pubs.acs.org/Biochemistry Published on Web 09/10/2010 r 2010 American Chemical Society Biochemistry 2010, 49, 8955–8966 8955 DOI: 10.1021/bi100988p Secondary Structure and Solvent Accessibility of a Calmodulin-Binding C-Terminal Segment of Membrane-Associated Myelin Basic Protein † Lopamudra Homchaudhuri, ‡ Miguel De Avila, § Stina B. Nilsson, § Kyrylo Bessonov, § Graham S. T. Smith, § Vladimir V. Bamm, § Abdiwahab A. Musse, §, ) George Harauz, § and Joan M. Boggs* ,‡ ‡ Department of Molecular Structure and Function, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada, and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada, and § Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada N1G 2W1 ) Present address: Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, CA 90095-1737. Received June 18, 2010; Revised Manuscript Received September 8, 2010 ABSTRACT: Myelin basic protein (MBP), specifically the 18.5 kDa isoform, is a peripheral membrane protein and a major component of mammalian central nervous system myelin. It is an intrinsically disordered and multifunctional protein that binds cytoskeletal and other cytosolic proteins to a membrane surface and thereby acquires ordered structure. These associations are modulated by post-translational modifications of MBP, as well as by interactions of MBP with Ca 2þ -calmodulin (CaM). Enzymatic deimination of usually six arginine residues to citrulline results in a decrease in the net positive charge of the protein from 19 to e13. This deiminated form is found in greater amounts in normal children and in adult patients with the demyelinating disease multiple sclerosis. In this paper, we examine the secondary structure of a calmodulin-binding domain, residues A141-L154, when associated with a lipid bilayer in recombinant murine 18.5 kDa forms rmC1 (unmodified) and rmC8 (pseudodeiminated). We demonstrate here by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy that the Y142-L154 segment in membrane-associated rmC1 forms an amphipathic R-helix, with high accessibility to O 2 and low accessibility to NiEDDA. In membrane-associated rmC8, this segment assumed a structure distorted from an R-helix. Spin-labeled residues in rmC1 in solution were more immobilized on binding Ca 2þ -CaM than those in rmC8. Furthermore, rmC8 was dissociated more readily from a lipid bilayer by Ca 2þ -CaM than was rmC1. These results confirm both a predicted induced ordering upon membrane association in a specific segment of 18.5 kDa MBP, and that this segment is a CaM-binding site, with both interactions weakened by deimination of residues outside of this segment. The deiminated form would be more susceptible to regulation of its membrane binding functions by Ca 2þ -CaM than the unmodified form. Myelin basic protein (MBP) 1 is a highly basic peripheral membrane protein and a major component of human central nervous system (CNS) myelin. It is the second most abundant protein in CNS myelin after proteolipid protein and accounts for 30% of the total protein content in myelin (1). The classic 18.5 kDa isoform of MBP, the major isoform in humans, is highly conserved in sequence in mammals (2) and occurs in the cytoplasmic leaflet of the oligodendrocyte, where it mediates myelin compaction by bringing together apposed cytoplasmic surfaces rich in acidic lipids (3, 4). In shiverer mutant mice that carry deletions in the MBP gene that prevent expression of MBP, compact myelin in the CNS is lacking (4, 5). MBP is an intrinsically disordered protein that undergoes extensive post-translational modifications (6-10). Like other intrinsically disordered proteins, MBP assumes some ordered structure upon interacting with its binding partners, e.g., upon interacting with lipid membranes or proteins (11), albeit with a good deal of polymorphism, or “fuzziness”, remaining (12). It has been shown by us previously, using the combination of site- directed spin labeling and electron paramagnetic resonance spectroscopy (SDSL-EPR), that a central segment of the protein (V83-T92) of recombinant murine 18.5 kDa MBP (rmMBP, with murine 18.5 kDa sequence numbering used henceforth unless otherwise indicated) forms an amphipathic R-helix upon association with lipid bilayers whose composition mimics that of the cytoplasmic leaflet of myelin (13). This structure was subse- quently confirmed by solution NMR spectroscopy of a synthetic † This work has been supported primarily by a grant to J.M.B. and G.H. from the Canadian Institutes of Health Research (CIHR MOP 86483) and a grant to G.H. from the Natural Sciences and Engineering Research Council of Canada (NSERC, Discovery Grant RG121541). L.H. and V.V.B. were the recipients of Postdoctoral Fellowships and A.A.M. and G.S.T.S. of Doctoral Studentships from the Multiple Sclerosis Society of Canada. *To whom correspondence should be addressed: Department of Molecular Structure and Function, Research Institute, Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G 1X8. Phone: (416) 813-5919. Fax: (416) 813-5022. E-mail: jmboggs@sickkids.ca. 1 Abbreviations: C1, most cationic charge component MBP; C8, least cationic charge component MBP; CaM, Ca 2þ -calmodulin; CNS, central nervous system; Cyt-LUV, LUV with the lipid composition of the cytoplasmic monolayer of myelin; DMPC, dimyristoylphosphatidylcho- line; DPPH, 1,1 0 -diphenyl-2-picrylhydrazyl; EAE, experimental allergic encephalomyelitis; EPR, electron paramagnetic resonance; HEPES, N-(2- hydroxyethyl)piperazine-N 0 -2-ethanesulfonic acid; LUV, large unilamellar vesicle; MBP, myelin basic protein; MS, multiple sclerosis; MTS-SL, [1-oxyl-2,2,5,5-tetramethyl-D-pyrroline-3-methyl] methanethiosulfonate spin-label; n-doxyl-PC, 1-palmitoyl-2-stearoyl(n-doxyl)-sn-glycero-3-phos- phocholine; NiEDDA, nickel ethylenediaminediacetate; PAD2, peptidy- larginine deiminase 2; PC, phosphatidylcholine; PE, phosphatidylethano- lamine; PI, phosphatidylinositol; PS, phosphatidylserine; rmC1, recombi- nant murine C1 18.5 kDa MBP variant; rmC8, recombinant murine C8 18.5 kDa MBP variant; rmMBP, recombinant murine 18.5 kDa MBP; SDSL, site-directed spin labeling; SM, sphingomyelin; TEMPO, 2,2,6,6- tetramethylpiperidine-1-oxyl; TEMPO-PC, 1,2-dipalmitoyl-sn-glycero-3- phospho(TEMPO)choline; TPX, tetramethylpentene polymer.