NUMBER SPACE CYTOPLASMIC FIG. 3. Predicted location of hydrophobic domains and of MEMBRANE turns in myelin proteolipid. Upper curve: the hydropathic constants of Kyte and Doolittle (11) were averaged over nine amino acids; FIG. 1. Hypothetical model for myelin proteolipid in a mem- hydrophobic domains are located above the dashed line. Lower brane lipid bilayer. Ti, T2, and T3 are homologous, a-helical trans- curve: relative probability of any four adjacent residues being a turn membrane segments; Cl and C3 are homologous cis-membrane seg- is plotted on the first residue. ments; and El, E2, E3, Cl', and C3' are located outside of the bi- layer. See text and ref. 10 for further description. Charged residues and 238-267 (30 residues). They contain no charged amino are indicated by + or -, and cysteine/cystine is indicated by e. E2 acids (except possibly His-65 in T1) and only one cysteine contains a covalently linked fatty acid chain, indicated by a zigzag residue (in T2). Although secondary structure calculations line. tend to predict ,3-sheet structure, we propose that T1, T2, and T3 are a-helical segments that span the lipid bilayer, in al. (19) suggest that at least 2 of the cysteine residues (at analogy with bacteriorhodopsin (13) and other membrane positions 6 and 9) in C1 are in the oxidized form and, in our proteins (21). The failure of hydrophobic domains in mem- own sequence studies (20), we found that the NH2-terminal brane proteins to follow secondary structure preduction tryptic peptide (Gly-1 to Arg-8) could only be isolated after rules has also been noted by Argos et al. (21). Consideration cleavage of the disulfide bonds, indicating that Cys-5 and/or of geometry also favors the helical structure. Given the stan- Cys-6 are crosslinked to other portions of the polypeptide dard a-helix parameter of 1.5 A pitch height per residue, one chain. In addition, our studies (7) showed that cysteine resi- can calculate that 31 residues would be needed to span the dues 183, 200, 219, and 227 are crosslinked within the cleav- 47-A thick myelin membrane bilayer. This is close to the seg- age fragment comprising residues 181-276, indicating that ment lengths of T1, T2, and T3. Finally, thermodynamic the cysteines in the segment E2 + C3 + C3' are not cross- considerations favor a-helical structures, wherein all poten- linked to other domains. tial hydrogen-bonding sites on the polypeptide backbone are Hydrophobic Trans-Membrane Domains T1, T2, and T3. internally bonded, in hydrophobic environments such as lip- The hydrophobic segments T1, T2, and T3 comprise, more ids (22). In a ,-sheet, most of these sites remain exposed, or less, residues 59-90 (32 residues), 151-177 (27 residues), resulting in a relatively high energy state.
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