Vladimir V. Bamm

Recognition Pliability Is Coupled to Structural Heterogeneity: A Calmodulin Intrinsically Disordered Binding Region Complex

Protein interactions within regulatory networks should adapt in a spatiotemporal-dependent dynamic environment, in order to process and respond to diverse and versatile cellular signals. However, the principles governing recognition pliability in protein complexes are not well understood. We have investigated a region of the intrinsically disordered protein myelin basic protein (MBP(145-165)) that interacts with calmodulin, but that also promiscuously binds other biomolecules (membranes, modifying enzymes). To characterize this interaction, we implemented an NMR spectroscopic approach that calculates, for each conformation of the complex, the maximum occurrence based on recorded pseudocontact shifts and residual dipolar couplings. We found that the MBP(145-165)-calmodulin interaction is characterized by structural heterogeneity. Quantitative comparative analysis indicated that distinct conformational landscapes of structural heterogeneity are sampled for different calmodulin-target complexes. Such structural heterogeneity in protein complexes could potentially explain the way that transient and promiscuous protein interactions are optimized and tuned in complex regulatory networks.

Divalent cations induce a compaction of intrinsically disordered myelin basic protein.

Central nervous system myelin is a dynamic entity arising from membrane processes extended from oligodendrocytes, which form a tightly-wrapped multilamellar structure around neurons. In mature myelin, the predominant splice isoform of classic MBP is 18.5kDa. In solution, MBP is an extended, intrinsically disordered protein with a large effective protein surface for myriad interactions, and possesses transient and/or induced ordered secondary structure elements for molecular association or recognition. Here, we show by nanopore analysis that the divalent cations copper and zinc induce a compaction of the extended protein in vitro, suggestive of a tertiary conformation that may reflect its arrangement in myelin.

Solid-State NMR Spectroscopy of Membrane-Associated Myelin Basic Protein—Conformation and Dynamics of an Immunodominant Epitope

Myelin basic protein (MBP) maintains the tight multilamellar compaction of the myelin sheath in the central nervous system through peripheral binding of adjacent lipid bilayers of oligodendrocytes. Myelin instability in multiple sclerosis (MS) is associated with the loss of positive charge in MBP as a result of posttranslational enzymatic deimination. A highly-conserved central membrane-binding fragment (murine N81-PVVHFFKNIVTPRTPPP-S99, identical to human N83-S101) represents a primary immunodominant epitope in MS. Previous low-resolution electron paramagnetic resonance measurements on the V83-T92 fragment, with Cys-mutations and spin-labeling that scanned the epitope, were consistent with it being a membrane-associated amphipathic alpha-helix. Pseudodeimination at several sites throughout the protein, all distal to the central segment, disrupted the alpha-helix at its amino-terminus and exposed it to proteases, representing a potential mechanism in the autoimmune pathogenesis of MS. Here, we have used magic-angle spinning solid-state NMR spectroscopy to characterize more precisely the molecular conformation and dynamics of this central immunodominant epitope of MBP in a lipid milieu, without Cys-substitution. Our solid-state NMR measurements have revealed that the alpha-helix present within the immunodominant epitope is shorter than originally modeled, and is independent of the pseudodeimination, highlighting the importance of the local hydrophobic effects in helix formation and stability. The main effect of pseudodeimination is to cause the cytoplasmic exposure of the fragment, potentially making it more accessible to proteolysis. These results are the first, to our knowledge, to provide atomic-level detail of a membrane-anchoring segment of MBP, and direct evidence of decreased MBP-membrane interaction after posttranslational modification.

Fuzzy complexes of myelin basic protein: NMR spectroscopic investigations of a polymorphic organizational linker of the central nervous system

The classic 18.5 kDa isoform of myelin basic protein (MBP) is central to maintaining the structural homeostasis of the myelin sheath of the central nervous system. It is an intrinsically disordered, promiscuous, multifunctional, peripheral membrane protein, whose conformation adapts to its particular environment. Its study requires the selective and complementary application of diverse approaches, of which solution and solid-state NMR spectroscopy are the most powerful to elucidate site-specific features. We review here several recent solution and solid-state NMR spectroscopic studies of 18.5 kDa MBP, and the induced partial disorder-to-order transitions that it has been demonstrated to undergo when complexed with calmodulin, actin, and phospholipid membranes.

Structured Functional Domains of Myelin Basic Protein: Cross Talk between Actin Polymerization and Ca2+-Dependent Calmodulin Interaction

The 18.5-kDa myelin basic protein (MBP), the most abundant isoform in human adult myelin, is a multifunctional, intrinsically disordered protein that maintains compact assembly of the sheath. Solution NMR spectroscopy and a hydrophobic moment analysis of MBPs amino-acid sequence have previously revealed three regions with high propensity to form strongly amphipathic α-helices. These regions, located in the central, N- and C-terminal parts of the protein, have been shown to play a role in the interactions of MBP with cytoskeletal proteins, Src homology 3-domain-containing proteins, Ca(2+)-activated calmodulin (Ca(2+)-CaM), and myelin-mimetic membrane bilayers. Here, we have further characterized the structure-function relationship of these three domains. We constructed three recombinant peptides derived from the 18.5-kDa murine MBP: (A22-K56), (S72-S107), and (S133-S159) (which are denoted α1, α2, and α3, respectively). We used a variety of biophysical methods (circular dichroism spectroscopy, isothermal titration calorimetry, transmission electron microscopy, fluorimetry, and solution NMR spectroscopy and chemical shift index analysis) to characterize the interactions of these peptides with actin and Ca(2+)-CaM. Our results show that all three peptides can adopt α-helical structure inherently even in aqueous solution. Both α1- and α3-peptides showed strong binding with Ca(2+)-CaM, and both adopted an α-helical conformation upon interaction, but the binding of the α3-peptide appeared to be more dynamic. Only the α1-peptide exhibited actin polymerization and bundling activity, and the addition of Ca(2+)-CaM resulted in depolymerization of actin that had been polymerized by α1. The results of this study proved that there is an N-terminal binding domain in MBP for Ca(2+)-CaM (in addition to the primary site located in the C-terminus), and that it is sufficient for CaM-induced actin depolymerization. These three domains of MBP represent molecular recognition fragments with multiple roles in both membrane- and protein-association.

Induced Secondary Structure and Polymorphism in an Intrinsically Disordered Structural Linker of the CNS: Solid-State NMR and FTIR Spectroscopy of Myelin Basic Protein Bound to Actin

The 18.5 kDa isoform of myelin basic protein (MBP) is a peripheral membrane protein that maintains the structural integrity of the myelin sheath of the central nervous system by conjoining the cytoplasmic leaflets of oligodendrocytes and by linking the myelin membrane to the underlying cytoskeleton whose assembly it strongly promotes. It is a multifunctional, intrinsically disordered protein that behaves primarily as a structural stabilizer, but with elements of a transient or induced secondary structure that represent binding sites for calmodulin or SH3-domain-containing proteins, inter alia. In this study we used solid-state NMR (SSNMR) and Fourier transform infrared (FTIR) spectroscopy to study the conformation of 18.5 kDa MBP in association with actin microfilaments and bundles. FTIR spectroscopy of fully (13)C,(15)N-labeled MBP complexed with unlabeled F-actin showed induced folding of both protein partners, viz., some increase in beta-sheet content in actin, and increases in both alpha-helix and beta-sheet content in MBP, albeit with considerable extended structure remaining. Solid-state NMR spectroscopy revealed that MBP in MBP-actin assemblies is structurally heterogeneous but gains ordered secondary structure elements (both alpha-helical and beta-sheet), particularly in the terminal fragments and in a central immunodominant epitope. The overall conformational polymorphism of MBP is consistent with its in vivo roles as both a linker (membranes and cytoskeleton) and a putative signaling hub.

Phosphorylation of Thellungiella salsuginea Dehydrins TsDHN-1 and TsDHN-2 Facilitates Cation-Induced Conformational Changes and Actin Assembly

Group 2 late embryogenesis abundant (LEA) proteins, also known as dehydrins, are intrinsically disordered proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperatures. These proteins are characterized by the presence of at least one conserved, lysine-rich K-segment and sometimes by one or more serine-rich S-segments that are phosphorylated. Dehydrins may stabilize proteins and membrane structures during environmental stress and can sequester and scavenge metal ions. Here, we investigate how the conformations of two dehydrins from Thellungiella salsuginea, denoted as TsDHN-1 (acidic) and TsDHN-2 (basic), are affected by pH, interactions with cations and membranes, and phosphorylation. Both TsDHN-1 and TsDHN-2 were expressed as SUMO fusion proteins for in vitro phosphorylation by casein kinase II (CKII), and structural analysis by circular dichroism and attenuated total reflection-Fourier transform infrared spectroscopy. We show that the polyproline II conformation can be induced in the dehydrins by their environmental conditions, including changes in the concentration of divalent cations such as Ca(2+). The assembly of actin by these dehydrins was assessed by sedimentation assays and viewed by transmission electron and atomic force microscopy. Phosphorylation allowed both dehydrins to polymerize actin filaments. These results support the hypothesis that dehydrins stabilize the cytoskeleton under stress conditions and further that phosphorylation may be an important feature of this stabilization.

MyelStones: the executive roles of myelin basic protein in myelin assembly and destabilization in multiple sclerosis

The classic isoforms of myelin basic protein (MBP, 14-21.5 kDa) are essential to formation of the multilamellar myelin sheath of the mammalian central nervous system (CNS). The predominant 18.5-kDa isoform links together the cytosolic surfaces of oligodendrocytes, but additionally participates in cytoskeletal turnover and membrane extension, Fyn-mediated signalling pathways, sequestration of phosphoinositides and maintenance of calcium homoeostasis. All MBP isoforms are intrinsically disordered proteins (IDPs) that interact via molecular recognition fragments (MoRFs), which thereby undergo local disorder-to-order transitions. Their conformations and associations are modulated by environment and by a dynamic barcode of post-translational modifications, particularly phosphorylation by mitogen-activated and other protein kinases and deimination [a hallmark of demyelination in multiple sclerosis (MS)]. The MBPs are thus to myelin what basic histones are to chromatin. Originally thought to be merely structural proteins forming an inert spool, histones are now known to be dynamic entities involved in epigenetic regulation and diseases such as cancer. Analogously, the MBPs are not mere adhesives of compact myelin, but active participants in oligodendrocyte proliferation and in membrane process extension and stabilization during myelinogenesis. A central segment of these proteins is pivotal in membrane-anchoring and SH3 domain (Src homology 3) interaction. We discuss in the present review advances in our understanding of conformational conversions of this classic basic protein upon membrane association, including new thermodynamic analyses of transitions into different structural ensembles and how a shift in the pattern of its post-translational modifications is associated with the pathogenesis and potentially onset of demyelination in MS.

Secondary Structure and Solvent Accessibility of a Calmodulin-Binding C-Terminal Segment of Membrane-Associated Myelin Basic Protein †

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(2+)-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 ≤13. 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 α-helix, with high accessibility to O(2) and low accessibility to NiEDDA. In membrane-associated rmC8, this segment assumed a structure distorted from an α-helix. Spin-labeled residues in rmC1 in solution were more immobilized on binding Ca(2+)-CaM than those in rmC8. Furthermore, rmC8 was dissociated more readily from a lipid bilayer by Ca(2+)-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(2+)-CaM than the unmodified form.

Misincorporation of the proline homologue Aze (azetidine-2-carboxylic acid) into recombinant myelin basic protein

We have evaluated the effects of the proline homologue Aze (1) (azetidine-2-carboxylic acid) on growth of Escherichia coli strains used to over-express recombinant forms of murine myelin basic protein (rmMBP), and on the degree of misincorporation. Addition of Aze to minimal media resulted in severe diminution of growth rate, but rmMBP could still be produced and purified. Mass spectrometry indicated that a detectable proportion of the rmMBP produced had incorporated Aze instead of proline (Pro), to a maximum of three of eleven possible sites. Molecular modelling of a proline-rich region of rmMBP illustrated that the misincorporation of Aze at any site would cause a severe bend in the polypeptide chain, and that multiple Pro-->Aze substitutions would completely disrupt a poly-proline type II structure that has been conjectured to be functionally significant.