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The myelin basic protein (MBP) family comprises a variety of developmentally-regulated members arising from different transcription start sites, differential splicing, and post-translational modifications. The "classic" isoforms of MBP include the 18.5 kDa form, which predominates in adult human myelin and facilitates compaction of the mature myelin sheath in the central nervous system, thereby maintaining its structural integrity. In addition to membrane-association, the 18.5 kDa and all other classic isoforms are able to interact with a multitude of proteins, including Ca(2+)-calmodulin, actin, tubulin, and SH3-domain containing proteins, and thus may be signalling linkers during myelin development and remodelling. All proteins in this family are intrinsically disordered, creating a large effective surface to facilitate multiple protein associations, and are post-translationally modified to various degrees by methylation, phosphorylation, and deimination. We have used spectroscopic (fluorescence, CD, EPR, and NMR) approaches to study MBP's conformational adaptability. A highly-conserved central domain presents an amphipathic alpha-helix in association with a phospholipid membrane, and contains a threonyl residue that is phosphorylated by MAP-kinases. In multiple sclerosis, this segment represents a primary immunodominant epitope. This helical structure is adjacent to a proline-rich region that presents a classic SH3-ligand, comprises a second MAP-kinase phosphorylation site, and forms a polyproline type II helix. This domain of the protein is thus essential to proper positioning of a protein-interaction motif, with the local conformation and accessibility being modulated by MAP-kinases. In addition, the C-terminus of 18.5 kDa MBP has been identified by NMR spectroscopy as a Ca(2+)-calmodulin-binding site, and is of note for having a high density of post-translational modifications (protein kinase C phosphorylation, and deimination). For the most part, any classic protein isoform functions as an entropic spring that interacts in its entirety with membranes and cytoskeletal proteins, but the central and C-terminal motifs may represent molecular switches.

The N-terminal region of the huntingtin protein, encoded by exon-1, comprises an amphiphilic domain (httNT), a polyglutamine (Qn) tract, and a proline-rich sequence. Polyglutamine expansion results in an aggregation-prone protein responsible for Huntington’s disease. Here, we study the earliest events involved in oligomerization of a minimalistic construct, httNTQ7, which remains largely monomeric over a sufficiently long period of time to permit detailed quantitative NMR analysis of the kinetics and structure of sparsely populated (≲2%) oligomeric states, yet still eventually forms fibrils. Global fitting of concentration-dependent relaxation dispersion, transverse relaxation in the rotating frame, and exchange-induced chemical shift data reveals a bifurcated assembly mechanism in which the NMR observable monomeric species either self-associates to form a productive dimer (τex ∼ 30 μs, Kdiss ∼ 0.1 M) that goes on to form a tetramer (τex≲25 μs; Kdiss ∼ 22 μM), or exchanges with a “nonproductive” dimer that does not oligomerize further (τex ∼ 400 μs; Kdiss ∼ 0.3 M). The excited state backbone chemical shifts are indicative of a contiguous helix (residues 3–17) in the productive dimer/tetramer, with only partial helical character in the nonproductive dimer. A structural model of the productive dimer/tetramer was obtained by simulated annealing driven by intermolecular paramagnetic relaxation enhancement data. The tetramer comprises a D2 symmetric dimer of dimers with largely hydrophobic packing between the helical subunits. The structural model, validated by EPR distance measurements, illuminates the role of the httNT domain in the earliest stages of prenucleation and oligomerization, before fibril formation.

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.

The mechanism whereby the prototypical chaperonin GroEL performs work on substrate proteins has not yet been fully elucidated, hindered by lack of detailed structural and dynamic information on the bound substrate. Previous investigations have produced conflicting reports on the state of GroEL-bound polypeptides, largely due to the transient and dynamic nature of these complexes. Here, we present a unique approach, based on combined analysis of four complementary relaxation-based NMR experiments, to probe directly the "dark" NMR-invisible state of the model, intrinsically disordered, polypeptide amyloid β (Aβ40) bound to GroEL. The four NMR experiments, lifetime line-broadening, dark-state exchange saturation transfer, relaxation dispersion, and small exchange-induced chemical shifts, are dependent in different ways on the overall exchange rates and populations of the free and bound states of the substrate, as well as on residue-specific dynamics and structure within the bound state as reported by transverse magnetization relaxation rates and backbone chemical shifts, respectively. Global fitting of all the NMR data shows that the complex is transient with a lifetime of <1 ms, that binding involves two predominantly hydrophobic segments corresponding to predicted GroEL consensus binding sequences, and that the structure of the bound polypeptide remains intrinsically and dynamically disordered with minimal changes in secondary structure propensity relative to the free state. Our results establish a unique method to observe NMR-invisible dynamic states of GroEL-bound substrates and to describe at atomic resolution the events between substrate binding and encapsulation that are crucial for understanding the normal and stress-related metabolic function of chaperonins. supramolecular machine | protein-protein interactions | conformational sampling

The 18.5 kDa isoform of myelin basic protein (MBP) is the predominant form in adult human central nervous system myelin. It is an intrinsically disordered protein that functions both in membrane adhesion, and as a linker connecting the oligodendrocyte membrane to the underlying cytoskeleton; its specific interactions with calmodulin and SH3-domain containing proteins suggest further multifunctionality in signaling. Here, we have used multidimensional heteronuclear nuclear magnetic resonance spectroscopy to study the conformational dependence on environment of the protein in aqueous solution (100 mM KCl) and in a membrane-mimetic solvent (30% TFE-d(2)), particularly to analyze its secondary structure using chemical shift indexing, and to investigate its backbone dynamics using (15)N spin relaxation measurements. Collectively, the data revealed three major segments of the protein with a propensity toward alpha-helicity that was stabilized by membrane-mimetic conditions: T33-D46, V83-T92, and T142-L154 (murine 18.5 kDa sequence numbering). All of these regions corresponded with bioinformatics predictions of ordered secondary structure. The V83-T92 region comprises a primary immunodominant epitope that had previously been shown by site-directed spin labeling and electron paramagnetic resonance spectroscopy to be alpha-helical in membrane-reconstituted systems. The T142-L154 segment overlapped with a predicted calmodulin-binding site. Chemical shift perturbation experiments using labeled MBP and unlabeled calmodulin demonstrated a dramatic conformational change in MBP upon association of the two proteins, and were consistent with the C-terminal segment of MBP being the primary binding site for calmodulin.

The prototypical chaperonin GroEL assists protein folding through an ATP-dependent encapsulation mechanism. The details of how GroEL folds proteins remain elusive, particularly because encapsulation is not an absolute requirement for successful re/folding. Here we make use of a metastable model protein substrate, comprising a triple mutant of Fyn SH3, to directly demonstrate, by simultaneous analysis of three complementary NMR-based relaxation experiments (lifetime line broadening, dark state exchange saturation transfer, and Carr-Purcell-Meinboom-Gill relaxation dispersion), that apo GroEL accelerates the overall interconversion rate between the native state and a well-defined folding intermediate by about 20-fold, under conditions where the "invisible" GroELbound states have occupancies below 1%. This is largely achieved through a 500-fold acceleration in the folded-to-intermediate transition of the protein substrate. Catalysis is modulated by a kinetic deuterium isotope effect that reduces the overall interconversion rate between the GroEL-bound species by about 3-fold, indicative of a significant hydrophobic contribution. The location of the GroEL binding site on the folding intermediate, mapped from 15 N, 1 H N , and 13 C methyl relaxation dispersion experiments, is composed of a prominent, surface-exposed hydrophobic patch.chaperonins | invisible states | dark state exchange saturation transfer | lifetime line broadening | relaxation dispersion C haperone networks have evolved to correctly fold native proteins and protect against the damaging effects of misfolding and aggregation on protein homeostasis (1, 2). The chaperonins, a ubiquitous subclass of chaperones, are barrel-shaped, multisubunit assemblies composed of two ring cavities, transiently capped by either an extrinsic cochaperone or a built-in lid domain, which assist protein folding in an ATP-dependent manner (3-6). Although the encapsulation mechanism and accompanying allosteric transitions driven by ATP have been extensively studied, the details of how chaperonins fold proteins remain elusive (3, 6, 7). Further, encapsulation does not appear to be an absolute requirement for successful re/folding (8). Moreover, hydrogen/deuterium exchange experiments on several protein substrates (9-12) and fluorescence-based refolding experiments (13) suggest that the prototypical chaperonin GroEL may possess intrinsic unfoldase activity. Here we take advantage of a monomeric, nonaggregating, well-defined system-a triple mutant of the Fyn SH3 domain that exists in dynamic equilibrium between the major native state and a sparsely populated folding intermediate (14, 15)-to directly demonstrate, using NMR relaxation-based methods (16), the ability of apo GroEL to accelerate the interconversion between these two states by almost three orders of magnitude. Simultaneous analysis of lifetime line-broadening (17), dark state exchange saturation transfer (DEST) (18), and Carr-Purcell-Meinboom-Gill (CPMG) relaxation dispersion (19) data permitted us to determine the cat...

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