Binding of the Proline-Rich Segment of Myelin Basic Protein to SH3 Domains:  Spectroscopic, Microarray, and Modeling Studies of Ligand Conformation and Effects of Posttranslational Modifications

Abstract

Myelin basic protein (MBP) is a multifunctional protein involved in maintaining the stability and integrity of the myelin sheath by a variety of interactions with membranes and with cytoskeletal and other proteins. A central segment of MBP is highly conserved in mammals and consists of a membrane surface-associated amphipathic alpha-helix, immediately followed by a proline-rich segment that we hypothesize is an SH3 ligand. We show by circular dichroic spectroscopy that this proline-rich segment forms a polyproline type II helix in vitro under physiological conditions and that phosphorylation at a constituent threonyl residue has a stabilizing effect on its conformation. Using SH3 domain microarrays, we observe that the unmodified recombinant murine 18.5 kDa MBP isoform (rmC1 component) binds the following SH3 domains: Yes1 > PSD95 > cortactin = PexD = Abl = Fyn = c-Src = Itk in order of decreasing affinity. A quasi-deiminated form of the protein (rmC8) binds the SH3 domains Yes1 > Fyn > cortactin = c-Src > PexD = Abl. Phosphorylation of rmC1 at 1-2 threonines within the proline-rich segment by mitogen-activated protein kinase in vitro has no effect on the binding specificity to the SH3 domains on the array. An SH3 domain of chicken Fyn is also demonstrated to bind to lipid membrane-associated C1, phosphorylated C1, and rmC8. Molecular docking simulations of the interaction of the putative SH3 ligand of classic MBP with the human Fyn SH3 domain indicate that the strength of the interaction is of the same order of magnitude as with calmodulin and that the molecular recognition and association is mediated by some weak CH...pi interactions between the ligand prolyl residues and the aromatic ones of the SH3 binding site. One such interaction is well-conserved and involves the stacking of an MBP-peptide prolyl and an SH3 domain tryptophanyl residue, as in most other SH3-ligand complexes. Lysyl and arginyl residues in the peptide canonically interact via salt bridges and cation-pi interactions with negatively charged and aromatic residues in the SH3 domain binding site. Posttranslational modifications (phosphorylation or methylation) of the ligand cause noticeable shifts in the conformation of the flexible peptide and its side chains but do not predict any major inhibition of the binding beyond somewhat less favorable interactions for peptides with phosphorylated seryl or threonyl residues.

Classic 18.5- and 21.5-kDa Myelin Basic Protein Isoforms Associate with Cytoskeletal and SH3-Domain Proteins in the Immortalized N19-Oligodendroglial Cell Line Stimulated by Phorbol Ester and IGF-1

“…Previously, MBP was predicted to bind to ZO-1 based on its binding to PSD-95 on an SH3-domain array [16]. Here we show that ZO-1-SH3 and 18.5-kDa MBP (specifically, rmMBP-C1) bind in vitro using a protein pull-down assay (Fig.…”

“…The 18.5-kDa isoform of MBP binds to the SH3-domain of the actin-binding protein cortactin [16]. Here, we immunostained PMA-stimulated N19-OLGs for cortactin, to examine whether it is complexed in the membrane-ruffled structures along with MBP and actin.…”

“…They also undergo combinatorial post-translational modifications including phosphorylation and deimination of arginine, resulting in a number of charge components [11]. They adapt structurally and bind to a variety of different polyanionic proteins such as actin and tubulin, and also have been shown to have other important interactions with calmodulin and SH3-domain proteins such as Fyn kinase, the actin-polymerizing protein cortactin, and PSD-95 (presynaptic density protein 95) [8,[10][11][12][15][16][17][18]. Although PSD-95, a membrane-associated guanylate kinase (MAGUK), is not known to be present in OLGs, its SH3-domain has 57% similarity to that of the MAGUK ZO-1 (zonula occludens 1) [19], which is present in OLGs and in peripheral myelin [20][21][22].…”

Invited review: Caseins and the casein micelle: Their biological functions, structures, and behavior in foods

“…Originally, Pro-rich PP-II sequences were known only as spacer or linker sequences between folded domains in multidomain proteins of known structure. However, as the study of unfolded proteins has expanded, it has become increasingly well recognized that their low sequence specificity is important in a large number of low affinity protein-protein interactions in signal transduction, protein transcription, and other pathways (Sudol, 1998;Macias et al, 2002;Musacchio, 2002;Hicks and Hsu, 2004;Polverini et al, 2008;Zhao et al, 2010). Common domains such as WW (named for a conserved Trp-Trp; Schleinkofer et al, 2004), GYF (conserved Gly-Tyr-Phe; Gu et al, 2005), and Src homology 3 (SH3) can bind and respond to a broad range of Pro-rich sequences.…”

Phosphorylation of U24 from Human Herpes Virus type 6 (HHV‐6) and its potential role in mimicking myelin basic protein (MBP) in multiple sclerosis

“…Here we propose that because of a stretch of sequence similarity between MBP and U24, PRTPPPS, there may be cases of mistaken identity, resulting in essential interactions with and post-translational modifications done on the viral as opposed to the self-protein. This sequence is also a PXXP SH3-target consensus sequence [29], and ultimately there may be more examples [30] in which U24 and MBP compete for molecular recognition based on their identity, perhaps additionally contributing to the destabilization of the myelin sheath and the pathogenesis of multiple sclerosis. Although there are clearly many membrane-associated phosphorylation targets in myelin (e.g.…”