Membrane-anchoring and Charge Effects in the Interaction of Myelin Basic Protein with Lipid Bilayers Studied by Site-directed Spin Labeling

Myelin basic protein (MBP) maintains the compaction of the myelin sheath in the central nervous system by anchoring the cytoplasmic face of the two apposing bilayers and may also play a role in signal transduction. Site-directed spin labeling was done at eight matching sites in each of two recombinant murine MBPs, qC1 (charge +19) and qC8 charge (+13), which, respectively, emulate the native form of the protein (C1) and a post-translationally modified form (C8) that is increased in multiple sclerosis. When interacting with large unilamellar vesicles, most spin-labeled sites in qC8 were more mobile than those in qC1. Depth measurement via continuous wave power saturation indicated that the N-terminal and C-terminal sites in qC1 were located below the plane of the phospholipid headgroups. In qC8, the C-terminal domain dissociated from the membrane, suggesting a means by which the exposure of natural C8 to cytosolic enzymes and ligands might increase in vivo in multiple sclerosis. The importance of two Phe-Phe pairs in MBP to its interactions with lipids was investigated by separately mutating each pair to Ala-Ala. The mobility at F42A/F43A and especially F86A/F87A increased significantly. Depth measurements and helical wheel analysis indicated that the Phe-86/Phe-87 region could form a surface-seeking amphipathic α-helix. Myelin basic protein (MBP) maintains the compaction of the myelin sheath in the central nervous system by anchoring the cytoplasmic face of the two apposing bilayers and may also play a role in signal transduction. Site-directed spin labeling was done at eight matching sites in each of two recombinant murine MBPs, qC1 (charge +19) and qC8 charge (+13), which, respectively, emulate the native form of the protein (C1) and a post-translationally modified form (C8) that is increased in multiple sclerosis. When interacting with large unilamellar vesicles, most spin-labeled sites in qC8 were more mobile than those in qC1. Depth measurement via continuous wave power saturation indicated that the N-terminal and C-terminal sites in qC1 were located below the plane of the phospholipid headgroups. In qC8, the C-terminal domain dissociated from the membrane, suggesting a means by which the exposure of natural C8 to cytosolic enzymes and ligands might increase in vivo in multiple sclerosis. The importance of two Phe-Phe pairs in MBP to its interactions with lipids was investigated by separately mutating each pair to Ala-Ala. The mobility at F42A/F43A and especially F86A/F87A increased significantly. Depth measurements and helical wheel analysis indicated that the Phe-86/Phe-87 region could form a surface-seeking amphipathic α-helix. The 18.5-kDa isoform of myelin basic protein (MBP) 1The abbreviations used are: MBP, myelin basic protein; q, quasi; SDSL, site-directed spin labeling; EPR, electron paramagnetic resonance; NiEDDA, nickel ethylenediaminediacetic acid; MTS-SL, [1-oxyl-2,2,5,5-tetramethyl-d-pyrroline-3-methyl]methanethiosulfonate; NTA, nitrilotriacetic acid; PC, phosphatidylcholine; PS, phosphatidylserine; LUV, large unilamellar vesicle; CW, continuous wave. is a stabilizing factor in the myelin sheath. A major function of MBP is to bind to the apposing cytoplasmic faces of the myelin membrane and maintain compaction for efficient nerve transmission (1Readhead C. Takasashi N. Shine H.D. Saavedra R. Sidman R. Hood L. Ann. N. Y. Acad. Sci. 1990; 605: 280-285Crossref PubMed Scopus (50) Google Scholar, 2Smith R. J. Neurochem. 1992; 59: 1589-1608Crossref PubMed Scopus (103) Google Scholar), but it may also be involved in signal transduction (3Dyer C.A. Philibotte T.M. Wolf M.K. Billings-Gagliardi S. J. Neurosci. Res. 1994; 39: 97-107Crossref PubMed Scopus (60) Google Scholar). Because of a diversity of post-translational modifications, MBP exists as a number of charge isomers denoted C1–C8 with a net positive charge decreasing from +19 to +13 at pH 7.0 (4Wood D.D. Moscarello M.A. Russell W. The Molecular Biology of Multiple Sclerosis. John Wiley & Sons, Inc., New York1997: 37-54Google Scholar, 5Moscarello M.A. Juurlink B.H.J. Devon R.M. Doucette A.J. Nazarali A.J. Schreyer D.J. Verge V.M.K. Cell Biology and Pathology of Myelin: Evolving Biological Concepts and Therapeutic Approaches. Plenum Publishing Corp., New York1997Google Scholar, 6Zand R. Li M.X. Jin X. Lubman D. Biochemistry. 1998; 37: 2441-2449Crossref PubMed Scopus (62) Google Scholar). The C8 component is characterized by the enzymatic deimination of arginine to citrulline. Each conversion results in the loss of one positive charge, and C8 is thus the least basic form of the protein and has a diminished ability to cause adhesion of lipid bilayers (7Bates I.R. Libich D.S. Wood D.D. Moscarello M.A. Harauz G. Protein Expression Purif. 2002; 25: 330-341Crossref PubMed Scopus (53) Google Scholar, 8Boggs J.M. Yip P.M. Rangaraj G. Jo E. Biochemistry. 1997; 36: 5065-5071Crossref PubMed Scopus (70) Google Scholar, 9Wood D.D. Moscarello M.A. J. Biol. Chem. 1989; 264: 5121-5127Abstract Full Text PDF PubMed Google Scholar). Component C8 occurs in greater amounts in patients with the demyelinating disease, multiple sclerosis (9Wood D.D. Moscarello M.A. J. Biol. Chem. 1989; 264: 5121-5127Abstract Full Text PDF PubMed Google Scholar, 10Moscarello M.A. Wood D.D. Ackerley C. Boulias C. J. Clin. Invest. 1994; 94: 146-154Crossref PubMed Scopus (285) Google Scholar). We have previously produced and characterized a recombinant murine 18.5-kDa MBP (11Bates I.R. Matharu P. Ishiyama N. Rochon D. Wood D.D. Polverini E. Moscarello M.A. Viner N.J. Harauz G. Protein Expression Purif. 2000; 20: 285-299Crossref PubMed Scopus (74) Google Scholar). Here, we will denote this protein quasi-C1 (qC1), because it is unmodified post-translationally (with the exception of an LEH6 tag) and emulates the least-modified, most basic charge isomer C1. We have also generated by site-directed mutagenesis a quasi-deiminated form of recombinant murine 18.5-kDa MBP that we call qC8, since it was designed to mimic the less cationic natural form C8. The recombinant qC8 consists of Arg/Lys → Gln substitutions at the same deimination sites in human MBP that predominate in chronic multiple sclerosis and has properties similar to those of natural C8 (7Bates I.R. Libich D.S. Wood D.D. Moscarello M.A. Harauz G. Protein Expression Purif. 2002; 25: 330-341Crossref PubMed Scopus (53) Google Scholar). The net charge of qC1 is +19 at neutral pH, whereas that of qC8 is +13 as for their natural counterparts. In this study, we investigated the electrostatic and hydrophobic components of MBP-lipid interactions by site-directed spin labeling (SDSL) of MBP and electron paramagnetic resonance (EPR) spectroscopy (12Hubbell W.L. Cafiso D.S. Altenbach C. Nat. Struct. Biol. 2000; 7: 735-739Crossref PubMed Scopus (740) Google Scholar, 13Hubbell W.L. Gross A. Langen R. Lietzow M.A. Curr. Opin. Struct. Biol. 1998; 8: 649-656Crossref PubMed Scopus (509) Google Scholar). The technique of SDSL involves replacement of residues at selected sites by cysteines, which are then labeled with a methanethiosulfonate spin label that can be probed by EPR spectroscopy. This approach enabled us to monitor the electrostatic lipid interaction profiles of qC1 and qC8 at numerous specific sites. The importance of hydrophobic interactions was evaluated by spin-labeling sites adjacent to each of the two Phe-Phe pairs and determining the effects of Phe-Phe → Ala-Ala substitution on spin label mobility and accessibility to lipid-soluble O2 and water-soluble nickel ethylenediaminediacetic acid (NiEDDA) as applied by Victor et al. (14Victor K. Jacob J. Cafiso D.S. Biochemistry. 1999; 38: 12527-12536Crossref PubMed Scopus (65) Google Scholar) to the myristoylated alanine-rich C kinase substrate effector region. The technique of SDSL is particularly well suited to MBP, because there are no native cysteinyl residues to be removed prior to mutagenesis and because the EPR spectrum is not affected by light diffraction and immobilization associated with MBP-induced lipid vesicle aggregation. Lipid vesicle aggregation is a powerful mimic of the in vivo function of MBP in the myelin sheath (15Jo E. Boggs J.M. Biochemistry. 1995; 34: PubMed Scopus Google Scholar, R.M. Moscarello M.A. PubMed Scopus Google Scholar), and the of interactions this natural spin label was from This spin label is to as in previously Lietzow M.A. K. W.L. Biochemistry. PubMed Scopus Google Scholar, K. Cafiso D.S. Biochemistry. 1998; 37: PubMed Scopus Google Scholar). The paramagnetic was as previously Altenbach C. W.L. Biol. 2000; Google Scholar). of the were of and were from The were from and were from The was the spin-labeled lipids from with and and and with to the of the lipids were in at of Site-directed of qC1 and qC8 quasi-deiminated of MBP was generated from qC1 by site-directed by and and murine the as previously (7Bates I.R. Libich D.S. Wood D.D. Moscarello M.A. Harauz G. Protein Expression Purif. 2002; 25: 330-341Crossref PubMed Scopus (53) Google Scholar). A of matching substitutions in each of qC1 and qC8 was generated for In in each of the two Phe-Phe pairs was separately by Ala-Ala. In the were generated in and The were generated in and The cysteinyl residues could then be spin-labeled by and of of qC1 and qC8 and spin labeling of recombinant murine 18.5-kDa were done a of a (11Bates I.R. Matharu P. Ishiyama N. Rochon D. Wood D.D. Polverini E. Moscarello M.A. Viner N.J. Harauz G. Protein Expression Purif. 2000; 20: 285-299Crossref PubMed Scopus (74) Google Scholar). of were one pH and and the with the exception of the of to the of The was the and and of the were done this the was with to the of spin labeling with pH were then the The were removed from the and in of spin labeling and a of was in of was to the The of the protein to the and was on a in a at The was the and with to MTS-SL, and the protein was The of each labeled protein was and the were pH and The labeling was for one spectroscopy (11Bates I.R. Matharu P. Ishiyama N. Rochon D. Wood D.D. Polverini E. Moscarello M.A. Viner N.J. Harauz G. Protein Expression Purif. 2000; 20: 285-299Crossref PubMed Scopus (74) Google Scholar, S. Harauz G. Moscarello M.A. Biochemistry. 2000; 39: PubMed Scopus Google Scholar) and was to be not The protein was by the at the (7Bates I.R. Libich D.S. Wood D.D. Moscarello M.A. Harauz G. Protein Expression Purif. 2002; 25: 330-341Crossref PubMed Scopus (53) Google Scholar). The protein were to in the same for EPR spectroscopy. of of the of the lipids were in the to form with a lipid similar to that for the cytoplasmic face of the myelin membrane and (15Jo E. Boggs J.M. Biochemistry. 1995; 34: PubMed Scopus Google Scholar, J. Full Text PDF PubMed Scopus Google Scholar). The was a of and the lipid was in a was in pH and The lipid was then a membrane a The lipid was a Chem. Scopus Google Scholar). EPR EPR spin-labeled qC1 and qC8 were to at a of of protein to of a of This is a less than the in were used to the of the by MBP and the were at to the of the was and the were in The of the was a and the were at for to a The was in the of the EPR of a The EPR were at a power of and a of G. the of MBP, the of the was also probed by EPR spectroscopy and no signal was We that of the MBP with the lipid and that there was no was from the of the is the of the and and are the of the and J.M. Moscarello M.A. J. Biol. 39: PubMed Scopus Google Scholar). the in the mobility of the were the of the greater a mobility of the spin Lietzow M.A. K. W.L. Biochemistry. PubMed Scopus Google Scholar, Cafiso D.S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Depth via The of of spin-labeled sites of of the were from power and analysis were as previously W. Biochemistry. 1997; 36: PubMed Scopus Google Scholar, Biochemistry. 1998; 37: PubMed Scopus Google Scholar) a with a Because of the increased of this the of used for saturation was of of the of it was a C.A. J. Scholar). The was and the power was from to The was by the A of the to is a is a of the of saturation of the resonance is the and is the power for to of its saturation was done on one of with and then with and on of with in the of The was by the in the in the and of and The accessibility was from as previously Altenbach C. W.L. 1992; PubMed Scopus Google Scholar) to The were used to to This the of the spin label in the to the of and O2 the The of on from the membrane was and with the at and The were with the lipids used for at a of in and were as The was done protein as well as two of qC1. The was a of lipid to protein of to that used for the and the was a of qC1 lipid to The were used in with the of the from the of spin-labeled S. Biochemistry. PubMed Scopus Google Scholar) to a for determining the of the in the protein to the The of the to the in was to be on by et al. Altenbach C. W.L. 1992; PubMed Scopus Google Scholar). results have that the of the with the and to M.A. N.J. Victor Cafiso D.S. Biochemistry. 2002; PubMed Scopus Google Scholar), is the from the of the label to the lipid A and the of in and and and C the of the and the This was then used to for the the of MBP in the spin-labeled residues of qC1 and qC8 a spectrum similar to that in were with with no sites that were in a hydrophobic Jacob J. Y. Cafiso D.S. Biochemistry. PubMed Scopus Google Scholar). The of similar were in used to of the protein Lietzow M.A. K. W.L. Biochemistry. PubMed Scopus Google Scholar). The of indicated that there was immobilization at of the sites probed with the The of each were also and to the associated with each is as from we that there is no in mobility for of the spin-labeled sites in The qC8 than the qC1 in the interactions with of qC1 and qC8 spin-labeled at sites and were from the EPR interaction of with the more and the were a in mobility and a and In especially and in a more component was as indicated by an on the in was no of MBP in of the The in a and qC1 and The mobility of most labeled residues in qC8 was greater as can be especially from the at was also a in the more component for qC1 at and the of the of the were and as a of means that the mobility is The spin at sites and in qC8 were more mobile than at the same sites in qC1. the the in qC1 and qC8 was more the the label was to an Arg/Lys → Gln was more mobile in qC8 than in qC1 especially from the loss of component from the spectrum in than from the in in there was no Arg/Lys → Gln substitution in the This that this might be more to its that and lipid a of the Phe-Phe in qC1 and qC8 in the spin at sites and to two Phe-Phe pairs at and Phe-86/Phe-87 were the most in qC1 and qC8 and suggesting that qC1 and qC8 in the The two Phe-Phe pairs were to a hydrophobic component to the of of MBP to lipid the importance of Phe-Phe and Phe-86/Phe-87 were by and in and The Phe-Phe → Ala-Ala substitutions in the of adjacent and and especially for In was the most mobile of of the sites probed as can be from the in In was with the Phe-Phe → Ala-Ala Depth via EPR of and could not on the of of the spin label the lipid but also on that can be in MBP by its interaction with this measurements of of of each spin label the membrane were power saturation W. Biochemistry. 1997; 36: PubMed Scopus Google Scholar, Biochemistry. 1998; 37: PubMed Scopus Google Scholar). the and for of the to and for qC8, and lipid spin label a to the of lipids in the of the the spin-labeled lipids was The accessibility of the lipid was in the than in the lipid used for measurements K. Cafiso D.S. Biochemistry. 1998; 37: PubMed Scopus Google Scholar, M.A. N.J. Victor Cafiso D.S. Biochemistry. 2002; PubMed Scopus Google Scholar, A. A. Biochemistry. 1994; PubMed Scopus Google Scholar). et al. A. A. Biochemistry. 1994; PubMed Scopus Google Scholar, A. Acad. Sci. S. A. 1989; PubMed Scopus Google Scholar) have that the and of the to of the of and more to a of which means that of the spin in MBP be more for than be in a The of the spin S. Biochemistry. PubMed Scopus Google Scholar) was the and a was then to the the function in This function was by et al. M.A. N.J. Victor Cafiso D.S. 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Takasashi N. Shine H.D. Saavedra R. Sidman R. Hood L. Ann. N. Y. Acad. Sci. 1990; 605: 280-285Crossref PubMed Scopus (50) Google Scholar, 2Smith R. J. Neurochem. 1992; 59: 1589-1608Crossref PubMed Scopus (103) Google Scholar) and may also be involved in signal transduction. by are the membrane to the by a MBP (3Dyer C.A. Philibotte T.M. Wolf M.K. Billings-Gagliardi S. J. Neurosci. Res. 1994; 39: 97-107Crossref PubMed Scopus (60) Google Scholar). it is to the specific sites of interaction of MBP with lipid bilayers and to this interaction is by lipid and post-translational of the MBP is to be a protein that is Biochemistry. 2002; PubMed Scopus Google Scholar) and is in but to lipids (11Bates I.R. Matharu P. Ishiyama N. Rochon D. Wood D.D. Polverini E. Moscarello M.A. Viner N.J. Harauz G. Protein Expression Purif. 2000; 20: 285-299Crossref PubMed Scopus (74) Google Scholar, E. A. P. P. J. 1999; PubMed Scopus Google Scholar, I.R. Harauz G. J. Struct. 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In this study, we have used site-directed spin labeling to the of MBP to of to that of the myelin sheath. of MBP with Lipid used a of recombinant murine MBP net charge +19 at pH as well as a less cationic form net charge +13 at pH to the charge affected interactions at sites. spin-labeled sites were to be more mobile in qC8 than in the accessibility measurements indicated that the C in qC8 was the most to the a interaction of qC8 sites with the lipid have that can bind to to and that the protein from the membrane is J.M. Rangaraj G. Biochemistry. 2000; 39: PubMed Scopus Google Scholar). 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PubMed Scopus Google Scholar) and to be that the is to its The hydrophobic of this of MBP it in the domain of surface-seeking A. A. S. G. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). this may be in as indicated by of MBP with which at Phe-Phe The pair is more than Phe-86/Phe-87 (7Bates I.R. Libich D.S. Wood D.D. Moscarello M.A. Harauz G. Protein Expression Purif. 2002; 25: 330-341Crossref PubMed Scopus (53) Google Scholar, L. R. Wood D. Moscarello M.A. Biochemistry. 1999; 38: PubMed Scopus Google Scholar). The power saturation accessibility of that this is the plane of the lipid is located on the of the amphipathic the that it is at the is The power saturation in the in the from the membrane of in the might have that this have more on the in mobility that was factor to is the that the spectrum of the spin-labeled is to L. J. K. W.L. Biochemistry. PubMed Scopus Google Scholar, L. W.L. Sci. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). 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We have used SDSL and EPR to the electrostatic and hydrophobic to MBP interactions with lipid The diminished electrostatic charge in quasi-deiminated MBP increased the mobility of most of the spin the especially at the C which dissociated from the The is that the natural C8 isomer will be more than the isomer to post-translational modifications, protein effects may be in multiple sclerosis. residues the and Phe-86/Phe-87 pairs were in the unmodified and their increased Phe-Phe was to Ala-Ala. The greater was adjacent to Phe-86/Phe-87 in a to be a for an amphipathic that bind to a membrane The spin-labeled was on the of this amphipathic with its of the region Phe-86/Phe-87 will its with lipid

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